Molecular Characterization Reveals Biodiversity and Biopotential of Rhizobacterial Isolates of Bacillus Spp

Abstract

Bacillus species appearas the most attractive plant growth-promoting rhizobacteria (PGPR) and alternative to synthetic chemical pesticides. The present study examined the antagonistic potential of spore forming-Bacilli isolated from organic farm soil samples of Allahabad, India. Eighty-seven Bacillus strains were isolated and characterized based on their morphological, plant growth promoting traits and molecular characteristics. The diversity analysis used 16S-rDNA, BOX-element, and enterobacterial repetitive intergenic consensus. Two strains, PR30 and PR32, later identified as Bacillus sp., exhibited potent in vitro antagonistic activity against Ralstonia solanaceorum. These isolates produced copious amounts of multiple PGP traits, such as indole-3-acetic acid (40.0 and 54.5 μg/mL), phosphate solubilization index (PSI) (4.4 and 5.3), ammonia, siderophore (3 and 4 cm), and 1-aminocyclopropane-1-carboxylate deaminase (8.1and 9.2 μM/mg//h) and hydrogen cyanide. These isolates were subjected to the antibiotic sensitivity test. The two potent isolates based on the higher antagonistic and the best plant growth-promoting ability were selected for plant growth-promoting response studies in tomatoe, broccoli, and chickpea. In the pot study, Bacillus subtilis (PR30 and PR31) showed significant improvement in seed germination (27–34%), root length (20–50%), shoot length (20–40%), vigor index (50–75%), carotenoid content (0.543–1.733), and lycopene content (2.333–2.646 mg/100 g) in tomato, broccoli, and chickpea. The present study demonstrated the production of multiple plant growth-promoting traits by the isolates and their potential as effective bioinoculants for plant growth promotion and biocontrol of phytopathogens.

Full text

Vol.:(0123456789)
Microbial Ecology (2024) 87:83
https://doi.org/10.1007/s00248-024-02397-w
RESEARCH
Molecular Characterization Reveals Biodiversity andBiopotential
ofRhizobacterial Isolates ofBacillus Spp
AlkaSagar1,2· ShaliniRai2,3· SoniaSharma1· KahkashanPerveen4· NajatA.Bukhari4· R.Z.Sayyed5,6·
AndreaMastinu7
Received: 5 January 2024 / Accepted: 4 June 2024
© The Author(s) 2024
Abstract
Bacillus species appearas the most attractive plant growth-promoting rhizobacteria (PGPR) and alternative to synthetic
chemical pesticides. The present study examined the antagonistic potential of spore forming-Bacilli isolated from organic
farm soil samples of Allahabad, India. Eighty-seven Bacillus strains were isolated and characterized based on their morpho-
logical, plant growth promoting traits and molecular characteristics. The diversity analysis used 16S-rDNA, BOX-element,
and enterobacterial repetitive intergenic consensus. Two strains, PR30 and PR32, later identified as Bacillus sp., exhibited
potent invitro antagonistic activity against Ralstonia solanaceorum. These isolates produced copious amounts of multiple
PGP traits, such as indole-3-acetic acid (40.0 and 54.5 μg/mL), phosphate solubilization index (PSI) (4.4 and 5.3), ammonia,
siderophore (3 and 4 cm), and 1-aminocyclopropane-1-carboxylate deaminase (8.1and 9.2 μM/mg//h) and hydrogen cyanide.
These isolates were subjected to the antibiotic sensitivity test. The two potent isolates based on the higher antagonistic and
the best plant growth-promoting ability were selected for plant growth-promoting response studies in tomatoe, broccoli, and
chickpea. In the pot study, Bacillus subtilis (PR30 and PR31) showed significant improvement in seed germination (27–34%),
root length (20–50%), shoot length (20–40%), vigor index (50–75%), carotenoid content (0.543–1.733), and lycopene con-
tent (2.333–2.646 mg/100 g) in tomato, broccoli, and chickpea. The present study demonstrated the production of multiple
plant growth-promoting traits by the isolates and their potential as effective bioinoculants for plant growth promotion and
biocontrol of phytopathogens.
