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Chapter 2
Characterization of Banyan endophytic Bacilli and
identification of their antifungal metabolites
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Characterization of Banyan endophytic Bacilli
27
2.1 Introduction
Endophytic bacteria colonize internal plant tissue without causing substantial
harm to the host plant and may either benefit the host or benefits may be
reciprocal (Bacon et al., 2002). The symbiotic association of host plants with
bacterial endophytes offers shelter and protection to the microbes against
physiological and environmental conditions while in return microbes provide
several benefits to the host plant such as protection against disease causing
pathogens, fitness by producing functional metabolites, improving soil quality,
increasing plant mineral uptake, inducing plant defense mechanisms, improve the
plant’s ability to withstand environmental stress (e.g. drought) or enhance N2
fixation (Kloepper, 1989; Sturz and Nowak, 2000; Malinowski et al., 2000;
Ciccillo, et al., 2002; Strobel and Daisy, 2003; Kloepper et al., 2004; Melnick et
al., 2008; Li et al., 2008). Recently, endophytes have also been investigated for
their potential application in biodegradation of pollutants in soil and
phytoremediation (Kuiper et al., 2004; Berg et al., 2005; Newman and Reynold,
2005). Bacterial endophytes colonize an ecological niche similar to that of
phytopathogenes, which makes them ideal candidates as biocontrol agents
(Hallmann et al., 1998; Azevedo et al., 2000; Coombs et al., 2004; Kloepper et
al., 2004; Cavaglieri et al., 2004; Berg et al., 2005; Senthilkumar et al., 2007;
Melnick et al., 2008). Endophytes belonging to several bacterial genera such as
Bacillus (Melnick, et al., 2008), Pantoea, Acinetobacter, Serratia (Li, et al.,
2008), Burkholderia (Compant, et al., 2005), Pseudomonas (Geramaine et al.,
2004), Phomopsis, Streptomyces, Enterobacter, Staphylococcus, Azospirillum,
Clavibacter, Herbasprillum (Ryan et al., 2008) have been reported and
investigated for their role in plant protection, plant growth promotion,
phytoremediation and production of novel bioactive compounds. The bioactive
compounds with diverse applications as antibiotics, insecticides,
immunosuppressants, antioxidants and antitumor agents of endophytic origin
have been reported. (Tan and Zou, 2001; Castillo et al., 2002; Strobel and Daisy,
2003; Strobel et al., 2004; Zang et al., 2006).
The plants growing in unique environmental settings with ethnobotanical value
and longevity or endemic location are likely to be a source of rarely occurring
novel microbial endophytes (Strobel, 2003). The novel microbial flora or flora
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Characterization of Banyan endophytic Bacilli
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from unique plants generally offers the bioactive and chemically novel
metabolites with huge medicinal and agricultural potential (Strobel and Daisy,
2003).
The matrix assisted laser desorption ionization time of flight (MALDI-TOF)
mass spectrometry has been used as an efficient method for rapid investigation of
complex mixture such as a cellular extract or culture filtrates originating from
microorganisms without purification (Saenz et al., 1999; Hitzeroth et al., 2005).
Thus, it permits the direct analysis of biomolecules from intact cells, tissues and
organelles (Vater et al., 2002; Hitzeroth et al., 2005). The intact cell MALDI
mass spectrometry (ICMS) technique has been developed for determining cell
surface associated as well as intracellular molecules. The most significant
application of ICMS has been demonstrated in mass spectrometric finger printing
and metabolic profiling of microorganisms, forming a novel chemotaxonomic
tool for rapid identification of bacteria and differentiation of closely related
strains. It has successfully been used for the rapid identification and taxonomic
characterization of intact bacterial cells based on their specific secondary
metaoblites, proteins, cell wall constituents (Krishnamurthy and Ross, 1996;
Leenders, et al., 1999; Evason et al., 2000; Lay, 2001; Pabel et al., 2003; Keys et
al., 2004; Price et al., 2007). Moreover, this method has been successfully used
for the rapid typing of vegetative cells and spores of bacilli (Leenders et al.,
1999; Williams et al., 2002).
Banyan tree (Ficus bengalensis) is ethanobotanically important Asian endemic
plant with a very long life span. Various parts of the Banyan tree like aerial roots,
latex, stem, bark, leaves, and fruits have been used in preparation of traditional
medicine for the treatment of various ailments like toothache, diarrhea, dysentery,
female sterility, leucorrhoea, rheumatism, skin disorders. The medicinal
properties of Banyan tree led us to work on endophytic flora of this plant.
The present study describes,
i) Isolation, identification and preliminary characterization of
endophytic bacilli from aerial roots of Banyan tree.
ii) In plantae visualization of endophytes in aerial roots of Banyan tree.
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Characterization of Banyan endophytic Bacilli
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iii) Isolation, purification and identification of antifungal compounds
produced by potential fungal antagonist endophytic bacterial strain, B.
subtilis K1
iv) Fingerprinting of all the endophytic bacilli isolates on the basis of
diversity of cyclic lipopeptides using intact cell MALDI TOF mass
spectrometry (ICMS).
2.2 Materials and methods:
2.2.1 Isolation of endophytes from hanging roots of Banyan tree:
The young aerial roots of Banyan tree were collected from Anand, Gujarat, India
and processed freshly within an hour for the isolation of endophytes. Bacterial
endophytes were isolated from surface sterilized budding aerial roots of Banyan
tree as described below. The aerial roots were dipped into solution of 15%
Savlon™ (v/v) for 15minutes followed by treatment with 70% ethanol for
2minutes. The ethanol treated roots were further immersed in 0.1% HgCl2 (w/v)
for 30 seconds followed by repeated (at least 6-8times) washes with sterile
distilled water to remove excess of HgCl2 from the surface of explants (Khyati et
al., 2009). The surface sterilized root tissues were cut into 8mm pieces and were
placed on PDA and Luria Agar (LA) plates (Himedia limited, Mumbai) for
growth of endophytes. The bacterial growth near surface sterilized aerial root
explants on plates was subjected to further isolation of pure cultures by streak
plate method. The pure cultures were repeatedly sub-cultured to check for purity
and then maintained on LA slopes at 4 ˚C as well as in form of glycerol stocks
stored at -20 °C.
