Growth Stimulation of Phaseolus vulgaris L
Plantules by Strain Bacillus amyloliquefaciens
Hla Producer of Beneficial Agricultural Enzymes
Slimane Mokrani1,2, Lakhder Belabid2, Billel Bedjaoui2 and Elhafid Nabti1*
1University of Bejaia, Algeria
2Department of Agronomy, Laboratory of Research on Biological Systems, University of Mustapha Stumbouli, Algeria
Submission: July 17, 2018; Published: August 27, 2018
*Corresponding author: Elhafid Nabti, FSNV, Laboratory of Renewable Energies, Group of Biomass and Environment, University of Bejaia, Targa Ouzemmour, Bejaïa, Algeria, Email: email@example.com University of Agriculture Abeokuta (FUNAAB), Nigeria;
How to cite this article: Slimane M, Lakhder B, Billel B, Elhafid N. Growth Stimulation of Phaseolus vulgaris L Plantules by Strain Bacillus amyloliquefaciens
Hla Producer of Beneficial Agricultural Enzymes. JOJ Hortic Arboric. 2018; 2(2): 555581.
The strain HLA was identified as Bacillus amyloliquefaciens. Colonies were small and growth rapidly on TSA medium. Microscopic observation with Gram staining and epi-fluorescence revealed Gram positive and long bacilli bacteria. Strain HLA 16S rDNA similarities to the respective species were greater than (79 %). The phylogenetic analysis grouped Strain HLA with strain Bacillus amyloliquefaciens NR 117946.1 (95 %) and two strains of Bacillus subtilis NR113265.1 and NR112116.1 (95%). Strain HLA showed positive results for production of lecithinase, gelatinase and amylase. Inoculation of Phaseolus vulgaris L seed with Bacillus amyloliquefaciens HLA resulted on significative growth stimulation of plantules compared to the control, within 21 days of culture in pots. Bacillus amyloliquefaciens HLA increased, stem length (34.08 %), leaves area (96.5 %), root fresh weight (46.15 %) and root dry weight (70.41 %).
In the face of increasing threats to global food security and pressure on natural resources, there is an urgent need to identify and market biologically based products as agricultural crop protectants and yield enhancers . The common bean (Phaseolus vulgaris L.) is the most important pulse crop in the world. It is an important source of calories, proteins, dietary fibers, minerals, and vitamins for millions of people in both developing and developed countries worldwide . Annually, more than 143.88 million nutrient tonnes of chemical fertilizers are used worldwide to increase the yield of crop plants . Despite their efficiency in promoting crop yield, they can, under certain circumstances, pollute the environment and contribute to a number of human and animal health problems . Excessive fertilization is a potential global threat to the soil and environment through soil salinity, acidification as well as neutralization of the soil, heavy metal accumulation, water eutrophication, and accumulation of nitrate . Therefore, studies were aiming to replace chemicals by biofertilizers microorganisms. Biofertilizers (fertilizers of a microbial origin) are eco-friendly and can reduce hazards effects caused by uncontrolled application of chemical inputs . Biofertilizers have been an alternative to mineral fertilizers to increase the yield and plant growth in sustainable agriculture . Plant growth promoting rhizobacteria (PGPR) play a vital role
in crop protection, growth promotion and in the improvement of soil health . Ninety-five percent of Gram-positive soil bacilli belong to the genus Bacillus. Members of Bacillus species are able to form endospores and hence survive under adverse conditions . Bacillus amyloliquefaciens were shown to produce volatile compounds that promote plant growth . Production of enzymes that interfere with fungal pathogenesis. Production of chitinases, glucanases, cellulases, lipases and other lytic enzymes may also contribute to effective biocontrol activity .
In this study, strain HLA was identified phenotypicaly and cell microscopy. In addition, qualitative production of beneficial agricultural enzymes. In vivo stimulation plantules growth traits of Phaseolus vulgaris L by strain HLA, as shoot and root system, were determined.
Strain HLA was isolated in 2010 from rhizosphere soil of Phaseolus vulgaris L (common bean) from Tighanif Mascara (35°24’ N 0°19’E). The strain HLA was stored in broth TSB with 25% glycerol, subcultured every 12-18 months.