Keywords Bacillus· Biocontrol· Phytohormone· Phytostimulant· Plant growth promotion· Siderophore
* Alka Sagar
alka.sagar@miet.ac.in; alka2011sagar@gmail.com
* R. Z. Sayyed
sayyedrz@gmail.com
* Andrea Mastinu
andrea.mastinu@unibs.it
Shalini Rai
shalinimicro09@gmail.com
Sonia Sharma
sonia.sharma@miet.ac.in
Kahkashan Perveen
kperveen@ksu.edu.sa
Najat A. Bukhari
najatab@ksu.edu.sa
1 Department ofMicrobiology andBiotechnology, Meerut
Institute ofEngineering andTechnology, Meerut, India
2 Department ofIndustrial Microbiology, Sam Higginbottom
University ofAgriculture, Technology andSciences,
Allahabad211007, India
3 Department ofBiotechnology, SHEPA, Varanasi, India
4 Department ofBotany & Microbiology, College ofScience,
King Saud University, P.O. Box-22452, 11495Riyadh,
SaudiArabia
5 Department ofMicrobiology, PSGVP Mandal’s S. I. Patil
Arts, G B Patel Science andSTKV Sangh Commerce
College, Shahada425409, India
6 Faculty ofHealth andLife Sciences, INTI International
University, Persiaran Perdana BBN, Putra Nilai, 71800Nilai,
NegeriSembilan, Malaysia
7 Department ofMolecular andTranslational Medicine,
Division ofPharmacology, University ofBrescia,
25123Brescia, Italy
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A.Sagar et al. 83 Page 2 of 14
Abbreviations
ACCD 1-Aminocyclopropane-1-carboxylate
deaminase
GI Germination index
HCN Hydrogen cyanide
IAA Indole-3-acetic acid
MOF Model Organic Farm
NA Nutrient agar
PGI Percent growth inhibition
PGP Plant growth promoting
PGPR Plant growth-promoting rhizobacteria
PSI Phosphate solubilization index
SD Siderophore
SHUATS Higginbottom University of Agriculture, Tech-
nology and Sciences
Introduction
Bacteria of the genus Bacillus are soil-borne, endospore-
forming, and stress-resistant bacteria from the phylum Fir-
micutes. They are ubiquitously present in many ecological
conditions [1]. Gram-positive Bacillus species are the most
promising plant growth-promoting rhizobacteria (PGPR),
ecologically sound, and economically viable alternative
to the pesticide usage in agriculture [2, 3]. These bacte-
rial strains colonize the crop rhizosphere, efficiently sup-
press phytopathogens, and promote plant growth. Several
researchers have reported the diversity, phylogeny, produc-
tion, and secretion of degradative enzymes to combat phy-
topathogens [4], the production of a wide array of secondary
metabolites and antibiotics [5, 6], and defense mechanisms
like plant-induced systemic resistance [7, 8]. Some of the
species of Bacillusaredescribed as endophytes that can
promote plant growth using varied mechanisms, including
colonization in roots, enlargement of root density, solubili-
zation of minerals, enhanced nutrient uptake, and induced
defense responses against abiotic and biotic factors [2, 9,
10]. Moreover, researchers employ Bacillus isolates that
exhibit multifarious potentials such as phosphorus solubi-
lization [11], production of indole-3-acetic acid (IAA) [12],
siderophore [13], and 1-aminocyclopropane-1-carboxylate
deaminase (ACCD) activity [14]. Bacillus species arewidely
used biocontrol agents against various phytopathogens as
commercially developed formulations are available [2, 15,
16].Commercially available forms of some Bacillus species
include phytostimulants [17], biopesticides, and biofertiliz-
ers [18]. It has been widely used on various plants, including
tobacco, soybean, cucumber, maize, rice, and watermelon
[18–21].
Bacterial wilt caused by Ralsatonia solanacearum
is a quarantine phytopathogen responsible for devastat-
ing agricultural losses worldwide [22]. It is a soil-borne
phytopathogen that infects various commercial crops and
can survive for long periods in the soil. During favora-
ble conditions, the dormant bacterium is activated, enters
through primary and secondary roots, and colonizes the host
plants’ xylem vessels, leading to a lethal wilt disease. This
disease causes enormous losses from the time of sowing till
maturity. The disease is characterized by the appearance of
yellow-colored leaves and light brown-colored lesions on the
shoot androot, leading toreduced crop yield [22].
The present study evaluatedthe diversity, plant growth-
promoting ability, and antagonistic activity of Bacillus sp.
against R. solanacearum in tomato, broccoli, and chickpea.
Materials andMethods
Soil Sample
Ten grams of each sandy-loam soil sample were collected
from the rhizosphere soil of tomato plants in Sam Higgin-
bottom University of Agriculture, Technology and Sciences
(SHUATS) Model Organic Farm (SMOF) Allahabad, India,
(25° 24′ 42" N, 81° 50′ 56" E) [23, 24] (Fig.1). Briefly, a
rhizosphere soil sample (1g) was transferred to 9mL of
sterilized phosphate buffer saline (PBS; 10mL in 100mL
flask; pH 7.2) for 30min, heated at 80°C for 20min in a
water bath. The soil suspensions were serially diluted (10–3
to 10–5), and 0.1mL of this suspension was spread on Nutri-
ent Agar (NA) plates comprising methyl red (0.2%). The
plates were incubated for 24h at 30°C. Single bacterial
colonies were streaked onto fresh NA plates. Endospore
staining was performed as described by Hamouda etal. [25].
Screening ofAntagonistic Bacillus Sp.
Seven selected Bacillus isolates were used for invitro antag-
onistic activity using disc diffusion against R. solanacearum
[26]. The growth inhibition of the pathogen was measured
as the percent growth inhibition (PGI) using the following
formula -
where C = measure of control group growth and T = measure
of treatment group.
The test was repeated 3 times with 5 independent
replications.
Molecular Identification ofBacillus Isolates
The standard microbiological protocols were followed for mor-
phological and biochemical characterization of isolates. DNA
PGI
=
C−T
C
×
100
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Molecular Characterization Reveals Biodiversity andBiopotential ofRhizobacterial Isolates…
Page 3 of 14 83
extraction, partial 16S rRNA gene amplification, PCR product
purification, and subsequent sequencing analysis of Bacillus
isolates were performed as previously described [27]. The
16S rRNAgenewas amplified using universal primers, PF [5′-
TGG CTC AGA TTG AAC GCT GGCGG-3′] and PR [5′-TGG
CTC AGA TTG AAC GCT GGCGG-3′], and the PCR products
were sequenced on ABI3100 Genetic Analyzer. The amplified
sequences were run in the BLASTnprogram and compared
with the NCBI database.
Using the multiple sequence alignment tool Clustal W, con-
sensus sequences of the 16S rRNA gene from Bacillus isolates
and reference sequences obtained from Genbank were aligned,
using MEGA version 5 to contruct a phylogenetic analysis
[28]. The unweighted pair group technique with the arithmetic
mean (UPGMA) approach was used to conduct the analysis.
The greatest composite likelihood approach was used to cal-
culate the evolutionary distances, and the evolutionary history
was constructed using the neighbor-joining method [29] with
2000 bootstrap replications, and the internal branches’ robust-
ness was evaluated.