2.2.2 Identification of Banyan endophytes:
The identification of bacterial endophytic isolates was done using MicroLog 1
(bacterial identification system) using GP2 plates procured from BioLog Inc.,
USA. The endophytes were grown on BioLog universal agar (BUG) media for 10
h at 30°C. The Biolog GP2 plates (gram positive2) were inoculated with 150 μL
bacterial suspension of 29% turbidity and incubated at 30 °C. The plates were
read after 18 h for utilization of carbon sources and identification was done on
the basis of carbon source utilization pattern from the gram positive database
using BioLog Microlog software (version 4.2; Biolog, Inc. USA). On the basis of
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Characterization of Banyan endophytic Bacilli
30
carbon source utilization pattern in BioLog GP2 microplates (Gram positive 2
microplates), the similarity coefficients among the endophytic isolates were
determined to construct dendogram using the NTSYS PC Version 2.0 software.
The identification of potential antagonist, endophyte K1, which was selected for
further investigation, was further confirmed by 16S rDNA gene sequence
analysis.
2.2.3 In planta localization/ visualization of endophytes:
2.2.3.1 Vital staining:
The surface sterilized hanging roots of Banyan tree were incubated in sterile 2, 3,
5-Triphenyl tetrazolium chloride (TTC) salts solution (for preparation see
appendix 2) under aseptic condition for 18 h at 30 ± 2 °C . The longitudinal or
cross sections of TTC treated roots were taken using Cryotome (Leica™) for thin
sectioning. The sections were taken on the glass microscopic slides and prepared
for the wet mount using glycerol phosphate buffer. The glycerol was used to
prevent drying of sections. The sections were then examined under light
microscope at a 100 X magnification.
2.2.3.2 Transmission electron microscopy (TEM):
The root tissues were fixed with Karnovsky’s (glutaraldehyde + formaldehyde)
fixative for 2-5 hours at room temperature under vacuum. The roots were then
removed from the fixative and washed thrice with cold solution of 0.1 M sodium
phosphate buffer (pH-6.8) each for 10 min. The roots after fixation were then
transferred into 2% (w/v) Osmium tetroxide (OSO4) and further incubated at
room temperature for 18 h. After the treatment with OSO4 the roots were washed
with buffer solution, twice followed by cold deionized water wash for three
times. The OSO4 treated roots were then stained by soaking the roots into the
cold solution of 1 % (w/v) uranyl acetate for 30 minutes. Once the roots were
fixed, the dehydration step was carried out. To remove the water content,
dehydration was carried out by using acetone water series. Roots were treated
with a series of acetone water solution starting from 10% (v/v) acetone to 100 %
(v/v) acetone and each treatment was carried out for 20 min.
The dehydrated roots were infiltrated with Spurr™ resin. The infiltration step was
carried out by treating the roots with increasing Spurr: acetone ratio for different
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Characterization of Banyan endophytic Bacilli
31
time intervals. The first treatment was carried out by using 3.1 Spurr: acetone
ratio for 2 h followed by treatment with equal proportion (2:2) Spurr: acetone for
12 h. Then the roots were incubated into 3:1 Spurr: acetone solution for 24 h. The
final step was carried out by using pure Spurr and samples were incubated for
24h. All the steps were carried out at room temperature. The infiltrated root
tissues were embedded into spurr resin by putting each infiltrated root piece into
the slot of rubber casting tray along with spurr resin. The spur resin with
embedded root tissues was allowed to polymerize at 70˚C in oven for 12-48 h till
the polymerized spurr became sufficiently hard. The block containing root tissues
were trimmed for ultra thin sectioning. The 1 μm and nm sections were taken
using ultra microtome (Leica™). The ultrathin sections were mounted on formvar
coated grids and with Sato’s lead solution. The coated grids containing root
tissues were then examined under electron microscope.
2.2.4 Characterization of endophytes:
2.2.4.1 In vitro fungal antagonism:
The antifungal activity of banyan endophytic isolates was investigated against
following fungal cultures: Aspergillus niger 40211, A. niger 16404, A. niger 181,
A. flavus, Chrysosporium indicum, Mucor indicus, Fusarium oxysporum f.sp.
lycopersicii, F. oxysporum f.sp. gingiberi, F. oxysporum 1072, Candida
albicans, Alternaria brunsii (1), A. brunsii (2), Cladosporium herbarum 1112,
Sclerotia rolfsii and Lasiodiplodia thoebromae ABFK1. The pure cultures of four
endophytic isolates were spot inoculated in four sectors on sterile potato dextrose
agar plate (3cm away from the center of the petri dish) and incubated at 30 ˚C for
48 h. After 48hrs of incubation, 9mm mycelial plug of each fungal pathogen
mentioned above was placed on the centre of agar medium on the Petri-plate and
further incubation was continued for 5-7 days.
