Sarmah et al.

Egyptian Journal of
Egyptian Journal of Biological Pest Control (2023) 33:119
https://doi.org/10.1186/s41938-023-00763-3 Biological Pest Control

RESEARCH Open Access

Endophytic Bacillus spp. from native chilli

cultivars and their effect against fruit rot
pathogen of Bhut Jolokia (Capsicum chinense
Jacq.)
Partha Pratim Sarmah1* , Hiranya Kumar Deva Nath2 and Tankeswar Nath3

Abstract
Background Fruit rot disease is one of the most important factors limiting the production potential of Bhut Jolokia
(Capsicum chinense Jacq.), which is known as one of the hottest chillies in the world. The management strategies are
highly dependent on synthetic chemicals which are causing a detrimental impact on the environment. Considering
the factors, this study focuses on exploring potential endophytic microflora from native chilli cultivars which can sup-
press the fruit rot pathogen.
Results Endophytic microflora occurs ubiquitously in plants that possess various plant-benefiting abilities. A total
of 34 endophytic isolates were obtained from different chilli cultivars. These endophytic isolates were subjected
to screening in vitro for their potential to suppress the incitant pathogen Colletotrichum gloeosporioides (the causal
agent of chilli fruit rot), which was confirmed based on cultural, morphological, pathogenicity and molecular stud-
ies. The preliminary screening yielded four bacterial endophytic isolates capable of suppressing the pathogen which
was found non-pathogenic to Bhut Jolokia plant. On the basis of morphological, biochemical and molecular identi-
fication, the four most promising isolates were identified as Bacillus velezensis, B. mycoides, B. altitudinis and B. cereus,
respectively, and used for further in vitro tests. B. velezensis showed the highest inhibition (68.67%) on mycelial growth
of C. gloeosporioides, followed by B. mycoides (65.33%), B. altitudinis (52.89%) and B. cereus (45.33%). Among the com-
patible combination, the highest efficacy (56.00%) was found in the combination of B. velezensis and B. altitudinis.
Conclusion From the present study, it can be concluded that B. velezensis and B. mycoides alone and in combination
can be used as potential biocontrol agent in managing the fruit rot of Bhut Jolokia considering their performance
in field conditions.
Keywords Antibiosis, Bacillus altitudinis, Bacillus cereus, Bacillus mycoides, Bacillus velezensis, Colletotrichum
gloeosporioides, Endophytic microflora

Background
India is known for its production of a variety of spices
*Correspondence:
Partha Pratim Sarmah
which add a plethora of flavours to Indian foods. Out of
parthz.sarmah23@gmail.com the spices, Chilli is one of the major components. India
1
Department of Plant Pathology, Assam Agricultural University, Jorhat, is known to be the world’s largest producer, exporter
Assam, India
2
AAU-Zonal Research Station, Shillongani, Nagaon, Assam, India
and consumer of Chilli and contributes a total of 36%
3
Department of Agricultural Biotechnology, Assam Agricultural to global production (Sahitya et al. 2014). Northeast
University, Jorhat, Assam, India India is blessed with a variety of chillies cultivars which