In order to determine the microscopic aspect of the strain HLA,
first subcultured onto TSA agar was carried out, Gram staining was
then performed, as well as observation by Fluorescence microscopy
(Leica DM 4000B) with camera (DFC310 FX), computer and image
processing software. Carried out by spreading a drop of strain HLA
suspension in sterile distilled water on a slide, then a microscopic
observation under a blue lamp and without coloration were done.
DNA extraction: Colonies in exponential phase were
suspended in 500μl of TE buffer containing (10 mM Tris-HCl
at pH= 7.4, 1 mM EDTA at pH=8). Followed by centrifugation
at 13.846xg for 3min. The pellet formed was re-suspended in
550μl of TE buffer. 17μl (35μg/ml) of lysozyme were added. The
mixture was incubated at 37 ℃ for 30 min. 30μl of SDS (10%)
were added. The mix was incubated at 37 ℃ for 30min. 100μl
NaCl (5 M) and 80μl CTABS/NaCl (10%/0.7M) were versed. The
whole compounds were mixed by inversion then incubated at 65
℃ for 10min. 800μl of CAI (chloroform/Isoamyl alcohol) were
added. The mixture was centrifuged at 13.846 xg for 30min.
The supernatant was recuperated and precipitated by addition
of 0.6ml of isopropanol and followed by incubation at -20 ℃ for
1-24 h. After centrifugation at 13.846xg for 5min, the pellet was
recovered and washed with ethanol (70%), then centrifuged at
13.846xg for 5min. After drying at 37 ℃, total DNA was dissolved
in 100μl of TE buffer and treated with ARNase 1μl (20μg/ml) and
incubated at 37 °C for 1 h. Finally, genomic DNA recuperated was
conserved at -20 ℃.
DNA amplification: PCR amplifications were performed
using the following primers: Amorce Formed 16SF (5’- AGA GTT
TGA TCC TGG CTC AG -3’) and Amorce reverse 16SR (5’- CTA
CGG CTA CCT TGT TAC GA-3’) for 16S rRNA gene. Amplification
was carried out using thermalcycler (BOECO TC PRO, Germany)
for microtubes containing volume 25μl: 1μl of ADN and 24μl of
relational mixture; each microtube contained: (2.5μL tampon,
2.5μL MgCl2 (20 mM), 0.5μL dNTP (25mM), 0.3μL amorce Formed,
0.3μL amorce Reverse, 0.2 μL Taq polymerase, 17.7μL H2O and
1μL DNA. 35 Cycle were applied: preheating at 94°C for 3min
denaturation at 94 ℃for 45s, hybridation at 55 ℃ for 1 min,
Elongation at 72°C for 2 min, final elongation at 72 ℃ for 7min
and final cooling at 14 ℃ for 7min.
DNAr16s sequencing: ADNr 16s sequencing was carried out
by the Laboratory of Microorganisms and Actives Biomolecules of
the Faculty of Sciences of Tunis (Tunisia) according to the Applied
Biosystems protocol on the automatic sequencer (ABI-model
3730xl). ADNr 16s sequences obtained were compared with the
nucleotide sequences of international databases using BLAST
(Basic Local Alignment Search Tool Program available on line. The
alignments of the nucleotide sequences were carried out by the
Clustal W algorithm. The phylogenetic tree was obtained by MEGA
7 software and the neighbor-joining algorithm. Bootstrap values
were determined from 100 replicates.
Lecithinase: The strain HLA was streaked on egg yolk agar.
Followed by incubation at 30 ℃ for 7 days. The appearance of
opaque zones around the colonies indicates the presence of
Lipase: To determinate lipase production, the strain HLA was
streaked on Tween 80 nutrient agar supplemented with CaCl2. The
Petri plate was incubated at 30 °C for 7 days. The appearance of
opaque areas around the colonies indicates the presence of lipase
Pectinase: Diluted nutrient agar (¼), which is equivalent to
1000ml/4, added of 0.5 % pectin was used to determine pectinase
production by the strain HLA. The bacterium was streak on the
medium and incubated at 30 ℃ for 48h. The appearance of a clear
halo around growth indicated the presence of a pectinase .
Gelatinase: The strain HLA was streaked in an epindorff
tube containing the gelatin agar medium with (12%). The control
consisted of an epindorff tube containing unincolated gelatin agar.
The two tubes were sealed with parafilm, then incubated at 20 ℃
for 15 days. The tubes are set at 4-5 ℃ for 15min. The liquefaction
of gelatin was revealed by the inclination of the agar in the test
tube and the solidification of the control tube.
