Annals of Agrarian Science 16 (2018) 413–415

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Annals of Agrarian Science

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Brevibacillus laterosporus as perspective source of new bioinsecticides T

∗
N.L. Ghazanchyan , M.H. Kinosyan, P.E. Tadevosyan, N.S. Khachaturyan, E.G. Afrikian
Microbial Depository Center of Scientific and Production Center, “Armbiotechnology” NAS of Armenia, 14, Gyurjyan Str., Yerevan, 0056, Armenia

A R T I C LE I N FO A B S T R A C T

Keywords: The paper discusses the main features of Brevibacillus laterosporus bacilli as bioinsecticides for pest control, as
Entomopathogenes well as perspective sources for searching the novel cancerolytic substances of microbial origin. It has been
Brevibacillus laterosporus established that the culture of B. laterosporus is widespread in Armenia as a highly active producer of protein
Bacillus thuringiensis inclusions against leaf beetles. Isolated strains of B. laterosporus can be used as an alternative to biological
Bioinsecticides
larvicides, which are produced on the basis of B. thuringiensis and B. sphaericus.
Parasporins
Antimicrobial activity

Introduction development of insecticides on the basis of these bacteria and wide

In connection with rapid development of biotechnology and the
need to ensure ecological conditions of life and industry there is a
growing interest in spore-forming bacteria. Bacillus subtilis and Bacillus
thuringiensis have been the main objects of basic and applied researches
during many years. В. subtilis strains are producers of enzymes, used as
prebiotics. В. thuringiensis strains are almost unique producers of bio-
logical agents of plant protection. Meanwhile the recent investigations
indicate that there are bacilli, which have a number of valuable prop-
erties in terms of applied microbiology. Bacteria Brevibacillus later-
osporus (previous Bacillus laterosporus) are the most important among
them. It has been shown that B. laterosporus produce insecticide agents,
enzymes, antibacterial and antifungal agents, they are used as effective
prebiotics [1,2].
One of the urgent tasks of biotechnology and ecology is to develop
safe and effective biological methods for pest control. Biological
methods for pest control include the use of industrial entomopathogenic
strain-producers of В. thuringiensis and Bacillus sphaericus as bioinsec-
ticides demonstrating selective activity against mosquitoes [3]. The
pathogenicity of these bacteria is driven by crystalline and non-crys-
talline toxins of protein origin. Nevertheless the spread of pests resistant
to products based on В. thuringiensis and В. sphaericus necessitates the
search for new bacterial species producing insecticidal toxins with
specific characteristics [4,5].
Entomopathogenic strains В. thuringiensis and В. sphaericus have
been well studied, while the entomopathogenic and other biological
features of В. laterosporus were not sufficiently studied. That is why the
usage of В. laterosporus in different fields of biotechnology are impeded.
In this work entomopathogenic spore-forming bacteria especially B.
laterosporus and related species have been studied.
Materials and methods
The strains of B. laterosporus used in this work were isolated from
insects, samples of soil and trees infected by leaf beetle of various re-
gions of Armenia.
The identification of isolated strains was carried out on the basis of
their morphological and physiological characteristics and by modern
methods of molecular genotyping based on the use of specific primers
[6–8]. The extraction of genomic DNA from bacilli has been done ac-
cording to benzyl chloride extraction method [7]. DNA samples have
been amplified by PCR technology and visualization of the results ob-
tained has been realized after agarose gel electrophoresis by further
exposure under UV transilluminator, what indicates high specificity of
used primers to B. thuringiensis and В. sphaericus.
Auxanographic method. Determination of needs for carbohydrates
was carried out by auxanographic method, using 24 carbon sources on
Petri dishes, containing Pridham agar medium [9]. Bacterial cells were
added to agar at 40–45 °C and sterile paper discs soaked in solution
containing 5 mg/ml carbon source were placed on the agar surface.
ͦ
Plates were incubated at 28–30 C. The bacterial growth around discs
was checked in 24–48 h later.

Peer review under responsibility of Journal Annals of Agrarian Science.