2.2.4.2 Production of extracellular enzymes:
The pure cultures of Banyan endophytic isolates were screened for xylanase,
cellulose, lipase and chitinase. The cultures were spot inoculated on the xylan
agar medium (Luria agar amended with 2.5g/L Birchwood xylan), CMC agar
medium (Luria agar amended with 10g/L carboxy methyl cellulose), chitin agar
medium (4g/L colloidal chitin in Luria agar) and tributyrin agar medium (10g/L
trybutyrin emulsion of Luria agar) for screening of xylanase, cellulase, chitinase
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Characterization of Banyan endophytic Bacilli
32
and lipase production, respectively. All the plates were incubated at 30 ˚C for 48-
72 h. The production of xylanase and cellulase were determined by appearance of
clear zone around colonies upon staining the medium with Congo red followed
by destaining with 2M NaCl. The ability of the cultures to produce chitinase was
determined by presence of dark zones around colonies against fluorescent
background upon treatment with calcofluor white, when observed under UV
light. The ability of the cultures to produce lipase was determined by presence of
clear zones of tributyrin hydrolysis around colonies against the opaque
background of tributyrin emulsion.
2.2.4.3 Hemolytic activity:
Endophytic bacteria were cultivated on sterile blood (5 % v/v) agar medium. The
plates were incubated at 30°C for 24-48 h. The plates were then observed for
zone of haemolysis upon incubation around colonies of bacterial isolates.
2.2.4.4 Profile of growth, antifungal activity as well as emulsifying activity of
Banyan endophytic isolates:
For inoculum preparation, cells from a single colony of a bacterial isolate was
inoculated into 50 mL of sterile Luria broth (LB) in 250 mL Erlenmeyer flask
and incubated at 30°C for 12 h (O.D. 1.9-2.0) on orbital shaker (150 rpm). For
investigation of antifungal as well as emulsifying activity, the inoculum was
added to 100 mL of sterile LB in 250 mL Erlenmeyer flasks to obtain an initial
O.D.600 nm ∼0.05. The flasks were incubated on orbital shaker (150 rpm) at 30°C
for 96 h and at regular interval of 24 h, one flask of each culture was removed,
cells were separated by centrifugation (10,062 X g for 20 min.) and supernatant
was collected separately. The cell pellet was used for measurement of growth by
gravimetric method while supernatant was subjected to analysis of antifungal and
emulsifying activity. The antifungal activity was assayed by agar cup diffusion
method as described below. The test plates were prepared by seeding 100 μL of
spore suspension (1 x 107spores/ml) of Aspergillus niger 40211 into 4.5ml of
molten soft agar (1% agar, w/v) and over-layered on sterile PDA plates and
allowed to solidify. The plate was divided into four sectors and four wells were
bored, one each in centre of each sector using sterile cup borer. To each well 100
μL aliquot of methanolic antifungal extract obtained from different cultures was
added and allowed to diffuse in medium. The plates were incubated for 48 h at
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Characterization of Banyan endophytic Bacilli
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30˚C. Upon incubation, the diameter of zone of inhibition was measured and
arbitrary antifungal activity units (AAU) were determined. One arbitrary
antifungal activity unit corresponds to the amount of antifungal active
metabolites which yielded 13 mm zone of inhibition on PDA plates seeded with
A. niger 40211. The emulsifying activity (E.A) was determined by using
modified emulsification assay described by Navon-Venezia et al., 1995. The 1mL
aliquot of culture supernatant was added to 6.5 mL of 20mM TM buffer (20 mM
Tris-HCl buffer [pH-7], 10 mM MgSO4) followed by addition of 0.1mL of 1:1
(v/v) mixture of 2-methyl naphthalene and hexadecane. The samples were
vigorously mixed for 2 min. and allowed to stand for 1 h at 30°C before
measuring turbidity at 600nm. One unit of emulsifying activity was defined as
amount of emulsifier that yielded an A600 nm of 0.1 in the assay mixture.
2.2.5 Isolation and Characterization of antifungal metabolite/s produced by
potential fungal antagonist:
2.2.5.1 Isolation of antifungal metabolites from Banyan endophyte, B. subtilis
K1:
The antifungal metabolites from culture supernatant were precipitated by
lowering the pH of broth to 2 using 6N HCL. The precipitates were harvested by
centrifugation of acidified broth at 10,062 X g for 20 min. The supernatant was
discarded while the pellet was solubilized in pure methanol. The methanolic
extract was then centrifuged to remove undissolved fraction, while supernatant
was collected and subjected to drying by rotary vacuum evaporation (Buchi,
Switzerland) at 30˚C. The yellowish brown sticky substance thus obtained was
dissolved into small volume of methanol for further analysis.
2.2.5.2 Effect of antifungal metabolites produced by B. subtilis K1 on
germination of conidia of A. niger 40211:
The conidia (1x103 spores/mL of potato dextrose broth) were incubated with
various concentrations of crude culture supernatant and methanolic antifungal
extract, for 10 h at 30°C. Upon incubation, the conidiospores were stained with
1% Lacto phenol blue, and observed under light microscope under oil immersion
lens (100 X) (Lawrence & Mayo, Kolkata). Each experiment was performed in
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Characterization of Banyan endophytic Bacilli
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triplicate. A conidium was considered as germinated if the germ tube was more
than half of the diameter of conidium (Chitarra et al., 2003).
2.2.5.3 Stability studies of antifungal metabolites produced by B. subtilis K1
The cell free culture (CFC) supernatant was adjusted to various alkaline and
acidic pH using 1N NaOH or 1N HCL and incubated at 30˚C for 30 min. The pH
of each treated sample was re-adjusted to pH 7.0. For thermal stability
determination, CFC was incubated at various temperatures varying from 25 to
121˚C for 30 min. The residual antifungal activity upon each treatment was
measured against A. niger as a test culture using agar cup diffusion method.