Molecular identification of Bacillus isolates was performed
using rep-PCR with the help of BOXA1R ERIC-1R and ERIC-
2F primers [28]. The PCR reaction mixture (25 μL) containing
5 × Gitschier buffer, 0.25μM of primer, 50ng DNA template,
1 U of Taq DNA polymerase (Bangalore Genie, India), and
2mM MgCl2 was amplified on thermal Cycler (Biorad, CA,
USA) and the amplified fragments were separated by agarose
gel electrophoresis with the help of aDNA ladder of 100bp
to 3kb.
Screening forPlant Growth‑Promoting Traits
Indole Acetic Acid Production
Indole acetic acid (IAA) production by the isolates was
screened in Luria Bertani (LB) medium containing 1g/L
tryptophan [30].
Phosphate Solubilizing Activity
The inorganic phosphate solubilizing (PS) activities
of bacterial isolates were perceived using the National
Botanical Research Institute’s phosphate growth medium
(NBRIP) agar medium at 37 ± 2°C for 7days. The abili-
tyof the isolates to solubilize inorganic phosphate was cal-
culated as a solubilization index (SI) using the following
formula [31].
Production ofAmmonia
Isolates were screened for ammonia (NH3) production
in peptone water (10mL) inoculated and incubated for
48–72h. Following the incubation, Nestler’s reagent
SI
=
Colony diameter (mm)+ Zone diameter (mm)
Colony diameter (mm)
Fig. 1 Map showing SHAUTS
farm (Courtesy: Google map)
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A.Sagar et al. 83 Page 4 of 14
(0.5mL) was added, and the tubes were observed to color
change from brown to yellow [32].
Siderophore Production
Screening of isolates for siderophore (SD) production was
performed on Chrome Azurol agar (CAS) medium [33] at
30°C. The isolates were grown on CAS agar medium at
30°C for 48h and observed for color change of medium
from blue to orange/golden.
1‑Aminocyclopropane‑1‑Carboxylate Deaminase Activity
According to the available protocol, isolates were screened
for 1-aminocyclopropane- 1-carboxylate deaminase (ACCD)
activity [30]. The ACCD activity was defined as the amount
of α-keto-butyrate produced per mg of protein per h.
Hydrogen Cyanide
Hydrogen cyanide (HCN) production by Bacillus isolates
was estimated according to Lork’s protocol [34].
Susceptibility toAntibiotics
Bacillus isolates were tested according to the method of
Bauer [35] for its resistance to various antibiotics (Table4).
All assays were performed three times with five replications.
Plant Growth Promotion Studies
In Vitro Study
The seeds of different vegetable crops such as tomato (vari-
ety-Lycopersicon; NTL-186), broccoli (variety-PalamSam-
ridhi), and chickpea (variety- Pusa 256) were surface-steri-
lized with ethanol (70%) for 2min and washed three times
with sterile distilled water. The following treatments were
applied.
T0- control (no bacterial inoculation),
T1 = B. subtilis PR30,
T2 = B. subtilis PR31.
The surface-sterilized seeds were transferred to bacterial
suspension (104cfu/mL), kept for 60min, placed on ger-
mination paper, and maintained for 15days at 25 °C [24].
Control (T0) seeds without bacterial inoculation were used
for comparison. Treated and control seeds were also checked
for the root and shoot elongation pattern under invitro con-
ditions. All the treatments were performed in triplicates, and
the average of triplicates was considered.
Percent Germination
The percent seed germination was calculated as follows
[36].
Germination Index
The germination index (GI) was calculated according to
the Association of Official Seed Analysts AOSA [37] using
the following formula–
Vigor Index
Vigor index was calculated according to Abdul and Ander-
son [38] and with the help of the following formula.
Green‑House Pot Experiment
Plant Seeds andTreatment
The bioefficacy of Bacillus isolates (PR30 and PR31)
was evaluated under a greenhouse pot experiment at Sam
Higginbottom University of Agriculture, Technology and
Sciences (SHUATS), Allahabad, India. Surface sterilized
broccoli, tomato, and chickpea seeds were treated with
endospore suspension of Bacillus isolates (~ 108 cells
mL−1). Treated seeds were sown in plastic pots contain-
ing autoclaved field soil and watered daily. Control seeds
were not treated with bacterial culture. Five seed pot−1 and
10 pot treatment−1 were maintained. The treatments for the
bioefficacy experiment were scheduled as (1) B. subtilis
PR30, (2) B. subtilis PR31, (3) and Control. Plant growth
parameters were measured 30, 60, and 90days after sow-
ing (DAS).
Measurement ofPlant Growth Parameters
Ten seedlings were harvested, root length, shoot length,
fresh weight, and dry weight (mg/plant) were measured.
The plant height (cm) was measured from the ground
level to the growing tip of the main shoot, and the aver-
age height was calculated in cm. One plant was randomly
selected from each pot, and the number of branches of
Seed germination
(%)=
Number of germinated seeds
Number of total seeds
×
100
GI
=
No. of germinated seed
Days of the first count
+
No. of germinated seed
Days of the final count
Vigor index =Percent germination ×Seedling length
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Molecular Characterization Reveals Biodiversity andBiopotential ofRhizobacterial Isolates…
Page 5 of 14 83
each plant was measured and averaged.Flowers from three
plants were counted and averaged. All the fruits from three
selected plants from each replication of all the treatments
were counted. All the fresh fruits from three selected
plants from each replication of all the treatments were
weighed after picking. Each replication’s average fresh
fruit weight per plant (g) was recorded and subjected to
statistical analysis. The diameter of the curd of three plants
was measured at the widest circumference in cm, and the
average diameter per curd from each pot was calculated.