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 2 of 14

are grown indigenously. Bhut Jolokia (Capsicum chin- (Singh et al. 2019). Endophytes are found to be involved
ense Jacq.) is one of the native cultivars considered one in the production of biochemicals same as plants which
of the hottest chillies in the world. It has been registered increase their survivability inside the plant ecosystem
in Guinness World Records 2006 as the world’s hottest (Caruso et al. 2020). These mutualistic association of
Chilli after being reported to have a Scoville rating of endophytes leads to the better survivability of plants in
1,001,304 SHUs (Bosland and Baral 2007). In the world the ecosystem, providing beneficial impact to plant life
now it stands in seventh position. without causing any apparent harm.
In Northeast India, Bhut Jolokia is used to make many There have been no to very less reports of fruit rot or
dishes. Starting from pickles to different cuisines to bev- anthracnose disease occurring in native chilli cultivars
erages, the people of the Northeast always find ways to of Northeast India. Also, there is not much informa-
spice up their food. Different start-ups are trying to tion available as to why the infection of fruit rot is less in
exploit the potential of Bhut Jolokia. Not only dishes native chillies of Northeast India. The presence of endo-
Bhut Jolokia is use for medicinal purposes since old phytic microflora can be a reason for the less occurrence
ages. Deorani and Sharma (2007) reported the beneficial of fruit rot disease in those chillies. Considering all these,
effect on ailments like boils, headaches and night blind- this research focused on the biocidal activities of endo-
ness. The capsaicin present in Bhut Jolokia helps relieve phytes isolated from local cultivars of chilli against fruit
asthma and gastrointestinal abnormalities and can dilate rot pathogen.
bronchial blood vessels and chronic congestion (Sarwa
et al. 2012).
Methods
Despite having so much potential it is always chal-
Isolation of the pathogen
lenged by various biotic factors. Diseases are among the
Fruit samples of Bhut Jolokia showing prominent symp-
factors due to which the crop’s full potential is never
toms of fruit rot disease were collected from Horticul-
achieved. Chilli plant suffers from nearly 83 different
tural Experimental Farm, Assam Agricultural University
kinds of diseases, out of which 40 are caused by fungi
(AAU), Jorhat, Assam. The infected Bhut Jolokia fruits
(Rangaswami et al. 1998). Fruit rot or anthracnose, a
were washed thoroughly with sterile water. Small bits
major fungal disease, alone contributes to a loss of about
were taken considering that the bits contained both
10–50% in both yield and quality of the fruit (Pakdee-
infected and healthy portions. The bits were surface steri-
varaporn et al. 2005). The disease is also responsible for
lized using 1% sodium hypochlorite (NaOCl) solution for
reducing the productivity and market value of the Bhut
30 s. To remove traces of sodium hypochlorite, the bits
Jolokia crop (Manandhar et al. 1995).
were then rinsed with sterile water for three times. Excess
The management perspective of the disease is limited
water was soaked using sterilized bloating paper. The bits
to synthetic chemicals and indiscriminate use of chemi-
were aseptically placed in a Petri plate containing potato
cals leads to irreversible detrimental impact on the envi-
dextrose agar media (PDA) supplemented with strepto-
ronment. The use of naturally occurring microbiomes
mycin @ 100 ppm. Incubation was occurred at 27 ± 1 °C
as an alternative management strategy and increasing
for three days to observe mycelial growth.
productivity can be a solution. The endophytic micro-
flora can play an important role in developing host plant
resistance against targeted diseases. Purification and preservation of pathogen
Endophytes are present ubiquitously in all living plants To obtain pure culture, hyphal tip from the mycelial
for at least a part of the life cycle without causing any vis- growth of the pathogen was aseptically transferred to a
ible symptoms of the disease (White et al. 2000). Endo- sterilized Petri plate containing PDA media. Incubation
phytes are known to have several effects or can change was at 27 ± 1 °C until full growth. The pure culture was
the functions of plants during their life cycle (Hardoim then preserved in slants at 4 °C refrigerated condition for
et al. 2008). They are considered potent plant inoculants further study.
that can enhance plant abilities to deal with biotic and
abiotic stresses (Singh et al. 2011). Their ability to man- Cultural, morphological and molecular studies of pathogen
age diseases is a research topic that has become popular Cultural studies
recently due to the need to substitute chemical alterna- For the cultural studies actively growing culture having
tives. The ability of endophytes can be classified mainly complete growth in a 9-cm Petri plate was taken. The
into one or more functional groups, i.e. microbes that observations on colony colour, elevation, type of mar-
can (1) reduce abiotic stress in plants, (2) shield the host gin, etc., were recorded. The colour of the colony was
plant from biotic stress, and (3) improve the plant’s nutri- recorded taking into account reference to the Royal Hor-
tional status by boosting nitrogen and phosphorus levels ticultural Society (RHS) colour chart.
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 3 of 14