2.2.5.4 Thin layer chromatography methanolic antifungal extract from B.
subtilis K1
The methanolic extract was spotted to pre coated silica G 60 F254 TLC plates
(Merck Darmstadt Germany) and developed in chloroform: methanol: water:: 65:
30: 5, v/v/v. The separated bands on TLC plate were developed by spraying 1 %
(w/v) Ninhydrin or Pauly’s reagent (Koppel et al. 1973). The antibiogram of
spots resolved on TLC plate was performed by over-layering it with molten soft
PDA agar seeded with 104 spores of A. niger. The over-layered TLC plate was
then incubated at 30˚C in moist chamber for 48 h.
2.2.6 Purification of antifungal metabolites from B. subtilis K1:
The cyclic lipopeptides in the extract were further separated by reverse phase
high performance liquid chromatography (RP-HPLC) using semi-preparative
Phenomenex (Torraance, CA, USA) C18 column (4.6 mm x 250 mm, 10m
particle size, 90 pore size) and MeOH/ H2O/ 0.1% TFA (tri-fluoro acetic acid) as
a mobile phase. The flow rate was maintained at 1mL/min with gradient of 60
min (80-95 %, v/v MeOH in 50 min; 95%, v/v MeOH for 5 min and 95 to 80 %,
v/v MeOH in 5minutes). The elution of metabolites was monitored using UV
detector at 226 and 280 nm. The metabolites eluted under individual peaks were
separately collected in different vials and concentrated using rotary vacuum
evaporator (Buchi, Switzerland) at 30˚C and lyophilized. The concentrated HPLC
peaks, thus obtained were used for the antifungal activity and sequence analysis.
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Characterization of Banyan endophytic Bacilli
35
2.2.7 Antifungal activity of purified HPLC fractions:
A stock solutions (1mg/mL) of metabolites eluted under major HPLC peaks P3,
P4, P5, P14, P15, P16, P17 were prepared by dissolving lyophilized fractions in
MeOH and analyzed for activity against A. niger 40211, A. flavus, A. parasiticus,
F. oxysporum1072, Chrysosporium indicum, Candida albicans, Trichosporon
sp.1110, Alternaria brunsii (2), Cladosporium herbarum1112, Helmethosporium
graminum1126, Lasiodiplodia theobromae ABK1, by paper disc method. The
5mm sterile paper (Whatmann filter paper no. 1) discs were dipped in
aforementioned stock solutions prepared from HPLC fractions and discs were
allowed to air dry under aseptic condition. The discs were then placed on the
PDA plates seeded with 100 μL (1 X 106 spores/mL) spore suspension of each
fungal culture.
Fungal spore suspension was prepared by harvesting spores into sterile distilled
water and the spore counts were determined using haemocytometer. In case of
Candida albicans and Trichosporon 1110, culture suspension was prepared by
growing the yeast cultures in 50 mL of potato dextrose broth under agitated
condition (150 rpm) at 30˚C for 10-12 h. The cell numbers were determined
using a hemocytomer and adjusted to 1 x 106 cells/mL by appropriate dilution.
The MIC and IC50 of HPLC fractions were determined by double dilution
technique against susceptible fungal cultures in sterile 96-well microtiter plates
with each well containing 100 μL of potato dextrose broth. After dilution ∼102
spores of test fungus were inoculated into each well. To control wells,
corresponding aliquot of MeOH instead of sample was added. The plates were
incubated for 24-48 h at 30˚C and MIC values for each fraction were determined
against susceptible test fungi on the basis of highest dilution showing no growth.
2.2.8 Mass spectrometry (MS) :
The HPLC fractions were subjected to MALDI-TOF MS analysis. The data were
acquired on Ultraflex TOF/TOF spectrometer (Bruker Daltonics, Germany)
equipped with 50 Hz pulsed N2 laser (337nm) operated in positive ion reflectron
mode using 90 ns delay time and 25 kV accelerating voltage. Samples were
prepared by mixing equal volume of purified HPLC fractions with α-cyano-4-
hydroxy-cinnamic acid or 2, 5 dihydroxy benzoic acid saturated in acetonitrile:
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Characterization of Banyan endophytic Bacilli
36
water (1:1, v/v) with 0.1% (v/v) trifluoroacetic acid and applied on the MALDI
sample plate. The sample spots on MALDI plate were allowed to air dry before
analysis.
2.2.8.1 Intact cell MALDI mass spectrometry (ICMS) :
Cells from a single colony of each bacterial isolate grown on Luria agar plate was
transferred into 100 μL of methanol: Water (1:1) and from that 1 μL was used for
mass spectrometric (MS) analysis using MALDI-TOF mass spectrometer as
mentioned above.
2.3 Results and Discussion:
Most of the studies on bacterial endophytes has been focused on agriculturally
important plants (Cavaglieri et al., 2004; Compant et al., 2005; Naik et al., 2006;
Melnick et al., 2008) while literature on bacterial endophytes from woody trees is
sparse (Wang et al., 2006). The young aerial roots of Ficus benghalensis (Banyan
tree) originates near the crown of the tree and grows down towards soil through
the air which appears to be an ideal plant organ to study endophytic flora
(Suryanarayan et al., 2001). Hence, we selected aerial roots tips of Ficus
benghalensis for the isolation of endophytic bacteria.
2.3.1 Isolation, identification, in plantae localization and characterization of
Banyan bacterial endophytes:
The surface sterilized tender aerial root tips of Banyan tree were placed on LA
and PDA plates and incubated at 30°C up to 10 days. On the fourth day of
incubation, bacterial growth was observed at the edges of surface sterilized
Banyan aerial root pieces. The bacterial growth thus obtained was then sub-
cultured on fresh sterile LA plates for isolation of pure cultures. Seven different
morphotypes could be isolated in pure form, which were designated as K1, A2,
A4, A11, A12, A13, and A32. All the seven isolates were found to be motile,
gram positive, spore forming bacilli.
