Scientia Horticulturae 325 (2024) 112645

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Seed coating with the synthetic consortium of beneficial Bacillus microbes

improves seedling growth and manages Fusarium wilt disease
Ali Raza a, Ali Hassan a, Waheed Akram a, *, Tehmina Anjum a, Zill-e-Huma Aftab a, Basharat Ali b
a
Department of Plant Pathology, Faculty of Agricultural Sciences, University of the Punjab, Lahore, Pakistan
b
Institute of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan

A R T I C L E I N F O A B S T R A C T

Keywords: Fusarium wilt disease of the pea crop caused by the infection of Fusarium oxysporum f. sp. pisi (FOP) is a serious
Pea soilborne disease. In this study, we developed a seed coating containing a synthetic consortium of beneficial
Fusarium wilt Bacillus microbes to manage the fusarium wilt disease. Three bacterial strains IS1, IS6, and IS7 isolated from the
Seed coating
rhizosphere of different healthy-looking crop plants, were selected for the seed coating based on antagonism
Synthetic consortium
Bacillus
against the FOP shown during in-vitro studies, and suppression of fusarium wilt disease when applied as seed
Antagonism priming in preliminary pot trials. The strains were identified as B. subtilis (IS1), B. amyloliquificiens (IS6), and
B. fortis (IS7) by 16 s rRNA gene sequencing. The seed coating with the consortium of all three bacterial strains
showed a significant positive effect on seed germination, seedling height, and biomass accumulation of pea
seedlings. Upon exposure to FOP, a > 50 % reduction in disease index was seen in pea plants raised from coated
seeds than the pathogen control along with the ameliorative effect on plant growth attributes. Besides, the
coating of consortium induced significant up-regulation of total phenolic compounds and enzymes of phenyl­
propanoid pathways. Additionally, the non-targeted metabolomic analysis indicated a large number of pertur­
bations in amino acids, sugars, carbohydrates, and phenylpropanoids in pea plants raised from consortium-
coated seeds and subsequently challenged with FOP. Based on the findings, consortium coating of Bacillus
spp. on pea seeds may have the potential to develop a bio-based remedy for the management of wilt diseases in
pea plants and improvement in growth attributes.

1. Introduction
Pea (Pisum sativum L.) is the most widely cultivated Fabaceae plant.
Pea is an important food crop that provides an ideal source of protein,
vitamins, minerals, and bioactive compounds beneficial to human
health (Han et al., 2023). Peas are a rich source of high-quality vegetable
protein in the human diet. Peas are cultivated in almost all countries
around the world. According to an estimate, 13.5 million metric tons of
peas are produced annually in the world (FAOSTAT, 2018). Numerous
fungal, bacterial, nematode and viral diseases infect the pea crop during
its growth cycle (Zakaria, 2023). Due to biotic and abiotic factors, losses
in pulse production vary from, 30 to 100 % based on the magnitude of
severity (Rana et al., 2016).
Soil-borne plant diseases are considered an important issue in agri­
cultural production around the world, which result in considerable
losses in the yield and quality of several essential crops (El-Sobky et al.,
2019). Fusarium wilt is the most destructive of all diseases and causes up
to 70 % reduction in yield (Gupta and Gupta, 2019). The estimated loss
of pea crop range between 30 and 40 percent in terms of monetary value
when attacked by Fusarium wilt disease (Rubiales et al., 2015). The
symptoms of fusarium wilt of pea disease are usually a yellowing of
leaves, stunting of plant growth, and ultimately death of wilted plants.
Fusarium oxysporum disseminates through the soil, and a range of rem­
edies are being applied to suppress this soil-borne pathogen including
the application of different fungicides (Aslam et al., 2019). The diffi­
culties associated with soil treatment and the poor delivery of toxic
fungicides to the vascular system of plants make chemical control
ineffective (Pazarlar et al., 2022). These fungicides have, nevertheless,
negative consequences on human health, increased environmental
pollution, loss of biodiversity, and the establishment of disease strains
that are resistant to their effects (Ahammed et al., 2020). The chla­
mydospores produced by Fusarium spp. are very infectious and able to
survive for longer periods, further making disease control a very chal­
lenging phenomenon (Hou et al., 2020)

* Corresponding author.
E-mail address: waheedakram.fas@pu.edu.pk (W. Akram).