Morphological studies database. The phylogenetic analysis of the sequence and

Morphological studies of the pathogen were done the phylogenetic tree was prepared using CLUSTALW
through modified slide culture technique described tool in MEGA X software.
by Riddell in (1950) and through microscopic studies.
Hyphal and conidial characteristics of the pathogen were Pathogenicity test of the pathogen
studied. The pathogenicity test was performed on detach healthy
fruit of Bhut Jolokia following the protocol described by
Tovar-Pedraza et al. (2020). Healthy Bhut Jolokia fruit
Molecular studies samples were brought to laboratory and washed in run-
Fungal genomic DNA was extracted using the protocol ning tap water. The sample were washed in running water
described by Aamir et al. (2015) with slight modifica- and then surface sterilized using 1% NaOCl for 40 s and
tion. For the extraction, 250 μl of pre-warmed extraction 70% ethanol for 1 min, followed by three times rinsing
buffer (containing 2% CTAB, 100 mM Tris–Cl, 20 mM with sterile water. A 5-cm mycelium disc from actively
EDTA and 5 M NaCl) was taken in 1.5-ml Eppendorf growing pathogen culture was placed on Bhut Jolokia
tube. A 100–300 mg fungal mat was taken from 7-day- fruit wounded with sterilized needle. For control non-
old culture and transferred it to an Eppendorf tube con- colonized PDA disc was transferred. The inoculated sam-
taining extraction buffer. The fungal mat was properly ples were incubated at room temperature (27 ± 1 °C) till
ground in the Eppendorf tube with the help of a sterilized development of symptoms.
micropestle till a homogenized mixture was obtained.
The mixture was vortex for 10 s and incubated at 65 °C Collection of chilli samples for endophyte isolation
for 1 h. The mixture was added with an equal volume For the isolation of the endophytic microflora, fruit, leaf,
of chloroform: Isoamyl alcohol (24:1) and centrifuged stem and root samples of different landraces of chillies
at 13,000 rpm for 15 min at 4 °C. The aqueous layer was were collected from different districts of Assam. Various
removed without disturbing the protein layer and trans- locally available landraces are considered for the present
ferred to a new Eppendorf tube. The aqueous layer was investigation, viz. Krishna Jolokia (Capsicum annum),
mixed with 0.1 volume of sodium acetate and 0.6 vol- Mem Jolokia (Capsicum frutescens), Dhan Jolokia (Capsi-
ume of ice-cold isopropanol. The reaction mixture was cum frutescens), and Black chilli (Capsicum annum). The
then incubated at − 20 °C overnight. The reaction mix- samples were thoroughly washed under tap water and
ture was centrifuged at 13,000 rpm for 10 min at 4 °C. used for isolation within 48 h of sample collection.
The obtained supernatant was discarded, and the pellet
was washed with 70% ethanol. The reaction was cen- Isolation of endophytes
trifuged at 10,000 rpm for 4 min at room temperature. For the isolation of the endophytes, the protocol of
The obtained supernatant was discarded, and the pellet McInroy and Kloepper (1995) was followed with slight
was left for air drying at room temperature. The pellet modification. Samples of fruit, leaf, stem and roots
obtained was resuspended in 100 μl T.E (Tris EDTA) and from different chilli cultivars were brought to the labo-
preserved at – 20 ℃ for further study. ratory of the Department of Plant Pathology, AAU,
Jorhat. The samples were washed properly in running
PCR amplification tap water to remove dust particles adhering to the sur-
The amplification of genomic DNA was used ITS-1 (5′- face. The samples were rubbed with 99.9% ethanol and
TCC​GTA​GGT​GAA​CCT​GCG​G-3′) and ITS-4 (5′-TCC​ 1 g of the sample was taken for isolation. The isolation
TCC​GCT​TAT​TGA​TAT​GC-3′) primers. The PCR (Poly- was occurred in laminar air flow system equipped with
merase chain reaction) cycle followed was 94℃ for 1 min, HEPA filter where the samples were washed with 70%
94 ℃ for 35 s, 57 ℃ for 1 min, 72 ℃ for 1 min and 72 ℃ ethanol and rinsed three times. The samples were dipped
for 10 min for 30 cycles. in 1% NaOCl + 0.1% Tween 80 for one min and rinsed
with double sterile water. From the final wash, 1 ml was
pipetted out and poured into a sterile Petri plate where
Sequencing and phylogenetic analysis sterile media was poured. This is served as the sterility
The extracted DNA was sent for partial sequencing at check. If any microbial growth was observed within four
Eurofins Analytical Services India Private Limited, Ben- days of incubation, the isolates obtained were discarded.
galuru, Karnataka. The result obtained was searched in After final wash, the samples were triturated in sterilized
public domain database. Basic Local Alignment Search pestle and mortar and diluted with 10 ml of 0.1 M potas-
Tool (BLAST) was used to find homolog sequences in the sium buffer (pH 7.0). The triturate was serially diluted in
National Centre for Biotechnology Information (NCBI) potassium buffer solution up to 1­ 07 times. 1 ml of diluted
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 4 of 14