All bacterial isolates except A32 could be identified on the basis of carbon source
utilization profile using GP2 plates of Biolog. The isolates designated as K1, A2,
A4 and A12 were identified as Bacillus subtilis with similarity coefficients of
0.86, 0.68, 0.77 and 0.65, respectively. The isolates A11 and A13 were identified
as Bacillus amyloliquefaciens with 0.78 and 0.74 similarity coefficients,
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Characterization of Banyan endophytic Bacilli
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respectively. The isolate A32 produced highly mucoid colonies preventing the
preparation of dense homogenous cell suspension, which is a pre-requisite for
identification using Biolog. The GP2 plate contains 95 different carbon
substrates, amongst which 48 substrates were not utilized by all the six isolates;
however these isolates exhibited significant variation in utilization of remaining
47 carbon sources (Table 2.1). The dendogram on the basis of carbon source
utilization profile for above six cultures grouped these cultures into two clusters
sharing more than 80% similarity. One of the cluster consisted of all four B.
subtilis isolates, amongst which K1 and A2 exhibited about 90% similar carbon
utilization profile. The other cluster grouped the two B. amyloliquefaciens
isolates, A11 and A13 which also exhibited high degree of similarity (90%)
(Figure 2.1). On the basis of carbon source utilization profile, it was confirmed
that all the seven isolates were different from each other.
Table 2.1: Utilization profile of 47 carbon sources by Banyan endophytes
No. Carbon source K1 A2 A4 A11 A12 A13 1 Dextrin + + + + + + 2 N Aacetyl ß-D glucosamine + + + + + + 3 Amygdalin + + + + + + 4 Arbutin + + + - + V 5 D-Cellobiose + + + + + + 6 D-Fructose + + + - + V 7 D-Galactose - - + - - - 8 Gentiobiose + + + - + + 9 D-Gluconic acid - - + V V - 10 Α-D-Glucose + + + - + V 11 m-Inositol - - - V - - 12 Maltose + + + + + + 13 Maltotriose V + + V V V 14 D-Mannitol + + + V + - 15 D-Mannose + + + + + B 16 3-Methyl glucose V V + V V + 17 Α-Methyl D-glucoside + + + + + + 18 ß-Methyl D-glucoside + + + + + + 19 Palatinose + + + + + - 20 D-psicose + + + V V + 21 Salicin + + + + + - 22 D-Sorbitol V + + + + - 23 Stachyose - - - - V - 24 Sucrose + + + + + - 25 D-Trehalose + + + + + + 26 Turanose + + + + + + 27 ß-Hydroxy butyric acid - - - - + -
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28 Γ-Hydroxy butyric acid + - - - - - 29 P-Hydroxy phenylacetic acid - - - - + - 30 Α-Ketovaleric acid - V V - + - 31 L-Lactic acid - - + - - - 32 L-Malic acid + V + - + - 33 Pyruvatic acid methyl ester + V + V + V 34 Pyruvic acid + V + V + V 35 N-Acetyl L-Glutamic acid - - - - + - 36 L-Alanine V + + - - V 37 L-Aspargine V V + - + V 38 L-Glutamic acid V + + - + V 39 Glycerol V + V + + + 40 Adenosine + + + - + V 41 2' Deoxyadenosine + + V - - - 42 Inosine + + + - + V 43 Thymidine + + + V + + 44 Uridine + + + V + + 45 Thymidine 5’-
monophosphate V - + - - - 46 D Glucose 6phosphate V V V V - - 47 DL α Glycerol phosphate - - - V - V
+, utilization of substrate as carbon source; -, substrate not metabolized; V, borderline reaction or weak positive.
Figure 2.1: Dendogram based on similarity coefficients calculated from carbon source utilization profile of six endophytic bacilli. The carbon source utilization profile was determined by employing GP2 plates of Biolog.
Chapter-2
Characterization of Banyan endophytic Bacilli
39
The transverse sections (T.S.) of hanging roots of Banyan tree upon staining with
tetrazolium dye revealed the presence of pink to purple colour stained bacteria
and fungi in the parenchyamtous cells of cortex and pith area. They appeared to
be localized in intercellular spaces as well as in paranchymatous cells of cortex as
well as around xylem vessels (figure 2.2a). The presence of rod shaped bacteria
was demonstrated in roots of Brassica sp. by Shefali et al., (1987) using
tetrazolium reducing dye. The presence of rod shaped bacterial cells could also be
demonstrated by transmission electron microscopy (figure 2.2b). Thus, light as
well as transmission electron microscopy supports the occurrence of bacterial
cells as endophytes in vascular as well as parenchymatous cells of Banyan aerial
roots. Furthermore, endophytes are known to produce extracellular hydrolyases
like cellulases, pectinases, xylanases in order to penetrate the host tissues for
colonization or as a resistance mechanism to overcome attack by host against
pathogenic invasion and/or to obtain nutrients from the host cells (Tan and Zou,
2001). All our endophytic isolates were found to hydrolyze xylan and cellulose
on solid media indicating their ability to produce xylanase/s and cellulose/s,
which are plant cell wall degrading enzymes. The ability to produce xylanase and
cellulase further supports their endophytic nature. None of the cultures exhibited
chitin hydrolysis while all the isolates hydrolyzed tributyrin. Thus, the
microscopic observations as well as their ability to produce hydrolyases, supports
the endophytic nature of our bacterial isolates.
Figure 2.2: Light micrograph (X 100) of T.S. cortical cells of TTC treated Banyan aerial roots (a); Electron micrograph of T.S. of Banyan aerial root (b).
Chapter-2
Characterization of Banyan endophytic Bacilli
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The endophytic isolates were then investigated for antifungal activity against 14
different fungal cultures. It was surprising to observe that all the endophytic
isolates exhibited antifungal activity (Table 2.2 and Fig. 2.3). The bacterial
cultures K1, A13, A2 and A4 were found to inhibit the growth of all the 14 test
fungal cultures, while A32 inhibited only 5 fungal cultures amongst 14 tested.