https://doi.org/10.1016/j.scienta.2023.112645
Received 10 September 2023; Received in revised form 15 October 2023; Accepted 2 November 2023
0304-4238/© 2023 Elsevier B.V. All rights reserved.
A. Raza et al.
The biological control of soil-borne disease has attracted consider­
able attention from researchers. Different beneficial microbes are found
associated with the rhizosphere of the crop plants without causing dis­
eases in the host plants. The utilization of antagonistic microbes also
known as biocontrol agents (BCAs), as a biological disease management
strategy has been shown effective against several soil-borne diseases of
plants along with plant growth promotion and improved seed quality
(Latz et al., 2012; Singh et al., 2018). For instance, the addition of
biocontrol agents in the rhizosphere of banana crops yielded very
promising results against Fusarium wilt disease in China (Xue et al.,
2015). Collimonas arenae strain Cal35 was able to effectively antagonize
F. oxysporum f.sp. pisi under in vitro conditions. This bacterial strain was
found to be more active when combined with some strains reducing
disease severity and the abundance of F. oxysporum in the root endo­
sphere of tomato plants under the field conditions (Doan et al., 2020).
Similarly, the findings from some previous research works have sug­
gested the ineffectiveness of beneficial microbes using single-strain in­
oculations, but mixtures of strains provided more consistency
(Adesemoye et al., 2009). A consortium of beneficial rhizobacteria
consisting of Bacillus pumilus, Bacillus subtilis, and Curtobacterium flac­
cumfaciens showed increased biocontrol activity against multiple cu­
cumber pathogens (Raupach and Kloepper, 1998).
Bacillus are the rhizospheric microbes that have also been used as a
disease control agent (Nandhini et al., 2012). Bacillus species are
capable of producing endospores, enabling them resistant to different
environmental stress conditions (Devi et al., 2022). Due to the wide­
spread production of antimicrobial chemicals and their capacity to in­
crease plant-induced systematic resistance (ISR) and stop the spread of
plant diseases by producing a wide range of secondary metabolites,
makes Bacillus a good choice for the biocontrol of plant disease. It was
reported that B. pumilus inhibited the mycelial growth of different fungi
including Fusarium sp. by producing extracellular antifungal metabolites
(Munimbazi and Bullerman, 1998). Similarly, B. pumilus strain CH4
inhibited the growth of F. oxysporum under in-vitro conditions using
chitinolytic activity (Nawangsih and Purba, 2013). Two Bacillus strains
“B. amyloliquefaciens strain YN0904 and B. subtillis YN1419” were iso­
lated from the rhizosphere. These strains effectively controlled the
Fusarium wilt disease of bananas up to 80 % under greenhouse condi­
tions along with the promotion of the growth of banana plantlets.
respectively. Furthermore, YN0904 promoted the growth of banana
plantlets (Fan et al., 2021). Munimbazi and Bullerman (1998) reported
on extracellular antifungal metabolites produced by B. pumilus which
inhibited the mycelial growth of different strains of Fusarium sp.
The inconsistent performance of beneficial microbes can be due to
the reduced population in the adjacent rhizosphere and poor survival
under extreme environmental conditions (Vejan et al., 2016). Hence, it
looks fascinating to discover different methods ensuring improved effi­
ciency of biocontrol methods. Seed coating is a method for applying
microbial inoculants such as biocontrol agents to the target plants. This
is considered a reliable technique to get maximum benefits using use
minor amounts of inocula in a precise application method and can
replace chemical seed treatments (Nagaraju et al., 2012; Tavares et al.,
2013). The beneficial microbes used in seed coatings increase the speed
and uniformity of germination, protect the seeds and seedlings of plants
from different seed and soil-borne diseases, and positively influence
plant growth by producing plant growth-promoting substances like
phytohormones (Haskett et al., 2021). In addition to direct antagonism,
microbes present in the seed coating effectively colonize the surround­
ing rhizosphere and induce host plants to develop systemic resistance to
protect from diseases. In a previous study, the application of Rhizobium
strains was performed as a seed coating and soil drenching to manage
root-knot nematode in soybean (Ahmed et al., 2016). The seed coating
was more effective in controlling the reproduction of Meloidogyne
incognita and increasing plant growth.
Research work describing the efficiency of the seed coating consist­
ing of beneficial microbes and/or biocontrol agents to manage Fusarium
2
Scientia Horticulturae 325 (2024) 112645
wilt disease is still scarce. Taking into consideration the above-
mentioned benefits of using the seed coating technique, the aims and
objectives of the present investigation were to create an effective seed
coating of Pea seeds using native antagonistic Bacillus strains to manage
Fusarium wilt disease.
2. Materials and methods
2.1. Procurement of Fusarium wilt Pathogen and in-vivo pathogenicity
analysis
The fusarium wilt pathogen Fusarium oxysporum f.sp. pisi (FOP)
strain FOP-IAGS1 (NCBI Accession No. OR493419) was procured from
the Fungal Biotechnology Laboratory, Department of Plant Pathology,
University of the Punjab, Pakistan. The pathogen was previously iso­
lated from infected pea plants showing typical wilt symptoms. FOP stock
culture was maintained on potato dextrose agar (PDA) medium plates at
4 ◦ C.
For in-vivo pathogenicity analysis, pea plants were raised from sur­
face sterilized seeds of the variety “Rachna” based on susceptibility
against Fusarium wilt disease (Raza and Akram, 2023), in pots con­
taining a sterilized commercial potting mix (Roshan Seeds LTD, Lahore,
Pakistan). The aqueous conidial suspension of FOP was prepared at a
concentration of 104 conidia/mL. Briefly, a stock agar plug containing
mycelia of FOP was cultured onto fresh PDA plates and incubated for 4
days at 25 ◦ C. The conidial suspension was collected from one-week-old
cultures by washing with distilled autoclaved water and concentration
was adjusted using a hemocytometer. After three weeks of pea seedling
emergence, 50 mL of spore suspension was poured into the rhizosphere
on the plants. The control group was provided with an amount of
autoclaved water. The plants were incubated in the greenhouse at 25 ±
2 ◦ C under natural daylight conditions. After two weeks of pathogen
application, disease rating was performed according to the scale devel­
oped by Hua et al. (2019) 0–4 [0 = no symptoms; 1 = less than 25 %
leaves wilted; 2 = 25–50 % leaves wilted; 3 = 51–75 % leaves wilted; 4
= more than 75 % leaves wilted or plants dead]. The disease index was
analyzed using the formula: DI = [Σ rating × number of plants rate­
d)/Total number of plants × highest rating] × 100.
2.2. Isolation and selection of antagonistic rhizospheric bacterial strains
The heat shock method was used for the selective isolation of bac­