triturate was pipetted into a sterile Petri plate and suit- Incompatible combinations obtained in the compatibility
able media was poured. The plates were then incubated test were excluded from further studies.
at 27 ± 1 °C (two days for bacterial endophytes and four
days for fungal endophytes). In vitro efficacy of mutually compatible isolates
Based on the cultural characteristics, representative To study the presence of synergistic effect in mutu-
colonies from the diluted plates were transferred to new ally compatible combinations of bacterial endophytes,
media plates (PDA for fungal endophytes and Nutrient in vitro test was done over the mycelial growth of path-
agar for bacterial endophytes). Pure culture of the colo- ogenic fungus following the protocol of Zegeye et al.
nies was preserved at 4 °C refrigerated condition for fur- (2011) with slight modification. The design of the experi-
ther study. ment followed completely randomized design (CRD)
with five replications.
Preliminary screening of endophytes
Initially, mycelial disc of pathogenic fungus was taken Pathogenicity test of promising endophytes
and transferred to the centre of fresh PDA plate and The pathogenicity test of the most promising endophytic
incubated at 27 ± 1 °C for 48 h. For screening of endo- isolates was done on the Bhut Jolokia plant to ensure
phytic bacteria for antagonistic properties, four differ- the endophytic isolates are non-pathogenic to the Bhut
ent isolates of endophytic bacteria were taken and made Jolokia plant. The test was conducted through spray-
streak leaving 1 cm away from the periphery on the same ing method, following the method suggested by Larran
48-h-old pathogenic fungus culture plate. Similarly, for et al. (2016) and the root inoculation method suggested
screening fungal isolates, four different mycelial discs of by Munif et al. (2011). The roots of one-month-old Bhut
endophytic fungal isolates were inoculated at four sides Jolokia plants were trimmed and washed with sterile
leaving 1 cm away from the periphery on the same 48-h- water. The plants were then dipped in bacterial suspen-
old pathogenic fungus culture plate. For control, only the sion 1 × ­106 CFU/ml for 60 min and replanted in pots
mycelial disc of pathogenic fungi was incubated alone. and kept in greenhouse conditions (22 ± 2 °C). The leaves
of the plant were spread with bacterial suspension after
In vitro screening of endophytes wounding. For control instead of bacterial suspension,
The most prominent bacterial endophytes were evalu- sterile water was taken. Observations were made from
ated for their ability to suppress the pathogen following the 3rd to the 14th days of inoculation.
the protocol of Utkhede and Rahe, (1983). Mycelial disc
of seven days old pathogenic fungus culture was inocu- Characterization of selected bacterial endophytic isolates
lated and incubated at 27 ± 1 °C for 48 h. The endophytic For the morphological and cultural characterization, col-
bacterial isolate was inoculated through line of streak on ony characteristics of selected bacterial endophytic iso-
both sides leaving 1 cm away from periphery of plate on lates were studied.
the same 48-h-old pathogenic fungus culture plate. For For the molecular characterization, the genomic DNA
control only mycelial disc of pathogenic fungus was incu- of bacterial isolates was extracted and partial sequencing
bated alone. The plates were then incubated at 27 ± 1 °C of 16 S rDNA was done at Eurofins Analytical Services
till the control plate attained full growth. India Private Limited, Bengaluru, Karnataka.
The per cent inhibition over control was calculated for
each treatment using the following formula (Nwachukwu Extraction of selected bacterial genomic DNA
and Umechuruba 2001). For the extraction of bacterial genomic DNA, the pro-
I = [(C − T )/C] × 100 tocol of Cardinal et al. (1997) was followed with modi-
fication. 1.5 ml of bacterial culture was taken in a 2-ml
where I = Per cent inhibition, C = Radial growth diameter Eppendorf tube from the broth culture prepared 24 h
of pathogen in control, and T = Radial growth diameter of before the start of the process. The Eppendorf tube
pathogen in treatment. containing the bacterial culture was subjected to cen-
trifuge at 12,000 rpm for 2–5 min. The supernatant
Mutual compatibility of selected endophytic isolates was discarded and 500 μl of Lysis buffer (containing
For the mutual compatibility test, perpendicular streaks 10 mM Tris–Cl, 1 mM EDTA and 0.5% SDS) was added
of two bacterial isolates were made in nutrient agar (NA) and vortex thoroughly. The homogenized mixture was
plates and incubated at 27 ± 1 °C for 48 h. Compatible then incubated at 65 °C for 20 min followed by shak-
isolates grow together, and incompatible combinations ing at an interval of 5 min. 250 μl of NaCl solution was
inhibit each other. The experiment followed completely added to the reaction mixture and incubated at − 20 °C
randomized block (CRD) design with five replications. for 20 min. After incubation, centrifugation was done at
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 5 of 14