The isolates A11 and A12 were also able to inhibit 13 out of 14 test fungal
cultures; they could not inhibit the growth of Sclerotia rolfsii.
Table 2.2 Spectrum of antifungal activity of cell free fermentation broth of
Banyan endophytes monitored by agar diffusion assay
Sr. No.
Fungal cultures Diameter of zone of inhibition (mm) K1 A11 A12 A13 A2 A4 A32
1 Aspergillus niger 40211
36 36 34 36 22-24 26 -
2 Aseprgillus niger 181
32-34 34 32-34 38 20 22 -
3 Aspergillus niger 16404
32 30 34 36 22 24 -
4 Aspergillus flavus 30 28 31 30 28 28 26 5 Alternaria brunsii (1) 32 22 30 20 24 30 20 6 Alternaria brunsii (2) 28-30 24 30 20 22 34 24 7 Chrysosporium
indicum 34-38 30-36 36-40 32-36 28 30 22-24
8 Fusarium oxysporum(1072)
32 28 24 20 28 24 -
9 Fusarium oxysporum lycopercisi
30 24 26 24 32 28 -
10 Fusarium oxysporum gingiberi
32 26-28 24 24 26 20 -
11 Cladosporium herbarum1112
30 28 25 25-28 28-30 28 25-27
12 Lasiodiplodia theobromae ABK1
32 28 24 20 28 24 -
13 Sclerotia rolfsii 30 - - 15 20 25 - 14 Mucor indicus 24 18 22 18 26 24 -
In some interactions between bacilli and sensitive fungi especially Aspergillus
species, a precipitation line was observed in the inhibitory zone between bacterial
and fungal growth. Similar observations have been documented by Cornea et al.,
(2003) in their in vitro antifungal assays of Bacillus sp. B209 against Sclerotinia
sclerotiorum. Several bacterial endophytes have been known for their fungal
Chapter-2
Characterization of Banyan endophytic Bacilli
41
antagonism. The bacterial endophyte, B. amyloliquefaciens ES-2 isolated from
Scultellaria baicalensis Georgi inhibited the growth of various plant pathogenic
fungi viz., A. niger, A. flvus, A. ficuum, A. oryzae, Mucor wuntungkiao, F.
culmorum, F. oxysporum, Maganporthe grisea and Botryodiplodia theobromae
(Sun et al., 2006). Similarly, Serratia marcescens isolated from Rhyncholacis
penicillata; Paenibacillus polymyxa isolated from wheat and Streptomyces sp.,
isolated from rice have been reported for their fungal antagonism (Beck et al.,
2003; Ezra et al., 2004; Strobel et al., 2004; Naik et al., 2006; Li et al., 2007).
Figure 2.3: Inhibition of growth of different fungal cultures by Banyan
endophytes on potato dextrose agar plates.
Furthermore all our endophytic isolates exhibited prominent haemolysis on sheep
blood agar plates. The haemolytic activity of Bacillus sp. has been correlated to
their ability to produce and secrete surface active metabolites which act on red
blood corpuscles (Thimon et al., 1992; Vanittanakom et al., 1986; Pabel et al.,
2008). The fungal antagonistic action and hemolytic activity exhibited by our
Chapter-2
Characterization of Banyan endophytic Bacilli
42
isolates suggest their ability to produce antifungal compounds that may also have
surface active properties. The antifungal compounds produced by some Bacilli
species have been reported for their surface active properties as well as their
antifungal activity (Vanittanakom et al., 1986; Peypoux et al., 1999; Vater et al.,
2002; Puja and Cameotra, 2004; Stein , 2005; Sanket et al., 2008; Tendulkar et
al., 2007).
Amongst all the Banyan endophytic isolates, B. subtilis K1 was found to be most
potent fungal antagonist based on in vitro inhibition assay and thus was selected
for further characterization. The B. subtilis K1 was also identified on the basis of
full length nucleotide sequence of 16 S rDNA (accession number EU056571).
Figure 2.4 shows the phylogenetic relatedness of B. subtilis K1 with other Bacilli
on the basis of 16 S rDNA sequence analysis using neighbor joining method.
Figure 2.4 Phylogenetic tree showing relatedness of Bacillus subtilis K1 with other Bacilli spp. on the basis of 16S r DNA analysis
Chapter-2
Characterization of Banyan endophytic Bacilli
43
2.3.2 Profile of growth and extracellular antifungal as well as emulsifying
activity of B. subtilis K1.
The extracellular emulsifying activity in fermentation broth of B. subtilis was
found to increase with growth, reaching maximum in mid log growth phase at
about 33 h of incubation and then onwards it started decreasing upto 50 h of
incubation (Fig. 2.5). This emulsifying activity again started slowly increasing
with further incubation upto 84 h before reaching plateau when further increase in
biomass ceased. In contrast to emulsifying activity, the extracellular antifungal
activity in the fermentation broth of B. subtilis K1 could not be detected upto 31
h of incubation. The antifungal activity started appearing after 33 h of incubation
i.e. approximately in the mid logarithmic growth phase and increased upto 51
hours of incubation (i.e. late logarithmic growth phase). This antifungal activity
then sharply decreased and then remained constant till further incubation upto 96
h of incubation (Fig. 2.5). This suggests that emulsifying and antifungal activities
of B. subtilis K1 are independent of each other and may be attributed to different
metabolites with varying production profiles.