teria belonging to the Bacillus genera (Senthilkumar et al., 2021). The
rhizosphere soil samples were collected from healthy-looking vegetable
crops including tomatoes (Solanum lycopersicum), cucumbers (Cucumis
sativus), and bottle gourd (Lagenaria siceraria) in the agricultural fields
situated in different areas of the Punjab Province, Pakistan. For isolation
purposes, one gram soil sample was mixed in distilled sterilized water
(10 mL) and incubated at 80 ◦ C for 10 min in the hot water bath. The
whole mixture was serially diluted up to 10− 7 dilutions using distilled
autoclaved water. Finally, 100 µL of the mixture from the last three
dilutions was spread on Luria Bertani (LB) agar media plates (g/L: Yeast
extract-5.0 g, Peptone-10 g, NaCl-10 g, Agar-15.0 g, pH 7). The plates
were incubated at 30 ± 2 ◦ C overnight. All single colonies were cultured
on LB plates using the streaking inoculation method.
Antagonistic bacteria were screened in-vitro on PDA medium plates
in a dual-culture assay in both qualitative and quantitative manners. The
5 mm diameter plugs from F. oxysporum cultured one-week-old culture
were transferred to one side of the media plates. The bacterial isolates
were inoculated on the other side of the media plate in a straight-line
manner. Each treatment was set to repeat three times. All plates were
then kept at 28 ◦ C for 5 days. The bacteria showing a zone of inhibition
were selected for quantitative analysis. For that purpose, the whole
experiment was repeated in the same manner. Hereafter the incubation
period, the pathogen colony diameters were measured and inhibition
rates were calculated to determine the inhibition percentage as follows:
A. Raza et al.
Inhibition (%) = (C − T) ÷ C × 100
where C is the radial growth of FOP (mm) alone (control); and T is the
radial growth of FOP (mm).
2.3. In-vivo analysis of the selected bacterial strains to manage Fusarium
wilt of pea
Seed priming of pea seeds was performed to analyze the potential of
selected bacterial antagonists to manage Fusarium wilt disease under in-
vivo conditions. Initially, aqueous suspension of bacterial strains was
made for priming purposes. Bacteria were raised in an LB broth medium
(g/L: Yeast extract-5.0 g, Peptone-10 g, NaCl-10 g, pH 7) overnight and
cells were collected by centrifugation. Bacterial suspension was pre­
pared in distilled autoclaved water and the concentration was adjusted
to 106− 7 c.f.u/mL by taking O.D of 1 at 600 nm. Surface sterilized Pea
seeds were dipped in spore suspension overnight. Water served as a
control treatment. Plastic pots were filled with sterilized potting mix and
pea seeds were cultivated in pots at the rate of five seeds per pot. After
emergence, thinning was performed and three seedlings were left in the
pots. Ten days after emergence, conidial suspension of FOP was pre­
pared and added to the rhizosphere of the pea seedlings. Plants raised
from only water-primed seeds and receiving FOP served as pathogen
control. Whereas, plants raised from only water-primed seeds without
receiving FOP inoculum served as non-treated control. Three replicate
pots were included in each treatment and the whole experiment was
repeated twice. Plants were irrigated with distilled sterilized water and
kept at green house under natural daylight conditions. After fifteen days
of pathogen application, the disease index was determined as described
in the in-vivo pathogenicity analysis section.
2.4. Molecular identification of best-performing bacterial strains
To identify the best-performing strains (IS1, IS6, and IS7), their 16S
rRNA motif were amplified and obtained sequences were analyzed.
Briefly, a pure culture of bacteria was grown in LB broth medium at 30
± 2 ◦ C, under continuous shaking (180 rpm) in a shaking incubator
overnight. Total genomic DNA was extracted with the help of Rapid
Bacterial Genomic DNA Isolation Kit (Sangon Biotech). Afterward,
routine PCR was performed to amplify the 16S rDNA sequence using the
universal primers 27F (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1492R
(5´-GGTTACCTTGTTACGACTT-3´) (Relman, 1993). After successful PCR,
the obtained PCR products were sequenced by the Sanger dideoxy
method at Macrogen Incorporation (Korea). The Phylogenetic trees were
constructed based on the 16S rRNA sequences with the MEGA 6.0
software. The neighbor-joining method was adopted to construct the
tree with 1000 bootstrap replications (Tamura et al., 2013).
2.5. Development of seed coating of pea plants
Three best-performance bacteria (IS1, IS6, and IS7) were selected for
coating. Firstly, all three strains were cultured on a single LB agar me­
dium plate in an overlapping streaking pattern to ensure their con­
sortium compatibility. Afterward, the aqueous suspension of the
bacterial consortium of all three strains was developed. Briefly, all the
bacteria strains were raised in 250 mL flasks, separately, containing LB
broth medium under shaking conditions overnight. The next day, the
cells were collected by centrifugation, and the pellets were washed with
distilled autoclaved water. Pallets were resuspended in distilled auto­
claved water to reach the OD of 1 at 600 nm to achieve the concentration
of 108 cfu/mL. A combined inoculum was prepared by mixing equal
volumes of the aqueous suspensions of the three bacterial isolates to
develop consortium suspension. Seeds of the Pea plant variety (Rachna)
were coated with a consortium of antagonistic bacterial strains as
described by Ehteshamul-Haque et al. (2007); and Singh et al. (2014).
Pea seeds were wetted with a consortium of beneficial bacteria (108 c.f.
3
Scientia Horticulturae 325 (2024) 112645
u/mL) in aqueous gum arabica solution (5 %). Sterilized talc was added
as filler material.
2.5. Effect of seed coating on seed germination and seedling growth
Coated seeds were sown in the plastic pots containing sterilized
potting mix. The experiment was divided into three treatments as fol­
lows: T1 = Untreated control, T2 = Coating Control only containing
binding material and filler, and T3 = Coating containing bacteria con­
sortium (IS1, IS6, and IS7). Germination data was taken daily to calcu­
late the germination index using the equation according to Zhang et al.
(2007).
( ) ∑
Germination index GI = Gt / Dt
where F is the number of seeds newly germinated at the time of X =
number of days from sowing. After 10 days of germination plants were
harvested and seedling dry weight and shoot length were observed.
Plants were irrigated with distilled sterilized water and kept at green
house under natural daylight conditions.
2.6. Effect of seed coating on suppression of Fusarium wilt disease and
plant growth properties
Another pot trial was performed and pea seedlings were raised from
coated seeds in plastic posts containing sterilized potting mix. The de­
tails of treatments are provided in Table 1. After 10 days of emergence,
FOP conidial suspension was prepared and inoculated in the rhizosphere
of the allotted pots as described in the upper section. Plants were irri­
gated with distilled sterilized water and kept at green house under
natural daylight conditions. Ten replicate pots were included in each