Amylase: The strain HLA was streaked on starch agar. Petri
plate awa then incubated at 30 ℃ for 2-7 days. After incubation,
the Petri plate was flooded with iodine solution. Appearance of
clear zones around the bacterial growth, indicated the presence
of an amylasic activity
Seed sterilization and soil preparation:
Bean seeds of
Phaseolus vulgaris L were desinfected by soaking in calcium
hypochlorite (2%) for 30min. Then seed were rinsed 3 times with
sterile distilled water. Soil was prepared by mixing it with peat
(1/3) then sterilized in the pastor oven at 100 ℃ for 3-4 days.
The soil is distributed in pots (Superior Ɵ: 7 cm, Inferior Ѳ: 4cm
Height: 8.5cm) containing approximately 136g of soil per pot.
Plantules culture and measure of growth traits: Two
treatments were prepared (four pots per batch); one traitement
was treated with the strain HLA and another control treatment.
Three bean seeds were sown per pot. The seeds of the first
treatment were soaked in a suspension of strain HLA at a
concentration of 108UFC/ml for 30min. The control treatment
consisted of seeds soaked in sterile distilled water. The seeds
treated, and the controls were coated with starch. The seeds were
sown, and the crop was followed for 21 days, under laboratory
conditions. The seedlings were watered every two to three days
with 10-20ml of sterile distilled water. To evaluate the effects of
strain HLA on plantules shoot and root systems growth, traits were
measured compared to the control. The measured traits were:
stems length, leaves area, fresh and dry weight roots (Burton,
1979). Leaves area were expressed using images of treated and untreated leaves by image J software. Percentages of plantules
growth were evaluated using the formula:
PGS = (t – c) /t ×100
Where, PGS: Percentage growth stimulation of each trait, t:
trait mean of plantules treated with strain HLA, c: trait mean of
Growth traits of treated plantules with strain HLA and control
were subjected to t- t using statistica.5.0 software at 5%. Traits
(SL and DWRB) were analyzed by two-tailed t-test for unequal
variances and (LA and WFRB) were analyzed by t-test for equal
variances. Normality of the distributions was verified by W test of
Shapiro-Wilk and variances homogeneity was verified by Levene
Macroscopic examination of strain HLA revealed that colonies
were creamy-white and rough and grew rapidly on TSA medium
(Figure1). Gram staining and microscopic examination of strain
HLA with and without coloration showed, Gram positive bacteria,
cells observed were long Bacilli (Figure 2).
The constructed phylogenetic tree using the 16s rRNA gene
sequences of the related strain HLA from Bacillus amyloliquefaciens
and Bacillus subtilis (Figure 3). The phylogenetic tree clearly
showed that the strain Bacillus HLA could be divided into two
closely related clades, separated by a long Branch length, which
indicates an unlimited number of nucleotide changes. Bacillus
amyloliquefaciens HLA belong to one clade that was closely related to the B. amyloliquefaciens strain NR 117946.1 (GenBank
accession). Four strains made another clade that comprised to
references strains Bacillus subtilis NR113265.1 and NR 112116.2.
These results were supported by the BLAST search analysis against
published 16S rRNA gene sequences in the GenBank database.
Characterization of beneficial agricultural enzymes: In
the present study the extracellular lecithinase, lipase, pectinase,
gelatinase and amylase activities of Bacillus amyloliquefaciens
HLA were qualitatively evaluated (Table 1). Strain HLA showed
positive results for production of lecithinase, gelatinase and
amylase. Negative production of lipase and pectinase.
In vivo study of effect of strain HLA on Phaseollus vulgaris L
revealed an increase growth of treated plantules, within 21days
(Figure 4). The application of strain HLA revealed showed clearly
that treated plantules were high developed, leaves and stems were
largest, compared to the control.
Stimulation of shoot system: Application of strain Bacillus
amyloliquefaciens HLA induced growth stimulation of shoots
system and stem length (SL) of Phaseolus vulgaris L seedlings
(Figure 5). Leaves area (LA) were characterized by significative
growth area compared to the control. The leaves area increased
to 3.2±0.566cm2 (96.5%) for the plantules treated with strain
HLA compared to the control which represented leaves area of
0.112±0.032cm2. Stem length of the treated pantules with strain
HLA, increased to 31.1±4.1cm (34.08%) control 20.5±1.4cm.