0
1
2
3
4
5
6
7
8
9
0 10 20 30 40 50 60 70 80 90 100
Time (h)
Gro
wth
(Abs
orba
nce
660
nm)
0
2
4
6
8
10
12
14
16
18
20
Emul
sify
ing
activ
ity (U
/mL)
A
ntifu
ngal
act
ivity
(AA
U/m
L )
Growth Antifungal activity Emulsifying activity
Figure 2.5: Profile of growth and production of extracellular antifungal as well as emulsifying activity by B. subtilis K1.
The haemolytic zone around the colonies of all our endophytic isolates on blood
agar plate may be attributed to their ability to produce compounds that can
Chapter-2
Characterization of Banyan endophytic Bacilli
44
penetrate into cell membrane and causes cell lysis. The fungal antagonistic action
and hemolytic activity exhibited by the isolates suggested that bacterial isolates
might be producing antifungal compounds with surface active properties. Bacilli
are known to produce surface active agents with antifungal and/or haemolytic
activity (Vanittanakom et al., 1986; Peypoux et al., 1999; Vater et al., 2002; Puja
and Cameotra, 2004; Stein, 2005; Tendulkar et al., 2007; Sanket et al., 2008).
2.3.3 Influence of antifungal extract on germinability of A. niger
conidiospores
In order to determine whether extracellular antifungal agents produced by B.
subtilis K1 affects fungal spores and its germination, conidiospores of A. niger
40211 were incubated with different dilutions of cell free crude fermentation
broth made with distilled water. The treatment of conidiospores with 10%, 25%
and 50% (v/v) of cell free culture supernatant of B. subtilis K1 obtained after 51 h
of incubation resulted in inhibition of A. niger conidiospores germination by
80%, 89% and 96%, respectively. Similar effect of methanolic extract from B.
subtilis YM 10-20 on germination of conidiospores of P. roquefortii has been
reported by Chitarra et al., (2003).
2.3.4 pH and Temperature stability of antifungal activity
The antifungal activity of crude extract from B. subtilis K1 was found to be stable
over wide range of pH (2-10) and temperature (30-121˚C). The antifungal
activity remained same upon 30 min. incubation at 121˚C. The stability against
high temperature and wide range of pH have been also been observed in the
antifungal compounds produced by B. licheniformis and B. subtilis (Tendulkar et
al., 2007; Nagorska et al., 2007). This type of pH and thermal stability of
antifungal metabolites have been reported for cyclic lipopeptides, produced
commonly by Bacilli sp.(Winkelmann et al., 1983; Chitarra et al. 2003; Stein,
2005; Tendulkar et al., 2007). Thus, it seemed that antifungal activity of B.
subtilis K1 might be due to its ability to produce and secrete cyclic lipopeptides
in environment.
Chapter-2
Characterization of Banyan endophytic Bacilli
45
2.3.5 Thin layer chromatography of antifungal compounds:
The methanol soluble antifungal active fraction obtained upon acid precipitation
from fermentation broth of B. subtilis K1 were resolved into 6 bands on silica gel
TLC plates using chloroform: methanol: water :: 65: 30: 5, v/v/v. and made
visible upon exposure to iodine vapors (figure 2.6 a). All the separated bands
could also be stained with ninhydrin reagent and showed positive Pauly’s test,
suggesting that the resolved metabolites consisted of peptides with aromatic
amino acid residues such as tyrosine (Kopple et al., 1973). These bands
fluoresced in UV upon development with Rhodamine, suggesting the presence of
lipid moiety as well in the compounds. In order to determine, which bands on
TLC had antifungal activity, the developed TLC plate was over-layered with
spores of A. niger 40211 seeded in molten 1% (w/v) PDA agar and upon 48 h of
incubation, zone of no growth was observed around bands with Rf values 0.51,
0.31 and 0.15. The complete inhibition of fungal growth was observed around
band at 0.51 Rf value, while only inhibition of sporulation was observed around
bands at 0.31 and 0.15 Rf (figure 2.6 (b)).
Figure 2.6: (a) TLC and (b) anti-biogram of methanolic antifungal extract
(AFK1) against A. niger 40211
Chapter-2
Characterization of Banyan endophytic Bacilli
46
2.3.6 Intact Cell MALDI-TOF mass spectrometry of Banyan endophytic
bacilli.
In this study, MALDI-TOF mass spectrometry technique was applied to
investigate the secondary metabolites produced by all seven endophytic bacilli
using intact cell as a target. The Intact Cell MALDI-TOF mass spectra (ICMS) of
all seven endophytic bacilli shows mass peaks ranging from m/z 551.0 to m/z
2047.3 which were compared with the reported m/z values of compounds
produced by other bacilli strains and from that three groups of mass peaks could
be identified (Figure 2.7, a-g; Table 2.3, a-c). These were putatively assigned
based on literature as surfactins (m/z, 979 to 1096.8), iturins (m/z, 1014.5-1123.5
and fengycins (m/z, 1422.2-1558.2), which represent the well-known families of
cyclic lipopeptides produced by Bacillus sp. (Leenders et al., 1999; Vater et al.,
2002; Yu et al., 2002; Pabel. 2003; Meng gong et al., 2006; Price et al., 2007;
Pyoung et al., 2010). Iturin is a cyclic heptapeptide and known for its strong
antifungal and hemolytic activity, while fengycin is cyclic depipeptide with 10
amino acids which also possess strong antifungal activity specific to filamentous
fungi with very limited hemolytic activity (Winkelmann et al., 1983;
Vanittanakom et al., 1986; Maget-Dana and Peypoux, 1994). Surfactin is a cyclic
heptapeptide which is known for its excellent surface activity and other biological
activities such as, antiviral, antitumor, antimycoplasma, mosquitocidal (Peypoux,
1997; Vollenbroich et al., 1997; Kim et al., 2007; Geeta et al., 2010). On the
basis of mass spectra profile, five isolates viz., B. subtilis K1, B. subtilis A2, B.