treatment and the whole experiment was repeated twice. The disease
index was noted 15 days after pathogen application in the in-vivo
pathogenicity analysis section, along with the plant growth parame­
ters like shoot length, root length, dry biomass, and total chlorophyll
contents by calorimetric method of Arnon (1949).
2.7. Analysis of the biochemical basis of induced defense responses in pea
plants against Fusarium wilt disease
2.7.1. Effect of seed coating to upregulate total phenolics and plant defense-
related enzymes
After five days of pathogen application, some plants were separated
and used for the analysis of the defense-related responses. Leaf samples
were removed and total phenolic compounds were analyzed by the Folin
ciaocaltue method (Singleton and Rossi, 1965), The peroxidase, poly­
phenol oxidase, and phenylalanine ammonia-lyase (PAL) activities were
estimated as described by Dalisay and Kuć (1995); Mozzetti et al. (1995)
and Cahill and McComb (1992) respectively.
2.7.2. Effect of seed coating on the metabolomic profile of pea plants
A UHPLC-ESI-QQQ-MS/MS analysis was performed to observe the
perturbations in the metabolomic profile of the pea plants receiving
different treatments. Leaves from plants of different treatments were
used for the analysis after five days of pathogen application. Leaves were
Table 1
Details of treatments.
Treatment Description
T1 Untreated control
T2 Pathogen alone (FOP)
T3 Coating control
T4 Coating control + FOP
T5 Coating with consortium
T6 Coating with consortium + FOP
A. Raza et al.
ground to powder material in the pestle mortar using liquid nitrogen.
The extraction was performed in a mixture of (methanol/water, 80/20,
v/v) containing 1 ng/µL of the internal standard as described by Jo et al.
(2022). The extract was centrifuged and passed by a microfilter as­
sembly. Chromatographic separation was performed on an Agilent 1200
ultra-performance liquid chromatography system (Agilent, USA) fitted
with a C18 analytical column (Agilent). The data for the identification
and quantification of compounds was obtained from a Triple Quad
tandem mass spectrometer (6470) system equipped with an Electrospray
Ionization Source (ESI). QC sample was made after pooling the samples
of all treatments in a single vial in equal quantities. The mobile phase A
consisted of 0.1 % formic acid (v/v) in deionised water and mobile phase
B consisted of 0.1 % formic acid (v/v) in methanol. The chromato­
graphic conditions were as following: 95 % A and 5 % B for the first 5
min, solvent A decreased to 45 % and B increased to 55 % up to 22 min,
solvent A 5 % and B 95 % over the course of 3 min and remained
un-changed for one-minute, solvent A 95 % and B 5 % for 3 min until the
end of the run. The MS scan range was 50− 1500 m/z with a 100 ms scan
time. The acquired mass data was converted into an mzxml format.
Afterward, the data was loaded onto MZmine 2.53 software and further
processed. Identification of compounds was performed using the NIST
MS/MS library operated by the MS search program coupled with the
MzMine software and previously published literature.
2.8. Statistical analysis
The data were analyzed statistically. The analysis of ANOVA and
DNMRT was performed by using the Excel addon “DSAASTAT” devel­
oped by Onofri (Italy).
3. Results
3.1. In-vivo pathogenicity analysis of FOP
A virulent isolate of Fusarium oxysporum f.s.p. pisi “FOP-IAGS1” was
obtained that was previously isolated from an infected pea plant. Before
performing further pot trials, the disease progression of FOP was
monitored in the Pea cultivar Rachna after infection with FOP. Nearly
five days after inoculation, lower leaves started turning yellow followed
by drying. The symptoms progressed to aerial parts of the plants causing
them to wither and plant death. The treated plants showed a disease
index of 79.36, hence conforming to the pathogenicity of FOP-IAGS1
(Fig. 1).
3.2. Isolation and selection of the antagonistic rhizospheric bacterial
isolates
Rhizospheric bacterial strains were isolated from soil using the heat
shock method with special emphasis on Bacillus genera. A library of 27
Fig. 1. Pathogenicity analysis of F. oxysporum f.sp. pisi isolate FOP-IAGS1 (FOP). (A) The disease index of Fusarium wilt, (B) Pea plants with fusarium wilt disease.
Left (infected planta) and right (Control plant). Vertical bars show the standard error between different biological replicates of a treatment. Capital letters present a
level of significance according to ANOVA and DNMRT at p < 0.05.
4
Scientia Horticulturae 325 (2024) 112645
purified bacteria was established by subculturing onto new media
plates. In qualitative analysis, eight bacterial strains displayed antago­
nism against FOP and coded as IS1 to IS8. In successive quantitative
antagonistic analysis, IS1, IS6, and IS7 displayed maximum significant
(p < 0.05) antagonistic activity against FOP and inhibited radial growth
of the fungi ranging from 69.28 to 48.36 % inhibition as compared to the
control treatment (Table 2).
3.3. Biocontrol efficacy evaluation of the bacterial antagonists against
Fusarium wilt of pea
A pot trial was performed to observe the biocontrol efficacy of the
potential antagonistic bacterial strains against the Fusarium wilt disease
in pea plants. At two weeks of pathogen application, typical Fusarium
wilt disease symptoms were evident on the pea plants, while healthy
control plants remained without symptoms (Fig. 2). The pathogen
control plants receiving only FOP showed more severe disease symp­
toms compared to the plants primed with antagonistic bacteria along
with the FOP (Fig. 2). The disease index in the pathogen control plants
was ~80 at the time of harvest, whereas the bacterial primed plants
receiving FOP displayed a significantly (p < 0.05) reduced disease index.
The disease index in the pathogen-inoculated plants primed with the
antagonistic bacterial strains ranged from 16.97 to 69.24 (Fig. 2).
Among the eight potential antagonistic bacterial microbes, the plants
primed with the strains IS1, IS6, and IS4 showed the significantly lowest
disease indexes, while the IS5-primed plants showed the highest disease
index when challenged with the FOP (Fig. 2). Overall, the biocontrol
behavior of the bacterial strains under investigation against Fusarium
wilt disease of PEA was in the order IS5< IS3….. > IS6< IS7< IS1
(Fig. 2). These findings indicated the fact that the antagonistic bacteria
screened under in-vitro experiments had variable disease control po­
tential against the Fusarium wilt of pea plants during pot trials. Based on
the findings of this preliminary pot trial, IS1, IS6, and IS7 were selected
Table 2
The antagonistic potential of rhizospheric bacterial
strains against F. oxysporum.
Strain Percentage inhibition
IS1 52.34 ± 3.12B
IS2 13.69 ± 0.97GH
IS3 22.58 ± 1.14EF
IS4 19.32 ± 0.82FG
IS5 31.07 ± 2.34D
IS6 69.28 ± 3.59A
IS7 48.36 ± 2.09BC
IS8 26.02 ± 1.13E
Values are mentioned as mean ± standard error. Capital
letters present the level of significance as governed by
ANOVA and DNMRT at p = 0.05.
A. Raza et al. Scientia Horticulturae 325 (2024) 112645