The Head or bud of three selected plants was weighted on
electrical balance in g, and the average was found to give
the bud weight per plant. The fresh weights of the three
selected plants were recorded in each pot, and the average
fresh weight was calculated. This calculated value was
assumed as the average weight of the rest of the remaining
plant per pot. The plants used to measure dry weight were
also subjected to dry weight analysis. The plants were
dried for 5–6h at a temperature of 50–60 °C. The dry
weights of all randomly selected plants in each pot were
added, and the average was calculated.
Biochemical Parameters
Chlorophyll, Carotenoid, and Lycopene Content Chloro-
phyll a, b, and carotenoid content were determined accord-
ing to Arnon's method [39]. The absorbance of the resulting
solution was read at 663, 645, and 480nm for chlorophyll
a, b, and carotenoids. The extraction and estimation of
lycopene content was performed according to the method
of Butnariuand Giuchici [40]. The absorbance of superna-
tant containing lycopene was read in a spectrophotometer at
472nm. The total lycopene content was measured as lyco-
pene mg per 100g of fruit tissues.
where E = extinction coefficient; W = weight (g).
Statistical Analysis
All the experiments were performed in triplicates, and the
average valueswere calculated. The standard errors were cal-
culated for all mean values and subjected to ANOVA followed
by DMRT. The BOX and ERIC-PCR product cluster analysis
was based on the binary matrix, presence (1), and absence
(0) of the band for each strain. Principle component analysis
(PCA) was performed using the XLSTAT software.
Lycopene
(mg∕100 g)=
E
3.45
×
20
w
Results
Isolation andInVitro Assessment ofBacillus Isolates
A total 7 potent Bacillus isolates, (PR30, PR31, PR35,
PR38, PR42, PR45, and PR55) were obtained from diverse
soilsamples. These isolates showed antagonistic activity
against Ralstonia solanacearum (Fig.2) (Table1).
Values are the mean of three replicates. According to
Duncan’s multiple range test, different letters in superscript
are significantly different (p ≤ 0.05).
Morphological andBiochemical Characterization
ofStrains
The Bacillusisolates were first characterized by their mor-
phological and biochemical attributes. The isolateswere
motile, spore-forming rods, forming white to creamy-white
colonies, endospore former, tolerated pH (5–9), temperature
(10–45°C), and NaCl (0–10%) (Table2).
They hydrolyzed starch and produced catalase.
Three strains (PR30, PR42, and PR45) produced H2S,
ammonia,siderophores IAA, HCN, and ACCD (Table3).
All strains showed intrinsic antibiotic resistance to variable
levels (Table4).
Identification ofantagonistic Bacillus isolates
Seven Bacillusisolates were recognized as the active antago-
nists. Their phylogenetic distribution and 16S rRNA gene
sequence individualities are presented in Table5. The
similarity values (≥ 97%) confirmed that all isolates (PR30,
PR31, PR33, PR38, PR42, PR45, and PR48) belong to the
genus Bacillus subtilis. The un-rooted phylogenetic tree
(Fig.3) presented the genotypic relationship of the isolates,
wherever all the bacterial isolates were clustered into two
major clades. The 16S-rRNA data showed that PR31and
PR35 (B. subtilis) isolate was more diverse than other strains
and grouped into distinct clusters.
Fig. 2 Antagonistic activity of isolated bacteria against R. solan-
acearum
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A.Sagar et al. 83 Page 6 of 14
ERIC andBOX‑PCR Analysis
ERIC's complex fingerprint patterns produced 81 polymor-
phic bands of variable range (250–3000bp) (Fig.4a). Princi-
pal component analysis (PCA) based on the first and second
coordinates showed a maximum Eigen value of 4.642 and
minimum value of 0.095 with a percent variation of 66.31
and 14.30, respectively (Fig.4b). Observation of the PCA
analysis revealed that four isolates (PR30, PR31, PR35, and
PR55) formed a major cluster (cluster I). Three isolates were
classifiedin cluster II (PR38, PR42, and PR45).
The BOX-PCR banding pattern of all seven strains
displayed 102 fragments in the 250–4000bp (Fig.4c).
The results of the PCA analysis are based on the first and
second coordinates and showed a maximum Eigen value
of 3.053 and minimum value of 0.162 with a percentage
Table 1 Antagonistic activity
of isolated bacteriaagainst
Ralstonia solanaceorum
Characteristics PR30 PR31 PR35 PR38 PR42 PR45 PR48
Bacterial growth inhibition (%) 59.00f63.29g 30.51a46.70b49.04bc 54.63e50.56d
Inhibition zone (mm) 16.90e18.10f6.70a6.90a8.66b12.36c13.96d
Table 2 Morphological and
biochemical characterization of
antagonistic isolates of Bacillus
sp. against R.solanaceorum
-It was not determined.Values are the average of three replicates analyzed by Duncan’s multiple range test
Characteristics Isolates
PR30 PR31 PR35 PR38 PR42 PR45 PR48
Endospore + + + + + + +
Colony colour Off-white Creamy white White White White White White
Pigmentation - - - - - - -
Motility + + + - + + +
Salt (%) 0–10 0–10 0–8 0–8 0–9 0–8 0–8
pH 5–9 5–8 6–9 6–8 5–9 5–9 6–9
Temperature (°C) 15–45 15–45 15–55 15–45 10–45 15–45 15–45
Starch hydrolysis + + + + + + +
Gelatin hydrolysis + + --- + +
H2S production + - -- + + -
Glucose fermentation + + + + - + +
Catalase + + + + + + +
Oxidase + + + + - + +
Indol - - - + + - -
MR test - - - + + + -
Citrate + + + --- +
Nitrate + - --- + -
Urease + + --- + +
VP test + + + --- +
Table 3 Plant growth-promoting traits exhibited by the Bacillus isolates
+ , presence; -, absence. (% + SD) (low significant) *p < 0.05, (moderate significant) **p < 0.01, (high significant) ***p < 0.001. Values are the
average of three replicates analyzed by Duncan’s multiplerange test
Characteristics PR30 PR31 PR35 PR38 PR42 PR45 PR48
Siderophore production 3.5 ± 0.05* 4.0 ± 0.04** - - 3 ± 0.09 3 ± 0.05 3.1 ± 0.06
Indole acetic acid 54.5 ± 0.05** 50.5 ± 0.03* - - 40.5 ± 0.15 41.5 ± 0.09 40.0 ± 0.15
Phosphorus solubilization activity 5.1 ± 0.05* 5.3 ± 0.02** -5.0 ± 0.03* 4.8 ± 0.06 4.4 ± 0.09 -
1-aminocyclopropane- 1-carboxylate
deaminase
9.1 ± 0.05* 9.2 ± 0.07** -8.9 ± 0.76 8.5 ± 0.08 8.1 ± 0. 08 9.0 ± 0.16*
Ammonia production + + - - + + +
Hydrogen cyanide production + + - - - - +
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Molecular Characterization Reveals Biodiversity andBiopotential ofRhizobacterial Isolates…
Page 7 of 14 83
variation of 50.05 and 13.90%, respectively (Fig.4d). PCA
analysis revealed that four isolates (PR30, PR31, PR38,
and PR55) formed a major cluster (cluster II), and three
isolates (PR35, PR42, and PR45) were classifiedin cluster
I.