12,000 rpm at 10 min at room temperature. The super- Statistical analysis

natant obtained was transferred to a new centrifuge tube. The experiment in Table No. 02 was analysed by using
An equal volume of chilled chloroform: isoamyl alcohol Fisher’s method of analysis of variance (ANOVA), and
(24:1) was added to the reaction mixture and was mixed data interpretation was carried out following Gomez and
till the mixture turned milky. The mixture was subjected Gomez (1984). Duncan’s multiple range test (DMRT) at
to centrifuge at 12,000 rpm for 10 min at 4 °C. The aque- p ≤ 0.05 was used to separate treatment means. Criti-
ous layer was transferred to a new centrifuge tube with- cal differences were calculated to compare the different
out disturbing the protein layer. To that, 2.5 volume of treatments among themselves.
chilled 99.9% ethanol was added and incubated at -20 °C
overnight. The reaction mixture was centrifuged again at Results
12,000 rpm for 10 min at 4 °C. The supernatant obtained Cultural, morphological and molecular studies
was discarded, and the pellet obtained was washed with of the pathogen
70% ethanol. The reaction mixture was centrifuged at For cultural studies, the pure culture of the pathogen
12,000 rpm for 3 min at room temperature. The superna- (PFC Jorhat) was studied on potato dextrose agar (PDA)
tant was discarded and the obtained pellet was dissolved media. The culture was observed to have flat to cottony
in 100 μl TE (Tris- EDTA) buffer and kept at − 20 °C for colonies with dull white to pale grey colour from the front
further studies. side and yellowish colour from the reverse side (Fig. 1(A)
and (B)). There was the presence of salmon-coloured
conidial masses near the centre (Fig. 1(C)). Morphologi-
PCR amplification cal studies were done using microscopy. The hyphae were
PCR amplification of the extracted bacterial genomic found to be hyaline, branched and septate. The conidia
DNA was studied using universal primers U16SF (5′- were found to be hyaline, cylindrical and obtuse at the
AGA​GTT​TGATCMTGG​CTC​AG-3′) and U16SR apex with one or two oil globules at the centre. The size
(5′-TAC​GGY​TAC​CTT​GTT​ACG​ACTT-3′). For ampli- of the conidia was found to be 12.5 μm (length) and 5 μm
fication, 94 ℃ for 3 min, 94 ℃ for 30 s, 55 ℃ for 1 min, (width). The presence of appressoria and characteristics
72 °C for 1 min 30 s and 72 °C for 7 min were followed for of conidia preliminarily confirmed the pathogen as Colle-
35 cycles. totrichum gloeosporioides (Fig. 2(A) and (B)). For further
confirmation, molecular studies were done.

Sequencing and phylogenetic analysis Molecular studies

The extracted bacterial DNA was sent for partial The concentration of the extracted genomic DNA of the
sequencing of 16S rDNA at Eurofins Analytical Ser- pathogen was found to be 154.79 ng/μl, and the OD value
vices India Private Limited, Bengaluru, Karnataka. The (260/280) was 1.92. The extracted genomic DNA was
result obtained was searched in the public domain data- subjected to PCR amplification, and the amplified prod-
base NCBI using the Basic Local Alignment Search Tool uct showed a band length of 600 bp in gel electrophoresis
(BLAST). The phylogenetic analysis of the sequence (Fig. 3). The sequence recovery of ITS region of the path-
and the phylogenetic tree was prepared using the ogen designated as PFC Jorhat isolate through partial
CLUSTALW tool in MEGA X software. sequencing was also 600 bp. The recovered sequence was

Fig. 1 Cultural characteristics of isolated pathogenic fungus. A Pure culture on PDA (front view), B Pure culture on PDA (reverse view), C Conidial
mass formation
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 6 of 14

Pathogenicity test
The symptoms of the lesion were observed from the
5th day of inoculation of the pathogen disc on detached
fruit (Fig. 5). There was no symptom development in the
control inoculated with sterile PDA disc. Re-isolation of
the pathogen was done which showed the development
of fungal colony with characteristic dull white to pale
grey colony with conidial mass formation at the centre.
The microscopic studies confirm the presence of the
Fig. 2 Morphological characteristics of isolated pathogenic fungus. pathogen.
A Conidia on culture B Appressoria
Isolation of chilli endophytic microflora
Healthy fruit, leaf, stem and root samples were collected
from different cultivars of chilli from four different dis-
compared with NCBI database using nucleotide BLAST
tricts of Assam, viz. Jorhat, Golaghat, Nagaon and Diphu.
tool and showed maximum homology (100%) with Colle-
A total of 34 endophytic microbes were isolated from
totrichum gloeosporioides Tezpur isolate accession num-
fruit, leaf, stem and root samples of chilli and character-
ber KF053200.1 (Fig. 4).
ized on the basis of colony characteristics, viz. texture,
growth pattern, color, shape of margin and viscosity of
microbe (Table 1).
Phylogenetic studies
Observing the table it can be said that the numbers of
The sequence PFC Jorhat isolate was aligned using endophytic isolates are relatively more in roots than the
CLUSTALW using Mega-X for the phylogenetic rela- stem, leaf and fruits. All the endophytic isolates were pre-
tionship. The phylogenetic tree was prepared using served as a pure culture for further study.
neighbourhood joining method with 1000 bootstrap rep-
lication which showed maximum homology with C. gloe- Preliminary screening
osporioides Tezpur isolate, accession number KF053200.1 All the endophytic isolates were subjected to prelimi-
(Fig. 4). nary screening against C. gloeosporioides to observe the

Fig. 3 Gel electrophoresis showing amplified DNA products of pathogen. L Ladder, P Positive control, N Negative control
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 7 of 14

Fig. 4 Phylogenetic tree of PFC Jorhat isolate based on its sequences made by neighbour joining with 1000 bootstrap replication which resembles
Colletotrichum gloeosporioides