Fig. 2. The potential of seed priming of different bacterial strains to suppress fusarium wilt disease. (A) The disease index of Fusarium wilt. Vertical bars represent
the standard error. Vertical bars show the standard error between different biological replicates of a treatment. Capital letters present a level of significance according
to ANOVA and DNMRT at p < 0.05. (B, C) Fusarium wilt symptoms of pea plants under the influence of different treatments.

for seed coating development and related experiments.

3.4. Molecular identification and characterization of selected bacterial

strains

The taxonomy of IS7, IS6, and IS1 was examined by the 16S rRNA
gene sequencing. BLAST analysis showed the 16S rRNA gene sequence
of the strains IS1, IS6, and IS7 had > 99 % homology with the B. subtilis
(accession no. LC178546, CP054177, MT491101), B. amyloliquificiens
(accession no: MH725637, LT745972, MF977354), and B. fortis
(accession no. KJ513350, KJ513346, KM502537), respectively. Hence,
these strains were identified as B. subtilis IS1, B. amyloliquificiens IS6 and
B. fortis IS7. The phylogenetic analysis closely clustered these strains
with their respective species (Fig. 3). The sequences were submitted in
the NCBI database under the accession number OR644612, OR644619,
and OR644625.

3.5. Effect of coating on germination and seedling growth of pea

The consortium of IS1, IS6, and IS7 were coated onto pea seeds using
gum arabica, and talc as binder and filler material, respectively. The
analysis of the germination data revealed that the coating of the con­
sortium positively affected the observed parameters (Fig. 4). The con­
sortium coating significantly (p < 0.05) increased the germination index
up to 27.3 % compared to untreated control plants (Fig. 4). Pea seedlings
emerged from coated seeds and grew well with dense root architecture.
These displayed a significant increase in shoot length and biomass
compared to the untreated control seeds. It is worth mentioning here
that coating material without consortium also has a positive effect on the
germination index which can be due to the water-retaining ability of the
coating material (Fig. 4).

3.6. Effect of seed coating on Fusarium wilt disease and plant growth
properties
Seed coating developed with the consortium of three selected strains
(IS1, IS6, and IS7) gum arabica, and talc remained significantly (p <
0.05) effective in reducing the disease index along with the positive
effect on plant growth parameters (Fig. 5 and Table 3). The statistical
analysis revealed very significant interaction among treatments for
Fig. 3. Maximum likelihood tree of phylogenetic relationship between selected
rhizospheric strains and reference strains from GenBank database.
disease index. The disease index reached up to 80 % in pathogen control
(T2) plants (Table 3). The coating of seeds with consortium reduced the
disease index of FOP in pea plants and displayed a 63 % lower disease

5
A. Raza et al. Scientia Horticulturae 325 (2024) 112645

Fig. 4. Effect of coating of the consortium of Bacillus bacterial strains on seedling growth of Pea plants. (A) Effect of coating of consortium of selected rhizospheric
bacterial strains on germination index of Pea plants, (B) Effect of coating of the consortium of selected rhizospheric bacterial strains on seedling length of Pea plants,
(C) Effect of coating of the consortium of selected rhizospheric bacterial strains on seedling dry weight of Pea plants. T1 = Untreated control, T2 = Coating Control,
T3 = Coating with Consortium. Vertical bars show the standard error between different biological replicates of a treatment. Capital letters present a level of sig­
nificance according to ANOVA and DNMRT at p < 0.05.

Fig. 5. Effect of seed coating containing consortium of Bacillus microbes on suppressing Fusarium wilt disease. (A) Coated Seeds of Pea. (B) Suppression of Fusarium
wilt disease of Pea plants raised from coated seeds. (i)= Untreated control, (ii)= pathogen control, (iii)= pea plants raised from seeds coated with a consortium of
beneficial microbes, and (iv)= Pea plants raised from seeds coated with a consortium of beneficial microbes in the presence of wilt pathogen.

length, dry biomass, and total chlorophyll contents were observed in

Table 3
pea plants raised from coated seeds (Table 3). Both in the presence and
Effect of seed coating with consortium of bacterial strains on growth parameters
absence of FOP, the consortium coating significantly (p < 0.05)
of pea plants.
increased the aforementioned growth parameters when comparisons
Treatment Disease Shoot Root Dry Total chl. were made with the respective control treatments (Table 3). The shoot-
index length length biomass (mg g− 1
(cm) (cm) (g) FW)
and root- lengths of pea plants raised from consortium-coated seeds (T5)
ranged from 13.0 cm and 17.5 cm, respectively, which were significantly
T1 08.26 ± 07.16 ± 0.21 ± 11.23 ±
–
higher as compared to those plants raised from non-coated (T1) seeds
0.61bc 0.31c 0.01bc 0.59bc
T2 82.38 ± 05.97 ± 03.02 ± 0.11 ± 07.36 ± (8.2 cm shoot-and 07. 1 cm root- length). Similarly, a significant in­
3.95a 0.34d 0.15de 0.00e 0.36d crease in growth parameters was seen when data were compared be­
T3 – 09.81 ± 08.67 ± 0.23 ± 12.13 ± tween the pea plants raised from consortium-coated seeds (T6) as
0.40b 0.52c 0.01ab 0.98b compared to the non-coated seeds (T2) in the presence of fungal wilt
T4 79.67 ± 06.33 ± 05.73 ± 0.12 ± 06.09 ±
5.21a 0.29d 0.33d 0.01e 0.54d
pathogen (Table 3). Here coating without consortium served as coating
T5 – 13.08 ± 17.52 ± 0.27 ± 16.56 ± control, which showed a non-significant impact in the case of all
0.13a 1.06a 0.02a 0.06a observed parameters.
T6 26.24 ± 11.72 ± 14.71 ± 0.18 ± 10.21 ±
01.94b 0.89ab 1.21b 0.01b-d 1.21bc