Evaluation forBioefficacy
Based on antagonistic activity and PGP traits, two isolates
(PR30 and PR31) were selected for plant growth parameters
of different vegetable crops under invitro and greenhouse
Table 4 Antibiotic resistance of
antagonistic isolates of Bacillus
sp
Nutrient agar media supplemented with antibiotics (µg/mL) represented by the different letters A = 20,
B = 40, C = 60, D = 80, and E = 100. Values are the average of three replicates analyzed by Duncan’s multi-
ple range test
Characteristics PR30 PR31 PR35 PR38 PR42 PR45 PR48
Ampicillin + B + C - + B + B + C + B
Cephataxime + C + B + B + B -- + C
Nalidixic + B + B -- + B + B + C
Neomycin + B -- + B + C + B + C
Kanamycin + C + C --- + D -
Tetracycline + C + C - + B - + B -
Gentamycin + B + B - + B + B - -
Chloramphenicol + C + C + B + B - + D -
Streptomycin + C + B - + B + B - -
Table 5 Identification of isolated bacteria based on 16S rRNA gene sequencing
Strain No. Sample collection field Sequence Size ana-
lyzed (bp)
Country % Match with
Bacillus sp.
NCBI accession No
PR30 Organic farm 1491 India 100 KP966505
PR31 Organic farm 1491 India 100 KP966499
PR35 Soil 1505 India 99 MT993603
PR38 Organic farm 1549 India 99 MT993356
PR42 Organic farm 1509 India 100 MT992787
PR45 Sugarcane field 1505 India 99 MT993414
PR48 Sugarcane field 1492 India 100 MT993345
A102 Plant 1503 China 98 AB526466.1
Bacillus sp. 19D1S38 Soil 1469 Korea 98 MN620404.1
Bacillus sp. S20609 Rhizosphere 1544 India 100 KF956597.1
Fig. 3 Phylogenetic tree based
on the 16S rRNA gene sequence
of potent Bacillus isolates
were generated from organic
farm soil samples based on
the 16S rRNA gene sequence.
Evolutionary distances were
calculated using the “neighbor-
joining” algorithm, based on
a bootstrap analysis of 2000
replicates (values on branches
denote % ofbootstrap support)
Bacillus subtilis PR45/MT993414
Bacillussubtilis gene for16S rRNA partialsequencecountry:VietNam
Bacillussp. (in: Bacteria)strai n19D1S38 16S ri bosomalRNA gene partialsequenc
e
Bacillus subtilis PR38/MT993356
Bacillussp. S20609 16Sribosomal RNA gene partialsequence
Bacillus subtilis PR30/KP966505
Bacillussubtilis gene for16S rRNA partialsequencestrain: A102
Bacillus subtilis PR42/MT992787
Bacillus subtilis PR48/MT993345
Bacillus subtilis PR31/KP966499
Bacillus subtilis PR35/MT993603
71
100
64
85
57
93
100
0.0000.0020.0040.0060.0080.010
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A.Sagar et al. 83 Page 8 of 14
conditions. The Bacillus isolates (PR30 and PR31) induced
a higher percentage (85–96%) of germination of seed com-
pared to control seeds (Table6).
The Bacillus spp. isolates (PR30 and PR31) showed the
best responses for plant growth promotion under greenhouse
conditions. The efficacy of selected Bacillus isolates var-
ied to induce germination, vigor index, and root and shoot
length in tomato, broccoli, and chickpea (Table6). Tomato
seedling treatment with B. subtilis PR30 and B. subtilis
PR31 significantly (p ≤ 0.05) enhanced seed germination
(27%), root length (20–50%), shoot length (20–40%), fresh
weight, and dry biomass (50–75%) compared to untreated
(Fig.5a) (Tables6, 7). In the case of broccoli, a significant
improvement in seed germination (15–24%), root length
(40–60%), shoot length (50–60%), fresh weight, and dry
biomass (50–60%) was evident over the control (Fig.5b)
(Tables7 and 8). While this inoculation also improved seed
germination (50–60%), root length (40–50%), shoot length
(20–40%), and vigor index (50–80%) in chickpea (Fig.5c).
Bacillus subtilis PR30 and PR31 isolates significantly
improved the chlorophyll and carotenoid content in tomato,
chickpea, and broccoli, whereas lycopene was considerably
enhanced in tomato plants (Table8).
Discussion
Increasing agricultural productivity with limited cultivable
land is the biggest challenge to growers around the globe.