∗
Corresponding author.
E-mail address: microbio@sci.am (N.L. Ghazanchyan).

https://doi.org/10.1016/j.aasci.2018.03.011
Received 18 February 2018; Accepted 29 March 2018
Available online 11 July 2018
1512-1887/ © 2018 Agricultural University of Georgia. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
N.L. Ghazanchyan et al. Annals of Agrarian Science 16 (2018) 413–415

Antimicrobial activity microorganisms has been carried out. According to the results of this
study, B. laterosporus strains have demonstrated antagonism in relation
Antagonistic features of B. laterosporus strains in relation to gram to other species of Bacillus, as well as gram positive and gram negative
positive and gram negative bacteria as well as to fungi were studied by bacteria (see Table 2).
using the following test strains: Micrococcus luteus 5270, Bacillus subtilis The tested strains of B. laterosporus did not have influence on the
1820, B. cereus 2111, Staphylococcus aureus 5233, Mycobacterium sp. growth of E. coli 5002 and C. albicans 8013, fungi A. flavus, P. expansum
5253, Serratia marcescens 5251, E. coli 5002, Candida albicans 10231, and A. alternata. The antagonistic action of B. laterosporus strains can be
Streptococcus faecalis 5254, Pseudomonas fluorescens 5248, Aspergillus explained by produced metabolites and enzymes such as endotoxins,
unilateralis 10648, A. flavus 10676, Penicillium funiculosum 8189, P. bacteriocins, antibiotics, phospholipases, proteases, etc. [12,13] (see
expansum 8134, Alternaria alternata 8126 (MDC, Table 3).
SPC″Armbiotechnology” NAS RA). Insecticidal features. The insecticidal activity of B. laterosporus strains
Test culture suspension was added to nutrient agar and placed in was tested on leaf beetle of Coleoptera detachment, of Chrysomelidae
Petri dishes. B. laterosporus strains were sown on the agar surface by family. The results of testing demonstrated that influence of the strain
loop. 199-3 caused 100% death of the insect. The insecticidal activity of B.
laterosporus towards Coleoptera, but not towards Lepidoptera was shown
earlier [14,15].
Results and analysis
Today the great interest has been given to the parasporal bodies
possessing anticancer activity. Such parasporal bodies are produced by
During long-term work the vast collection of entomopathogenic
species and subspecies of Bacillus thuringiensis, L. sphaericus and related
bacilli (more than 5000 strains) isolated from various sources has been
species [16,17]. Our investigations of entomopathogenic Bacilli have
created in MDC. The physiological, biochemical and genetic char-
revealed that formation of crystalline protein bodies (parasporins) is
acteristics of these strains were thoroughly studied. The number of
indicative characteristic for these bacteria. Searching for new para-
strains were used in industrial production of insecticides. The collection
sporins derived from bacteria of genus Bacillus, having specific effect on
includes entomopathogenic strains active against a wide range of pest
certain tumor cell lines was provided [18].
and infection spreading mosquitoes, mites and other insects.
Study of parasporins produced by entomopathogenic bacteria paves
The new species of enthomopathogenic bacilli producing inclusions
the way to reveal novel anticancer drugs.
of protein nature were revealed by serological and modern methods of
molecular genotyping. 30 strains were isolated from various samples.
Isolated bacteria were identified as Brevibacillus laterosporus according
Conclusion
to morphological and physiological features and molecular genotyping.
All strains did not produce crystalline inclusions.
The species specificity of entomopathogenic Bacilli producing crys-
Morphological features. B. laterosporus is a rod-shaped, endospore-
talline toxins reflects their genetic patterns and is of basic importance
forming bacterium morphologically characterized by the production of
for directed screening of the strains needed. So, the strategy for ob-
a typical canoe-shaped parasporal body (CSPB) firmly attached to one
taining of new insecticides from Bacilli has to be based on isolation and
side of the spore, which determines its lateral position in the spor-
use of their new species and subspecies.
angium [10,11]. On agar surface B. laterosporus forms flat, smooth
Nevertheless among bacilli there are many other species to be stu-
(plain), white or beige colonies with rhizoid margins.
died and used as perspective source of new insecticides: many sub-
Utilization of carbohydrates. The utilization of 24 carbohydrates by
species or serovars of L. sphaericus, B. thuringiensis, B. lentimorbus, B.
strains of B. laterosporus was studied (Table 1).
laterosporus and related Bacilli producing specific entomocidal crystal-
According to auxanographic data, all tested strains of B. laterosporus
line toxins of proteinous nature or having infectious action on the in-
could not utilize 17 carbohydrates from 24: L-, D-arabinose, L-, D-lyxose,
sect.
D-fucose, L-rhamnose, L-sorbose, sucrose, D-melibiose, D-melezitose, D-
Today entomopathogenic aerobic spore-forming B. laterosporus
galactose, L-lactose, D-raffinose, inulin, D-sorbitol, D-mannitol, D-xy-
strains having high level of parasporal body production are very at-
litol. All tested strains were able to utilize fructose, glucose, trehalose,
tractive from the scientific and industrial point of view. The wide range
maltose. Four strains could not utilize ribose and mannose. The ob-
of entomopathogenic features of these strains should be used for pest
tained results have indicated the diversity of B. laterosporus species.
control. Biocide effect (antibacterial, fungicidal) allows recommending
Antagonistic features. Since the data on antagonistic features of B.
B. laterosporus strains as producers of biological means of plant pro-
laterosporus are limited, the study B. laterosporus strains antimicrobial
tection.
activity and spectrum of their influence on the other groups of