Values are mentioned as mean ± standard error. Capital letters present a sig­
nificance level as governed by ANOVA and DNMRT at p = 0.05. T1 = Untreated
control, T2 = Pathogen Alone, T3 = Coating control, T4 = Coating control +
FOP, T5 = Coating with consortium, T6 = Coating with consortium + FOP.
index compared to pathogen control (Table 3).
Additionally, the beneficial effect of seed coating on pea plants was
ascertained based on data. Significantly increased shoot- and root-
3.7. Analysis of the biochemical basis of induced defense responses in pea
plants against Fusarium wilt disease
3.7.1. Effect of seed coating to upregulate total phenolics and plant defense-
related enzymes
Coating of consortium on pea seeds elicited the plants for increased
production of total phenolics and three defense enzymes when chal­
lenged with the pathogen (T6) as compared to the pathogen control

6
A. Raza et al. Scientia Horticulturae 325 (2024) 112645

treatment (T2). Plants raised from coating showed a 2.13-fold increase
in quantities of total phenolics in plants under T6 treatment (coating +
FOP) compared to the T2 (pathogen control) plants. Likewise, the T6
(coating +FOP) treatment represented significantly higher quantities of
peroxidase (PO), polyphenol oxidase (PPO), and phenylalanine
ammonia-lyase (PAL) compared to the pathogen control (T2) treatment
(Table 4). For this, the induced accumulation of PO, PPO, and PAL was
2.02-, 2.43, and 1.87-fold higher in plants of T6 treatment than path­
ogen control (T2), respectively. Similarly, the coating of consortium
alone (T5) significantly incited pea plants for increased production of
total phenolics and defense-related enzymes (PO, PPO, and PAL)
compared to the control plants (T1) raised without pathogen challenge.
Compared to other treatments, plants treated with coating material
alone (T4) serving as coating control showed non-significant changes for
the total phenolics and enzymes studied (Table 4).
quantities of some phenolic compounds were seen in T6 (consortium
coating + FOP) compared to the rest of the treatments (Fig. 6).
Together, the findings of metabolomic analysis showed that (1)
exposure to fusarium wilt pathogen decreased the production of a large
number of metabolites belonging to sugars, amino acids, and carbohy­
drates along with the increase in the production of some organic acids;
(2) coating material alone serving as coating control (T3) exerts no
significant/detectable changes in pea plants; and (3) the coating of the
consortium has a positive influence on metabolic profiles, including the
increased production of different metabolites in pea plants and
amelioration/restoration of the production of some metabolites nega­
tively affected by the wilt pathogen. Thus, these findings suggest that
consortium coating has an important role in disease resistance in pea
plants, which is dependent on the re-modulation of the metabolic
process.