It is necessary to improve agricultural productivity to
nourish and feed the growing world population. Crop yield
and productivity can be enhanced in two ways: by increas-
ing crop productivity through fertilizers or biofertilizers
and by preventing crop losses caused by phytopathogens.
Fig. 4 Genotypic patterns of
bacterial strains obtained after
ERIC (4a) and BOX-PCR (4b)
fingerprinting. Principal compo-
nent analysis score plot of seven
bacterial isolates based on ERIC
(4c) and BOX-PCR (4d) data
ab
d
c
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Molecular Characterization Reveals Biodiversity andBiopotential ofRhizobacterial Isolates…
Page 9 of 14 83
Using PGPR that possesses the dual potential of plant
growthpromotion and biocontrol is expected to play this
dual role [24].
The diverse potential of Bacillus spp. makes it a prom-
ising plant growth-promoting rhizobacterium and BCA in
various crops. Inoculation of crops with Bacillus spp. pro-
motesseed germination, seedling vigor, leaf index, root and
shoot growth, and photosynthetic ability. The plant growth
promotion due to Bacillus spp. inoculation is due to the
production of various PGP substances [2, 14]. Inhibition of
phytopathogens results from the secretion of a wide range
of antagonistic substances [17, 26]. Members of the Bacillus
genus produce multiple PGP traits, such as phytohormones,
and they help in nutrient mobilization (iron, P, etc.) [7, 14],
which improves the growth of inoculated crop plants. Bacil-
lus spp. is one of the major biological control agents (BCA)
and antagonistic soil bacterium [21, 26]. Bacillus sp. pro-
duces various antagonistic substances, such as hydrogen
cyanide, siderophore, and hydrolytic enzymes to inhibit the
growth of phytopathogens.
Developing biofertilizers/formulation strategies using
spore-forming Bacillusbioagents is an emerging area in
crop protection. A total of 87 Bacillus strains were iso-
lated from organic farm soil samples and examined by per-
forming a disc diffusion approach to raise the possibility
of using antagonistic bacteria asBCAs against Ralstonia
solanacearum. Using this strategy, seven potent B. subtilis
strains (PR30, PR31, PR35, PR38, PR42, PR45, and PR55)
were specifically selected as an effective biocontrol agent
against R. solanacearum. Similar strategies have been used
effectively to isolate potential BCAs, such as Bacillus strains
exhibiting antimicrobial activity towards phytopathogens
[41].
Molecular identification based on 16S rRNA gene
sequences of Bacillus isolates indicates phylogenetic clus-
tering between bacteria at inter- and intra-species levels [42].
At the same time, the present study perceived that identifi-
cation based on 16S rRNA gene sequences is limited and
incapable of distinguishing along with bacterial strains.
Consequently, polyphasic gene-based fingerprinting tools
(BOX-PCR and ERIC-PCR] were used to discriminate intra-
species unevenness between the bacterial strains. The results
presented that isolates could not be distinguished by partial
16S rRNA gene sequence were different regarding ERIC and
BOX-PCR patterns. Besides, these tools help separate the
ecologically diverse Bacillus strains into a distinct group,
which is otherwise tricky through 16S rRNA gene analysis
[23].
Seedling treatment among isolates significantly sup-
pressed the pathogen growth in invitro conditions and
enhanced plant height and biomass compared to con-
trol. Inhibition of phytopathogens is attributed to various
antagonistic substances,viz.siderophore, hydrogen cyanide,
other volatile compounds, and a wide range of antibiotics.
Siderophore-producing PGPR prevents iron nutrition and,
hence, the growth of phytopathogens [43]. HCN is a volatile
organic compound (VOC) synthesized by a wide range of
PGPR. Many bacterial genera, including Bacillus, can pro-
duce HCN [6, 20]. HCN exerts a potent toxic effect on many
phytopathogens, forming stable complexes with Cu2+, Fe2+,
and Mn2+ and causing disruption in protein functions [25,
44] (Reference?). It also inhibits electron transport and dis-
rupts the energy supply to the cell, leading to living organ-
isms’ death. Besides their biocontrol ability to produce HCN
and antibiotics, Bacillus species produce phytohormones,
increase uptake of phosphate and iron, produce ammonia,
Table 6 Inoculation effect
of B. subtillis on percent
seed germination and growth
parameters of vegetable crops
under invitro condition
T0- control T1 = B. subtilis PR30, T2 = B. subtilis PR31 (% + SD) (low significant) *p < 0.05, (moderately
significant), **p < 0.01, (highly significant),***p < 0.001. Values are the average of triplicates analyzed by
Duncan’s multiple range test
Treatments Percent seed
germination
Elongation in cm (± SD) Index (± SD)
Root Shoot Germination Vigor
Tomato (variety-NTLI86)
T0 60 ± 1.1 4 ± 0.1 5 ± 0.1 1 ± 0.5 541 ± 1.5
T1 97 ± 1.5* 5 ± 0.3 7 ± 0.4 7 ± 0.5*** 1141 ± 1*
T2 96 ± 1.7* 7 ± 0.4* 8 ± 0.4 9 ± 0.4*** 1521 ± 1.0**
Broccoli (variety-PalamSamridhi)
T0 70 ± 0.1 3 ± 0.1 4 ± 0.1 2 ± 0.1 490 ± 0.5
T1 94 ± 0.5* 5 ± 0.4 8 ± 0.4** 9 ± 0.5** 1172 ± 1.5**
T2 85 ± 0.5 7 ± 0.2* 9 ± 0.4** 7 ± 0.5** 1051 ± 1.0**
Chickpea(variety-Pusa-256)
T0 60 ± 1.1 3 ± 0.1 4 ± 0.1 3 ± 0.4 421 ± 1
T1 93 ± 1* 5 ± 0.2 6 ± 0.4 7 ± 0.4* 992 ± 1.5*
T2 94 ± 0.5* 6 ± 0.3** 6 ± 0.4 8 ± 0.5* 1263 ± 1.0**
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

A.Sagar et al. 83 Page 10 of 14
and protect cells from oxidative damage by producing cata-
lase enzyme. Rahman etal. [43] reported the inhibition of
Agrobacterium tumefaciensby HCN-producing Bacillus
megaterium strain CtST3.5.