Stimulation of root growth: Strain Bacillus amyloliquefaciens
HLA induced also growth of plantules root system (Figure
6). The root systems were visually affected, revealing clearly
that root treated with a strain HLA were high compacted and
condensated compared to the control. The dry and fresh weight
roots of plantules treated by strain Bacillus amyloliquefaciens
HLA increased significative (Figure 7). The fresh weight of the
root biomass (WFRB) increase to 1.3±0.3g (46.15%) compared to
the control of 0.7±0.4g. Also, the dry weight of the root biomass
(DWRB) increased to 0.1893±0.0445g (70.41%) compared to the
control 0.0560±0.0158g .
The use of PGPR to enhance plat crops is an environment
friendly approach and an effective alternative to toxic chemical
fertilizers. Microorganisms that presents several biologic activities
of agricultural interest, practically, can make them subject of
biofertilizing object, for this the enzymatic tests play a crucial role in
the microbial screening . Those bacteria produce extracellular
enzymes as strategies to join skills to get ecological advantages
. Pectinase is a group of enzymes that known to catalyse the
pectic substance through depolymerisation and deexrification
reaction. These enzymes have the role in preventing plant from
infection caused by pathogen . Protease, which can degrade
cell wall proteins; and lipase, which can degrade some of the cell
wall-associated lipid, all of which can to some extent individually
lyse fungal cells . Bacillus spp. strains are an effectiveness
soil bacterium characterized by various enzymatic activity with
implications either in cell wall degradation of plant pathogenic
microorganisms, or in metabolism of various substrates. Those
enzymes included cellulases, chitinases, proteases (gelatinases)
and lipases . Production of chitinases, glucanases, cellulases,
lipases and other lytic enzymes may also contribute to effective
biocontrol activity .
In this study, inoculation of Phaseolus vulgaris L seed with
strain Bacillus amyloliquefaciens HLA increased plants growth.
Growth traits of shoot and root system were signivicativelly
increased there is no single mechanism for promoting plant
growth . The mechanisms by which bacteria can influence
plant growth differ among species and strains, so typically PGPR
exsers their effects on root tissues, can modify the physiology
and functioning of plant tissues located at a substantial distance
from the colonized sites, such as shoots. Two types of mechanisms
are involved. On the one hand, some PGPR can enhance nutrient
availability/uptake for plant roots. On the other hand, certain
PGPR trigger specific systemic responses, mostly by unknown
signaling mechanisms .
Sufficient densities of PGPR in biofertilizer provide a beneficial
role in creating a proper rhizosphere for plant growth and
converting nutritionally important elements through biological
process, for example increasing the availability of N, P, K, as well
as inhibiting pathogen growth . The high availability of N, P,
and K could enhance soil fertility, improve antagonistic isolates’
bio-control effects, and extend microorganisms’ survival rates
in soil . By far the most evidence for the positive effects of
biofertilizing-PGPR points to bacteria-mediated changes in root
growth and morphology. Bacterial mediated increases in root
weight are commonly reported responses to GPR inoculations.
Sabaté et al.  reported that B. amyloliquefaciens in
greenhouse experiments showed that the black common bean
cv. Nag 12 seeds inoculated with B14, had increased germination
of 10%, as well as an increase in root length of 2cm and in shoot
length of 6 cm compared with the non-inoculated control seeds.
PGPR treatments increased fresh and dry shoot, stem diameter,
seedling height, chlorophyll reading values, and leaf area of
cabbage seedlings. Such an improvement might be attributed to
the N2-fixing and phosphate-solubilizing capacities of bacteria,
as well as the ability of these microorganisms to produce growthpromoting
Strain Bacillus amyloliquefaciens HLA, identified and used in
this present study as a biofertilizer of growth of Phaseolus vulgaris
L shows that it is a promising natural antagonist. It exhibited
sufficient enzymes with beneficial agriculture application, that
can play as the same time role on suppressing pathogens and
stimulation plant growth by degradation of macromolecules/
nutriments in soil.
Research funding from Laboratory of Biotechnology and Bio-
Geo Resources Valorization, Higher Institute for Biotechnology,
University of Manouba, Ariana, (Tunisia) and Laboratory of Microorganisms and Actives Biomolecules of the Faculty of
Sciences, Tunis (Tunisia).