3.7.2. Effect of seed coating on the metabolomic profile of pea plants 4. Discussion
This experiment was performed to uncover the mechanism(s) un­
derlying disease suppression in pea plants raised from consortium- Biological control of plant diseases using biocontrol agents has been
coated seeds. Detailed metabolomics analysis was performed to profile of great importance (Bisen et al., 2020). Along with the devastating
metabolic changes in pea plants raised from coated seeds subsequently pathogens, plants also have evolved beneficial symbiotic relations with
challenged with the wilt pathogen at five days post-pathogen inocula­ some soilborne microbes, which protect them from diseases either by
tion. The analysis was performed using three independent replicates direct antagonism or by inducing systemic resistance in plants (Pieterse
from each treatment. The comparison of different treatments (T2, T3, et al., 2014). Several bacteria microbes can provide an environmentally
T4, T5, T6) was performed with the untreated control treatment (T1) to friendly alternative to rescue plants and seeds from many diseases.
uncover the changes in different metabolites belonging to amino acids, Bacilli are among the well-studied microbes, particularly for plant
organic acids, sugars, flavonoids, phenolic, etc. A general trend observed growth-promoting properties and their ability to produce spores capable
was that the pathogen negatively affected the production of most of the of surviving environmental stresses (Rayavarapu and Padmavathi,
metabolites including amino acids, sugars, organic acids, etc. (Fig. 6). 2016). Little information is available regarding their potentiality as
Whereas, the presence of consortium ameliorated the negative impact of agricultural formulation, leaving a major aspect unexploited.
the pathogen and elicited pea plants for increased production of a range F. oxysporum is a devastating plant pathogen that causes severe irre­
of metabolites (Fig. 6). Plants raised from consortium-coated seeds (T5) versible damage to different crops across the globe. Initially, potential
in the absence of wilt pathogen displayed the highest quantities of most Bacillus bacteria were isolated using the heat shock method and purified
of the metabolites belonging to sugars, amino acids, and organic acids as from the rhizosphere of different plants looking healthy in the fields.
compared to the control treatment (T1). Significant (p < 0.05; p < 0.01) Both qualitative and quantitative studies were performed to screen eight
changes in the quantities of phenolic compounds like cinnamic acid, antagonistic bacterial strains from a library of 27 bacterial strains. The
caffeic acid, ferulic acid, gallic acid, etc. were also observed among biocontrol activity of these eight bacterial strains was evident with the
different treatments (Fig. 6). The plants raised from consortium-coated formation of a clearing zone, successfully suppressing the growth of
seeds and subsequently challenged with Fusarium wilt pathogen (T6) F. oxysporum f.sp. pisi (FOP). In previous studies, various Bacillus strains
showed 0.68-, 3.19-, 2.13-, 1.17- fold increase in the quantities of cin­ have been used against the Fusarium wilt pathogen. Bacillus subtilis
namic acid, caffeic acid, ferulic acid, gallic acid, respectively, as strain SG6 showed a significant antifungal effect and inhibited the
compared to the non-treated control plants (Fig. 6). Importantly, the mycelium growth and sporulation of F. graminearum up to 87.9- and
production of caffeic (2.64-fold), ferulic (2.13-fold), and gallic acid 95.6 %, respectively (Zhao et al., 2014). Chan et al. (2003) showed in
(0.98-fold) was positively influenced by the pathogen alone as compared another study that B. subtilis strain D1/2 inhibited the growth of multiple
to the non-treated control treatment (Fig. 6). Maximum inducible Fusarium species including F. graminearum, F. subglutinans, and
F. verticilliodes. The antagonistic potential of the bacterial microbes can
be due to the owing of competition for nutrients, antibiosis, and pro­
Table 4 duction of secondary metabolites acting as lethal weapons against fungi
Analysis of changes in the defense-related biochemicals of Pea plants developed
(Fira et al., 2018). Additionally, the varying antagonistic potential
from coated seeds.
among different bacterial strains for the same fungi is representative of
Treatment Total phenolics PO (ΔOD PPO (ΔOD PAL (cinnamic the involvement of multiple genes governing more than one mechanism
(mg/g FW) g− 1 min− 1 g− 1 min− 1 acid mg− 1 FW)
of antagonism (Slama et al., 2019).
FW) FW)
The use of microbial inoculants is permissible in the organic pro­
T1 1.13 ± 0.25d 1.28 ± 0.16 ± 0.49 ± 0.02c-e duction system. A preliminary pot trial was conducted to evaluate the
0.01d 0.00ef
T2 1.79 ± 0.12bc 2.49 ± 0.21 ± 0.52 ± 0.03cd
potential of eight antagonistic bacterial strains for the suppression of
0.03b 0.01b-d Fusarium wilt disease of pea plants. Seeds were primed separately with
T3 1.16 ± 0.09d 1.26 ± 0.18 ± 0.02e 0.51 ± 0.02cd the bacterial strains and the plants raised from primed seeds were
0.01d challenged with the FOP. Plants raised from the seeds primed with IS1,
T4 1.82 ± 0.12b 2.52 ± 0.25 ± 0.57 ± 0.02c
IS6, and IS7 displayed a significant reduction in disease index. Based on
0.02b 0.01bc
T5 1.62 ± 0.07bc 2.47 ± 0.29 ± 0.79 ± 0.04ab the sequencing results of gene encoding 16S rRNA, the best-performing
T6 2.34 ± 0.16a 0.03bc 0.01ab 0.86 ± 0.05a bacteria (IS1, IS6, and IS7) were identified as B. subtilis, B. amyloliqui­
4.55 ± 0.02a 0.33 ± 0.02a ficiens, and B. fortis respectively. Bacillus species have been attractive
Values are mentioned as mean ± standard error. Capital letters present a sig­ due to their potential role in the biological control of plant diseases
nificance level as governed by ANOVA and DNMRT at p = 0.05. T1 = Untreated caused by fungal diseases.
control, T2 = Pathogen Alone, T3 = Coating control, T4 = Coating control + Beneficial microbes and synthetic chemicals can be coated onto seeds
FOP, T5 = Coating with consortium, T6 = Coating with consortium + FOP. to control diseases. The coating formulation facilitates direct contact