Fig. 5 Pathogenicity test on detached fruit

presence of antagonistic properties. Out of 34 isolates, In vitro efficacy of selected endophytic isolates
four bacterial endophytic isolates, viz. PBEDL1, PJE5B, against mycelial growth of C. gloeosporioides
PGE4B and PM5A, were found to be the most promis- The four most promising bacterial endophytic isolates were
ing which were then subjected to further study. subjected to in vitro screening to observe their potential
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 8 of 14

Table 1 Diversity of endophytic microflora isolated from

different parts of chilli plant
Sl. No Plant part used Number of isolated
endophytes
Bacteria Fungi

1 Fruit 7 0
2 Leaf 6 0
3 Stem 3 0
4 Root 15 3
Total 31 3

to suppress the mycelial growth of C. gloeosporioides. The

bacterial endophytes significantly differ in their potential
to inhibit the radial growth of pathogenic fungi. The iso-
late PBEDL1 showed maximum inhibition (68.67%) on
mycelial growth of C. gloeosporioides over control followed
by PJE5B (65.33%), PGE4B (52.89%) and PM5A (45.33%)
(Fig. 6).

Mutual compatibility
From the various combinations of selected bacterial endo-
phytic isolates, three combinations, viz. PBEDL1+PGE4B,
PBEDL1+PM5A and PGE4B+PM5A, showed, viz.
PBEDL1+PJE5B, PGE4B compatible reaction, while other
three combinations +PJE5B and PM5A+PJE5B showed
incompatible reaction.

In vitro efficacy of mutually compatible isolates

Out of the three compatible reactions, the highest inhi-
bition (56.00%) of mycelial growth of C. gloeospori-
Fig. 6 Efficacy of effective bacterial endophytes (individually
oides over control was recorded from the combination and in combination) on mycelial growth of Colletotrichum
PBEDL1+PGE4B, followed by PBEDL1+PM5A (48.22%) gloeosporioides. A PBEDL1, B PJE5B, C PGE4B, D PM5A, E
and PGE4B+PM5A (44.67%) (Fig. 6). There was non-syner- PBEDL1+PM4B, F PBEDL1+PM5A, G PGE4B+PM5A, H Control
gistic effect recorded from the combinations (Table 2).

Pathogenicity test of promising endophytes

As the bacterial endophytes were isolated from different
chilli cultivars apart from Bhut Jolokia, the pathogenicity
test of the endophytic isolates was tested to check whether
the isolates possess any pathogenic ability towards Bhut
Jolokia. From the observations recorded, there was no Morphological and cultural characterization of selected
development of lesions, necrotic spots, hallowing or any bacterial endophytes
kind of hypersensitive reaction in the inoculated plants The morphological and cultural characteristics of the bac-
along with the control. The lack of symptoms in the inocu- terial endophytes were recorded by observing various col-
lated plants showed the non-pathogenic nature of the iso- ony characteristics which are detailed in the table below
lated bacterial endophytes. (Table 3).
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 9 of 14

Table 2 Effect of bacterial endophyte (individual and Molecular characterization of selected bacterial
in combination) on mycelial growth of Colletotrichum endophytic isolates
gloeosporioides For the identification of bacterial endophytes, molec-
Isolate Mycelial growth (mm) Per cent ular characterization was studied. The concentra-
inhibition over tion of the extracted genomic DNA was found to be
control 230.65 (PBEDL1), 186.19 (PJE5B), 331.02 (PGE4B) and
PBEDL1 28.20a 68.67 230.12 ng/μl (PM5A) and OD value (260/280) was 1.79
PJE5B 31.20b 65.33 (PBEDL1), 1.86 (PJE5B), 1.92 (PGE4B) and 1.84 (PM5A).
PGE4B 42.50d 52.89 The PCR and gel electrophoresis using U16SF and U16SR
PM5A 49.20f 45.33 primers results showed the band length of all the iso-
PBEDL1+PGE4B 39.60c 56.00 lates were nearly 1450 bp approximately (Fig. 7). The
PBEDL1+PM5A 46.60e 48.22 sequence recovery after partial sequence of selected bac-
PGE4B+PM5A 49.80f 44.67 terial isolates was PBEDL1 (1416 bp), PJE5B (1,304 bp),
Control 90.00 g – PGE4B (1,437 bp) and PM5A (1,174 bp). On compar-
SEd(±) 0.413 ing the sequence in the public domain of NCBI using
C.D (0.05) 0.846 nBLAST showed maximum homology of PBEDL1
isolate with Bacillus velezensis, China isolate, acces-
Data are mean of 5 replications. Means followed by different superscript letters
within the same column denote significant variation and means followed by sion number MT626060.1 (99.65%) followed by isolate
the same superscript within the same column denote non-significant variation PJE5B with Bacillus mycoides (96.98%), Himachal iso-
in relation to each other at P = 0.05 (level of significance) as determined by
Duncan’s Multiple Range Test (DMRT)
late, accession number KF475859.1, isolate PGE4B with