Many PGPR produce IAA, a crucial phytohormone asso-
ciated with root elongation and initiation.Plants provide
tryptophan to PGPR to synthesize IAA, an essential phyto-
hormone for plant growth promotion. Bacillus spp. produced
copious amounts of IAA, and this IAA has promoted root
ramification.
Phosphorus is one of the vital elements for plant growth
and development. It is regarded as a limiting nutrient for
plant growth as it is usually present in insoluble forms. P
solubilizing PGPR can potentially solubilize P and make it
available forplant growth promotion [45].
Liu etal. [14] also suggested that the B. amyloliquefa-
ciens strain displays maximum invitro inhibitory activity
towards multiple plant pathogens. Sudha etal. [45] reported
the production of volatile compounds in Streptomyces rochei
that inhibited the growth of sorghum pathogen. Sayyed and
Patel [46] presented siderophore production in Alcaligenes-
faecalis and found that this siderophoregenic culture inhibits
the growth of a wide range of fungal phytopathogens. They
found more antifungal activity in siderophoregenic culture
than in a chemical fungicide. The present study also dem-
onstrated that B. subtilis PR31 colonized more frequently
than other test strains in tomato and broccoli rhizosphere,
while B. subtilis PR30 was assessed more in chickpea.
These findings justify the previous biocontrol reports that
emphasize the proficient colonization of BCAs in the host
rhizosphere,which is expected to enhance plant growth pro-
motion and disease management.
Biocontrol efficacy of Bacillus spp. has been confirmed in
the greenhouse and field conditions and at the post-harvest
stage for fruit diseases [47]. It has been established primarily
to resist gram-negative bacteria invitro and under controlled
conditions and to reduce diseases caused by these pathogens.
A single strain can act against numerous bacterial pathogens.
For example, B. velezensis LS69 has been shown to display
antibacterial activities against Erwinia carotovora and Ral-
stonia solanacearum [43, 48]. Production of plant growth-
promoting traits (PGPT) is the characteristic feature of all
PGPR. These PGPT promote plant growth through direct
mechanisms as green biostimulants [2, 49]. PGPR promotes
plant growth through an indirect mechanism, such as the
production of antibiotics [43] and the production of hydro-
lytic enzymes. The induction of resistance in plants and
production of siderophore [46, 50] and phosphate solubiliz-
ing ability in different cultures of PGPR isolated from the
rhizosphere have been reported. Kapadia etal. [51] reported
the production of multiple plant growth-promoting traits
in Bacillus sp., Klebsiella variiocola, and Mesorhizobium
sp., respectively, and found that this multipotent culture
improves growth in wheat and maize. The Bacillus sutilis
isolates identified and used in the present study possessed
all the plant growth promoting traits and hence are ideal
candidates for biological control of R. solanacearum.
The production of ammonia is one of the major traits of
PGPR that helps promote plant growth. The production of
ACCD in PGPR is one of the best mechanisms involved
in plant growth promotion under oxidative stress. PGPR
a
b
c
Fig. 5 Plant growth promotion–greenhouse pot assay (5a). Tomato
with control, (5b) Broccoli with control, and (5c). Chickpea with con-
trol
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Molecular Characterization Reveals Biodiversity andBiopotential ofRhizobacterial Isolates…
Page 11 of 14 83
lowers the ACC level in root exudates, decreasing the con-
centration of ethylene in the plant roots and thus helps
in root length for better absorption of nutrients.Through
antioxidants and other mechanisms, PGPR induces resist-
ance to protect crop plants, thus helping plant growth [47,
48, 52–54].
Bacillus spp. suppressing the development of a wide
range of phytopathogens while promoting plant growth in
various crops can make this culture a multipotent PGPR for
sustainable plant disease management and an eco-friendly
biocontrol agent [2].
The present study successfully screened the Bacillus
strains such as B. subtilis PR30 and B. subtilis PR31 associ-
ated with tomato rhizosphere that could stimulate growth
in various crops (tomato, chickpea, and broccoli). These
strains are identified as potent antagonists to suppress the
growth of R. solaniunder invitro conditions. They can be
used in integrated disease management of tomato root rot
and damping off. The combined studies, comprising bio-
chemical and molecular technologies, are essential to select
indigenous antagonistic Bacillus strains that can be used in
combinations of other strains under different environmental
Table 7 Effect of Bacillus subtilis on growth promotion of vegetable crops under greenhouse conditions
T0- control (no bacterial inoculation, T1 = B. subtilis PR30, T2 = B. subtilis PR31 Note: Mean ± SE value of three independent experiments.