Table 1
Utilization of carbohydrates by B. laterosporus.
Strain number D- ribose D- glucose D- fructose D- mannose D- cellobiose D - trehalose D- maltose

59–1 – 2+ + + 3+ + +
69–1 2+ 2+ 3+ 3+ 3+ 3+ 3+
69-3-5 – + + + + + +
101–5 + 2+ 2+ + + + +
233–2 – 2+ 2+ – – + +
670–1 + + 2+ 2+ – + +
688–2 2+ 2+ 2+ + + 2+ 2+
194–1 2+ 2+ 3+ 2+ – 2+ 2+
199–3 + + + + + 2+ +
105–1 2+ + 2+ 2+ – 2+ 3+
209–3 2+ 2+ 3+ 2+ + 2+ 2+
186–1 – 2+ 2+ – 2+ + 2+
186–2 + 2+ 2+ – 2+ + –

Notes: ”+” - growth 1–10 mm; “2+” - 11–20 mm; “3+” - more than 21 mm; ” - ” no growth.

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N.L. Ghazanchyan et al. Annals of Agrarian Science 16 (2018) 413–415

Table 2
Antimicrobial activity of B. laterosporus species.
No. of Species and No. of test-cultures
B. laterosporus
strains E.coli 5002 St.aureus S.marcescens Myc.sp. St.faecalis Ps. fluorescens B.subtilis B.cereus Mic.luteus C.albicans
5233 5251 5253 5254 5248 1820 2111 5270 8013

186–1 – 2+ + 3+ 3+ + + 3+ 3+ –
688–2 – 2+ + 3+ 3+ 2+ + 3+ 3+ –
69–1 – 2+ + 3+ 3+ + + 3+ 3+ –
184–2 – 2+ + 3+ 3+ 3+ 2+ 3+ 3+ –
194–1 – 2+ + 3+ 3+ 3+ + 3+ 3+ –
105–1 – 2+ + 3+ 3+ 2+ 2+ 2+ 2+ –
200–4 – + + 2+ 2+ + + – + –
199–3 – + – 2+ 3+ + + 3+ 2+ –
59–1 – + + 2+ 2+ 3+ + – + –
233–2 – + + 2+ 2+ 3+ + – + –
561–1 – + + 2+ 2+ 3+ + – + –
55–2 – + + 2+ 2+ 3+ + – – –
69-3-5 – 2+ + 2+ 2+ + 3+ 2+ 2+ –
209–3 – + + 2+ 2+ 3+ 3+ 2+ 2+ –

Notes: growth inhibition: “+” - 1–3 mm, “2+” - 4–7 mm, “3+” - 8–10 mm; no inhibition: “-”

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