Table 3 Morphological and cultural characteristics of bacterial endophytes

Characters Particulars PBEDL1 PJE5B PGE4B PM5A

Morphological characters Shape Rod Rod Rod Rod

Cultural characters Colony shape Irregular Filamentous Circular to irregular Circular
Surface Rough Thread like Smooth Flat
Edge Undulated Filiform Entire Entire
Colour Creamy white Pale white White Pale white
Elevation Raised Flat Convex Convex
Opacity Opaque Opaque Translucent Opaque

Fig. 7 Gel electrophoresis showing amplified DNA products of endophytic bacteria. L Ladder, P Positive control, N Negative control
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 10 of 14

Bacillus altitudinis (96.11%), China isolate, accession MN710447.1 and isolate PM5A with the B. cereus Karna-
number MN710447.1 and isolate PM5A with Bacillus taka isolate, accession number MW227464.1 (Figs. 8, 9,
cereus (95.52%), Karnataka isolate, accession number 10 and 11).
MW227464.1 (Figs. 8, 9, 10 and 11). On the basis of morphological, cultural, biochemical
and molecular characteristics, the four bacterial endo-
Phylogenetic analysis phytic, viz. PBEDL1, PJE5B, PGE4B and PM5A, were
The sequence obtained was aligned in CLUSTALW identified as Bacillus velezensis, B. mycoides, B. altitudi-
using MEGA X software for phylogenetic analysis. The nis and B. cereus, respectively, which were found effec-
phylogenetic tree was prepared using neighbourhood tive in suppressing the mycelial growth against fruit rot
joining using 1000 bootstrap replications. The phyloge- pathogen of Bhut Jolokia C. gloeosporioides. The pre-
netic tree of isolate PBEDL1 showed maximum similar- sent investigation showed the in vitro efficacy of endo-
ity with B. velezensis Portugal isolate, accession number phytes where B. velezensis showed the highest inhibition
MZ234619.1, isolate PJE5B with B. mycoides Himachal (68.67%) followed by B. mycoides (65.33%), B. altitudinis
isolate, accession number KF475859.1, isolate PGE4B (52.89%) and B. cereus (45.33%). From the compatible
with the B. altitudinis China isolate, accession number reaction, the combination of (B. velezensis+B. altitudinis)

Fig. 8 Phylogenetic tree of PBEDL1 Diphu isolate based on 16S rRNA sequences made by neighbour-joining with 1000 bootstrap replication which
resembles Bacillus velezensis

Fig. 9 Phylogenetic tree of PJE5B Jorhat isolate based on 16S rRNA sequences made by neighbour-joining with 1000 bootstrap replication which
resembles Bacillus mycoides
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 11 of 14

Fig. 10 Phylogenetic tree of PGE4B Golaghat isolate based on 16S rRNA sequences made by neighbour-joining with 1000 bootstrap replication
which resembles Bacillus altitudinis

Fig. 11 Phylogenetic tree of PM5A Jorhat isolate based on 16S rRNA sequences made by neighbour-joining with 1000 bootstrap replication which
resembles Bacillus cereus

Table 4 The accession number of isolates (pathogen and endophytes)

SL. No Isolate Organism Plant parts used Cultivar GenBank
accession
number

1 PFC JORHAT Colletotrichum gloeosporioides Fruit Bhut Jolokia OM202512

2 PBEDL1 DIPHU Bacillus velezensis Leaf Dhan Jolokia OM202620
3 PJE5B JORHAT Bacillus mycoides Root Mem Jolokia OM203143
4 PGE4B GOLAGHAT Bacillus altitudinis Root Mem Jolokia OM203114
5 PM5A JORHAT Bacillus cereus Root Mem Jolokia OM203119

showed the highest efficacy (56.00%), followed by B. All the sequences of isolates were deposited in Gen-
velezensis+B. cereus (48.22%) and then (B. cereus+B. alti- Bank for accession number. The obtained accession num-
tudinis) (44.67%). bers are presented in Table 4.
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 12 of 14