7
A. Raza et al.
Fig. 6. Change in the levels of indigenous metabolites in pea plants raised from coated seeds. (A) Ellagic acid, (B) Cinnamic acid, (C) Ferulic acid, (D) Sinnapic acid,
(E) Tryptophan, (F) Carboxylic acid, (G) Caffeic acid, (H) Heat map showing comparative levels of different metabolites in pea plants raised from coated seeds. T1 =
Untreated control, T2 = Pathogen Alone, T3 = Coating control, T4 = Coating control + FOP, T5 = Coating with consortium, T6=Coating with consortium + FOP. The
asterisk represents the level of significance among different treatments as governed by ANOVA. p < 0.05 (*), p < 0.01(**).
between the beneficial microbes and the seed, thus helping microor­
ganisms colonize the plants at early stages of growth. This improves the
plants’ ability to better assimilate the nutrients (Tavares et al., 2013).
Secondly, a single microbe does not necessarily exploit all the mecha­
nisms to rescue plants against disease and promote plant growth
(Mesa-Marín et al., 2019). The use of a consortium of beneficial mi­
crobes is of current research interest. Hence the consortium of
best-performing bacterial strains (IS1, IS6, and IS7) were coated onto
pea plants using gum arabica and talc as binding and filler material,
respectively. Seed coating can improve the emergence, seedling vigor,
and establishment (Zeļonka et al., 2005). The same was observed in our
study as the coating of the consortium significantly affected seed
germination index, seedling length, and seedling dry matter.
Afterward, plants raised from consortium-coated seeds were chal­
lenged with the fusarium wilt pathogen (FOP). The study showed that
consortium coating significantly rescued the pea plants from fusarium
wilt disease and increased plant growth and biomass under pathogen-
attached conditions. The study confirmed that seed coating of con­
sortium provided better protection against fusarium wilt disease as
compared to the priming of pea seeds with individual bacterial strains.
Previously, B. amyloliquefaciens strain BLB369, B. subtilis strain BLB277,
and Pseudomonas polymyxa strain BLB267 were used to protect drum
wheat from F. graminearum infection (Zalila-Kolsi et al., 2016). The
consortium of all three bacterial strains led to the highest protection
which could be due to the synergistic effects. In a recent study, the
consortium of Trichoderma virens strain Gl006 and B. velezensis strain
Bs006 effectively suppressed Fusarium wilt disease of Physalis peruviana
plants under greenhouse and field conditions with an efficacy compa­
rable to chemical control (Izquierdo-García et al., 2021). Our findings
further reinforce the effective role of the consortium of beneficial mi­
crobes in protection against plant diseases.
The seed coating with bacterial consortium might have delivered
higher bacterial concentration right adjacent to the plant and conse­
quently in the adjacent rhizosphere. Secondly, different components/
chemicals produced by different microbes in the consortium can addi­
tively rescue the pea plant from the fusarium wilt disease. Some
8
Scientia Horticulturae 325 (2024) 112645
previous studies also prove that seed coating or seed biopriming is an
effective approach to protect plants against diseases. Müller and Berg
(2008) applied Serratia plymuthica strain HRO-C48 by different methods
to control Verticillium dahliae in oilseed rape. The bio-priming of seeds
was found effective among all formulations. Wilson and Jackson (2013)
showed that the use of Bacillus firmus as a seed treatment allows a
much-reduced load to be applied per unit area in contrast to the soil
drench method. Seed coating of wheat with Trichoderma harzianum
strain T-22 resulted in improved seedling growth and a significant
reduction in crown rot disease caused by Fusarium culmorum (Vitti et al.,
2022). Similar types of multiple benefits as better seedling growth and
reduced disease index were observed in this study when pea plants were
raised from coated seeds. The multiple beneficial effects observed on pea
plants can be due to the direct antagonism of the pathogenic fungus,
increased nutrient uptake, and the presence of nutrient solubilization
capabilities and phytohormones biosynthesis by bacterial microbes
(Anckaert et al., 2021).
The current study also indicates that beneficial microbes may induce
systemic resistance in plants by upregulating the plant biochemical
defense responses of plants. Our study confirmed and displayed similar
results to some previous studies that increased production of total
phenolic compounds and upregulated activities of enzymes of the phe­
nylpropanoid pathway, can help rescue the plant from the disease. From
these findings, it is clear that besides direct antagonism, plant disease
ameliorative effects can be regulated by the symbiosis of beneficial
microbes. Under biotic stress conditions, phenolic compounds and
phenylpropanoid pathways play a vital role in plant protection against
invading pathogens (Yadav et al., 2020).
Similarly, we observed significantly increased quantities of photo­
synthetic pigments e.g. chlorophyll a, b in plants raised from coated
seeds. Photosynthesis is directly related to plant productivity (He et al.,
2019). The findings demonstrate the effectiveness of consortium to
protect the chlorophyll a, and b of pea plants from the damaging effects
of FOP, and present it as one of the possible mechanisms of rescuing the
growth of pea plants protective in disease conditions. This also dictates
the ability of beneficial microbes to positively affect the photosynthetic
A. Raza et al.
machinery of pea plants.
Metabolic regulations are important actions ensuring plants’ survival
under stress conditions (Mundim and Pringle, 2018). Accordingly, in
this present study, we observed that the seed coating with the con­
sortium of beneficial microbes extensively re-modulated the metab­
olomic profile of pea plants. This was made possible by
UPLC-ESI-QQQ-MS/MS analysis that uncovered altered levels of a
range of endogenous metabolites belonging to amino acids, sugars,
carbohydrates, organic acids, etc. The infection of FOP decreased the
production of different metabolites related to sugars, and amino acids.
Whereas, the production of some phenolic acids was increased in this
scenario. The coating of consortium showed a restoring/ameliorative
effect by increasing the production of a range of metabolites under
pathogen stress. GABA has a positive role during plant response to stress
conditions through the maintenance of the metabolism, and regulation
of plant growth (Carillo, 2018). Similarly, phenolic acids and flavonoids
are plant metabolites that play a role in defense mechanisms and
plant-microbe interactions (Mierziak et al., 2014). The induction of
phenolic accumulation by beneficial microbes is due to the stimulation
of the phenylpropanoid pathway (Mendoza et al., 2018). These findings
are in agreement with observations where the application of Rhizo­
bacteria combined with the chemical elicitors increased the production
of phenolic contents in the Peppermint plant (Cappellari et al., 2019). In
another study, two rhizobacteria viz., Pseudomonas fluorescens strain Pf4
and P. aeruginosa strain Pag, increased production of phenolic acids in
Cicer arietinum plants including gallic, ferulic, chlorogenic, and cinnamic
acids when applied as a seed treatment (Singh et al., 2003).
We also observed increased quantities of GABA in plants raised from
coated seeds. Similarly, altered accumulation of secondary metabolites
of phenolic compounds like flavonoids and hydroxycinnamic derivatives
was seen in pea plants under different treatments. These compounds
play important roles in plants like building blocks of nucleic acids and
energy intermediates. Pathogen showed remarkable effects on the
quantities mentioned compounds, whereas the treatment of consortium
attenuated these effects. The same was observed by Chamam et al.
(2013). They reported the potential of rhizobacteria belonging to Azo­
spirillum genera to re-modulate plant secondary metabolites under
stress conditions. Further work is needed by combining molecular
strategies to elucidate how beneficial microbes stimulate abiotic stress
tolerance and cost-effective methods for the production of microbial
biomass. This will help to develop biomarkers helpful for plant breeding
and selection of beneficial microbes to predict efficacy on different
crops.
5. Conclusion
The present study showed that the coating of the consortium of
beneficial microbes is effective in rescuing plants from pathogenic
infection. Seed coating can ameliorate the damage to plants from disease
and increase plant growth. These findings further strengthen the role of
Bacillus species as plant-growth-promoting and biocontrol agents. These
beneficial microbes can be effectively used to develop different formu­
lations for conventional agriculture.
CRediT authorship contribution statement
Ali Raza: Methodology. Ali Hassan: Methodology. Waheed Akram:
Methodology, Project administration, Writing – original draft. Tehmina
Anjum: Resources, Visualization. Zill-e-Huma Aftab: Writing – review
& editing. Basharat Ali: Resources, Formal analysis.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
9
Scientia Horticulturae 325 (2024) 112645
Data availability
The original contributions presented in the study are included in the
article.
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