Each experiment was conducted in five replicates. *p < 0.05 = low significant, **p < 0.01 = moderately significant,,***p < 0.001 = highly signifi-
cant. Values are the average of triplicates analyzed by Duncan’s multiple range test
Treat-
ments
Length (cm) Fresh weight (g) Dry weight (g)
Root Shoot Root Shoot Root Shoot No. of fruits/
plant
Fresh fruit
weight (g/plant)
Root coloniza-
tion (log cfu/g
root)
Tomato (variety-NTLI86)
T0 15.47 ± 0.14 33.54 ± 0.17 6.901 ± 0.04 15.324 ± 0.10 2.042 ± 0.06 4.204 ± 0.06 7.30 ± 0.15 340.40 ± 9.18 -
T1 17.02 ± 0.09* 42.01 ± 0.32 7.891 ± 0.03** 19.022 ± 0.04 3.277 ± 0.13 4.950 ± 0.02 9.543 ± 0.11 393.16 ± 2.36 6.76 ± 0.08
T2 17.80 ± 0.07** 44.11 ± 0.15** 8.175 ± 0.03*** 21.205 ± 0.29** 3.539 ± 0.02** 5.659 ± 0.08** 9.823 ± 0.10** 420.33 ± 0.60** 7.21 ± 0.13*
Broccoli (variety-PalamSamridhi)
T0 8.51 ± 0.06 16.52 ± 0.05 31.666 ± 0.36 287.80 ± 1.07 5.410 ± 0.13 32.266 ± 0.17 4.22 ± 0.04 108.35 ± 0.17 -
T1 9.38 ± 0.09 18.62 ± 0.07* 38.270 ± 0.38 421.32 ± 0.98 6.690 ± 0.05 39.0567 ± 0.17* 5.76 ± 0.04 182.59 ± 0.40 6.17 ± 0.02
T2 9.99 ± 0.04** 19.41 ± 0.06** 41.026 ± 0.18** 4*45.45 ± 0.65* 7.680 ± 0.05** 40.670 ± 0.04** 6.61 ± 0.04** 196.28 ± 0.58** 6.72 ± 0.02*
Chickpea (variety-Pusa-256)
T0 7.05 ± 0.32 16.51 ± 0.13 2.270 ± 0.04 8.210 ± 0.20 0.692 ± 0.04 1.651 ± 0.04 10.57 ± 0.04 25.37 ± 0.02 -
T1 10.51 ± 0.10** 22.86 ± 0.04** 2.947 ± 0.01*** 10.076 ± 0.21** 0.984 ± 0.006** 2.133 ± 0.02** 12.99 ± 0.03** 31.86 ± 0.06*** 6.92 ± 0.00*
T2 9.21 ± 0.16* 21.13 ± 0.15 2.530 ± 0.02 9.815 ± 0.02 0.743 ± 0.007 1.945 ± 0.02 12.42 ± 0.10 29.93 ± 0.07 6.57 ± 0.04
Table 8 Effect of Bacillus subtilis on Biochemical traits of vegetable crops under greenhouse condition
T0- control (no bacterial inoculation, T1 = B. subtilis PR30, T2 = B. subtilis PR31. Values are the average of three replicates ana-
lyzed by Duncan’s multiple range test. Mean ± values are SD. (Low significant) *p < 0.05, (moderately significant), **p < 0.01, (highly
significant),***p < 0.001. Values are the average of triplicates analyzed by Duncan’s multiple range test
Treatments Biochemicals
Chlorophyll ‘a’(mg./g)FW Chlorophyll ‘b’(mg./g) FW Total chlorophyll (mg/g? Carotenoid (mg/g) FW Lycopene (mg/100g)
Tomato (variety-NTLI86)
T0 1.04 ± 0.004 0.733 ± 0.027 1.646 ± 0.950 0.806 ± 0.007 2.333 ± 0.027
T1 1.396 ± 0.051 1.083 ± 0.014 2.103 ± 1.214 1.243 ± 0.090 2.716 ± 0.082**
T2 1.413 ± 0.015** 1.236 ± 0.009** 2.493 ± 1.439* 1.313 ± 0.0381* 2.646 ± 0.060
Broccoli (variety-PalamSamridhi)
T0 0.51 ± 0.020 1.696 ± 0.044 3.673 ± 0.030 0.533 ± 0.027 -
T1 0.666 ± 0.007 2.573 ± 0.094** 4.456 ± 0.126 0.686 ± 0.010 -
T2 0.686 ± 0.002* 2.376 ± 0.064 4.506 ± 0.072* 0.716 ± 0.015* -
Chickpea(variety-Pusa-256)
T0 1.09 ± 0.0478 0.36 ± 0.036 1.263 ± 0.051 0.543 ± 0.011 -
T1 1.643 ± 0.170 0.473 ± 0.0237** 1.6 ± 0.012 0.766 ± 0.0272 -
T2 1.766 ± 0.0272** 0.456 ± 0.0151 1.62 ± 0.024** 1.733 ± 0.0544** -
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

A.Sagar et al. 83 Page 12 of 14
conditions (greenhouse and field conditions to obtain
resistance against pathogens in various crops). Without the
extraordinary effect of genetic resistance in tomato cultivars,
these biocontrols may be a potential candidate for handling
vascular wilt infection and minimizing losses in enhanced
fruit quality and yield. However, additional investigations
are required to conclude the efficiency of these strains under
diverse cultivar varieties and locations to understand the
interaction behavior with the pathogen, host plants, and soil
factors.
Acknowledgements The authors sincerely thank the ICAR-National
Institute for Plant Biotechnology, New Delhi, India, for providing the
facilities. The authors would like to acknowledge the support provided
by Researchers Supporting Project Number RSP2024R358, King Saud
University, Riyadh, Saudi Arabia.
Author Contribution Conceptualization and supervision: AS and RZS;
writing: AS, SR, and SS; writing—review and editing: RZS, PP, NAB,
and KP; formal analysis: AS and SR; fund acquisition: NAB and KP.
All authors contributed to editorial changes in the manuscript. All
authors read and approved the final manuscript.
Funding Open access funding provided by Università degli Studi di
Brescia within the CRUI-CARE Agreement. This work was funded by
Researchers Supporting Project Number RSP2024R358, King Saud
University, Riyadh, Saudi Arabia.
Data Availability No datasets were generated or analysed during the
current study.
Declarations
Informed Consent Not applicable.
Institutional Review Board Statement Not applicable.
Competing Interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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