Discussion Phytophthora rot of soybean (Lu et al. 2017) and potato

In the present investigation, the causal agent of the fruit common scab (Li et al. 2019).
rot disease of Bhut Jolokia was identified as Colletotri- Chauhan et al. (2016) studied the antifungal activities
chum gloeosporioides on the basis of morphological, of Bacillus cereus and found two lipopeptides, surfactin
cultural and molecular studies. This was in conformity and fengycin which are effective against many plant path-
with the findings of many researchers (Dutta 2020) who ogens. Huang et al. (2005) also reported the presence of
confirmed C. gloeosporioides as the most common and two chitinases (ChiCW and ChiCH) showing inhibitory
dominant one causing fruit rot disease of Bhut Jolokia activity of conidial germination of major fungal pathogen
in Northeast India. The cultural characteristics are in causing lily leaf blight, i.e. Botrytis elliptica.
conformity with the reports of Dev et al. (2017) who
described the colony characteristics as flat mycelium Conclusion
with regular margin and zonation. Kimaru et al. (2018) The present investigation concluded that the potential
reported that the colony colour of the fungus varied of four bacterial endophytes, viz. Bacillus velezensis, B.
from white to dark grey and the growth of the colony mycoides, B. altitudinis and B. cereus, respectively, sup-
varied from flat, fluffy, raised and sparse on cultured pressed the fruit rot pathogen of Bhut Jolokia. The result
media. Ganagadevi and Muthumary (2008) reported of the present investigation explores the possibility of
that the conidia are hyaline one-celled, straight, cylin- using endophytes as an eco-friendly and alternative solu-
drical, obtuse at ends and measuring 9 to 24 × 3 to tion against synthetic chemicals. Considering the per-
4.5 μm. Kimaru et al. (2018) also reported similar formance of endophytes in field conditions, the bacterial
results where they found that the conidia of the fungus endophytes B. velezensis and B. mycoides can be used as
were cylindrical and hyaline measuring about 10.3 to potential biocontrol agent in managing the fruit rot dis-
18.2 × 3.0 to 5.8 μm. Both the results were in tune with ease of Bhut Jolokia.
the present investigation.
The present investigation showed the non-pathogenic
nature of the isolated bacterial endophytes. The lack Abbreviations
% Per cent
of hypersensitive reaction in plants inoculated with °C Degree Celsius
endophytes was also reported by Larran et al. (2016) on Approx. Approximately
30-day-old wheat plants inoculated with 10 endophytic BLAST Basic Local Alignment Search Tool
bp Base pair
microbes. Similarly, Bacilio-Jiménez et al. (2001) also CD Critical difference
reported that two endophytic isolates Corynebacterium CFU Colony forming unit
flavescens and Bacillus pumilus from rice rhizoplane CRD Completely randomized design
CTAB Cetyl trimethyl ammonium bromide
showed a lack of hypersensitivity in the tobacco plant. DMRT Duncan’s multiple range test
From the present study, the four most promising bac- DNA Deoxyribonucleic acid
terial endophytes that can suppress the mycelial growth E East
EDTA Ethylene diamine tetraacetic acid
of C. gloeosporioides all belong to the genus Bacillus et al. Et alia
and are identified as B. velezensis, B. mycoides, B. alti- Fig. Figure
tudinis and B. cereus, respectively, on the basis of mor- g Gram
h Hours
phological, cultural, biochemical and molecular studies. HEPA High efficiency particulate air
The antimicrobial properties of the genus Bacillus were i.e. Id est (That is)
reported by many researchers. Chitarra et al. (2003) IAA Indole acetic acid
ITS Internal transcribed spacer
reported the presence of a number of antifungal com- kb Kilo base pair
pounds produced by Bacillus spp. which disrupt the l Litre
fungal membrane-like release of chitin-binding pro- LC-MS Liquid chromatography–mass spectrometry
M Molar
teins and Chitinase affecting the cell polarity. min Minute
Bacillus velezensis reported producing a wide range ml Millilitre
of antimicrobial compounds as secondary metabolites N North
NaCl Sodium chloride
in the form of Surfactin, Plantazolicin, Bacilysin, and NaOCl Sodium hypochlorite
Iturin which are effective against many microorganisms NCBI National Centre for Biotechnology Information
(Kim et al. 2021). ng/μl Nanogram per microliter
nm Nanometre
The efficacy of B. altitudinis as a biocontrol agent Nos. Numbers
has been established against various diseases includ- OD Optical density
ing root rot disease of Vigna radiate (Sunar et al. 2015), PBEDL1  Bacillus velezensis
PCR Polymerase chain reaction
PDA Potato dextrose agar
Sarmah et al. Egyptian Journal of Biological Pest Control (2023) 33:119 Page 13 of 14

PFC Jorhat Colletotrichum gloeosporioides Cardinal MJ, Meghrous J, Lacroix C, Simard RE (1997) Isolation of Lactococcus
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TBE Tris Borate EDTA An antifungal compound produced by Bacillus subtilis YM 10–20 inhibits
Tris-Cl Tris Hydrochloride germination of Penicillium roqueforti conidiospores. J Appl Microbiol
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This study was done as a Post-graduate degree research work under the endophytes and their proposed role in plant growth. Trends Microbiol
Department of Plant Pathology at Assam Agricultural University supported by 16(10):463–471
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Availability of data and materials 38(1):82–88
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