subtilis A4, B. amyloliquefaciens A11 and B. subtilis A12 seemed to produce
higher proportion of iturin homologues in comparison to surfactins. All these five
isolates produced fengycin homologues but the intensity of fengycin m/z peaks
were significantly lower in comparison to the intensity of iturin peaks (Fig. 2.7 a-
e). Similarly on the basis on MALDI-TOF M/S data, isolate A32 seemed to
produce higher proportion of fengycins in comparison to surfactins and iturins
(Fig. 2.7 g). In surfactin-iturin cluster of ICMS of Bacillus sp. A32, three peaks
corresponding to iturins and seven mass peaks of surfactins were assigned (Table
2.3 a-c). Furthermore, peaks at m/z 1220.9, 1234.0, 1248.0 observed in ICMS
spectra of isolates B. subtilis A2, B. amyloliquefaciens A13 and Bacillus sp. A32
differed from each other by 14 Da, suggesting that the corresponding metabolite
Chapter-2
Characterization of Banyan endophytic Bacilli
47
belonged to the same family varying from each other in mass by multiples of 14
da. The peak at 1270.0 may be assigned as sodium adduct of m/z 1248.0. There
are no reports in literature on bacilli producing cyclic lipopeptides with m/z
1220.9 to 1270.0. The mass peaks with m/z 1901.3 in ICMS of A4 and m/z
2047.3 in ICMS of A12 could not be assigned. The molecules at m/z 551.0, 614.7
and 660.8 in ICMS of A13 also could not be assigned. These unassigned m/z
peaks may belong to new molecules produced by the strains of endophytic bacilli
but their low intensity makes it difficult to select and fragment them further for
their structural elucidation. On the basis of ICMS profile, the similarity
coefficients among these isolates were determined and used to construct a
dendogram (Figure 2.8). The similarity coefficients of B. subtilis A2, B. subtilis
A4, B. amyloliquefaciens A11, B. subtilis A12, B. amyloliquefaciens A13;
Bacillus sp. A32 with Bacillus subtilis K1 were calculated to be 0.64, 0.55, 0.50,
0.51, 0.64 and 0.54, respectively. Similarity coefficient values of ICMS pattern of
all seven bacilli suggested their variability in production of metabolites as none
of them shared 100% similarity. The isolates B. subtilis K1, B. subtilis A2, B.
subtilis A4, B. amyloliquefaciens A11, B. subtilis A12 and B. amyloliquefaciens
A13 exhibited higher heterogeneity as well as intensity of mass peaks
corresponding to iturins and fengycins, in comparison to isolate A32, which may
be correlated with their spectrum and potency of antifungal activity. The Bacillus
sp. A32, which produced more of surfactins and fengycins, exhibited relatively
weaker antifungal activity with narrow spectrum. According to literature, most
strains of Bacilli, have been reported to produce cyclic lipopeptides of a single
family (Vanittanakom et al., 1986; Winkelmann et al., 1983; Beson et al., 1987;
Sen and Swaminathan, 1997; Yu et al., 2002; Cho et al., 2003; Bais et al., 2004;
Meng-gong et al., 2006; Mizumoto and Shoda, 2007). Nevertheless, there are
reports of Bacilli producing mixture of lipopeptides belonging to two different
families such as surfactins + iturins (Ohno et al., 1995) or iturins + fengycins
(Pryor et al., 2007; Cazorla et al., 2007; Ongena et al., 2007) or fengycins +
surfactins (Sun et al., 2007; Cazorla et al., 2007). However, reports of Bacilli co-
producing lipopeptides of sufactin, Iturin as well as fengycin families, with high
degree of microheterogeneity are sparse (Vater et al., 2002; Toure et al., 2004;
Price et al., 2007; Romero et al., 2007). More significantly such strains have
Chapter-2
Characterization of Banyan endophytic Bacilli
48
been found to exhibit broader range as well as higher potency of antifungal
activity, suggesting synergism between members of different families of cyclic
lipopeptides (Thimon et al., 1992; Ongena et al., 2007; Romero et al., 2007). It is
noteworthy to mention here that all the endophytic Bacilli exhibiting antifungal
activity that could be isolated from Banyan aerial roots were found to be co-
producers of surfactins, iturins and fengycins. This implies that, these organisms
must be playing a definite biological role while residing as endophytes in Banyan
aerial roots, which would be worth investigating.
Table 2.3(a) Assignment of mass peaks belong to iturins from ICMS spectra of Banyan endophytic bacilli cells Assignments of
cyclic lipopeptide
Mass
peak
(m/z)
Banyan endophytic bacilli
K1 A2 A4 A11 A12 A13 A32
C12 Iturin [M+H+] 1014.6 + - - - - - -
C13 Iturin [M+H+] 1028.9 + - - - + - -
C14 Iturin [M+H+] 1043.6 + - - - + + -
C15 Iturin [M+H+] 1057.6 + + - - + + -
C16 Iturin [M+H+] 1071.7 + + - - - - -
C17 Iturin [M+H+] 1084.7 + + - - - - -
C14 Iturin [M+Na+] 1065.6 - - - + - + -
C15 Iturin [M+Na+] 1079.7 + - - - - + -
C17 Iturin [M+Na+] 1107.7 + + + - - - -
C18 Iturin [M+Na+] 1121.7 - + + + - - -
C19 Iturin [M+Na+] 1134.7 - - + - - - -
C20 Iturin [M+Na+] 1150.8 - + + - - - -
C21 Iturin [M+Na+] 1165.9 - - - + + - +
C 15 Iturin [M+K+] 1095.7 - - + + + + +
C 17 Iturin [M+K+] 1123.8 - + + + - - -
Chapter-2
Characterization of Banyan endophytic Bacilli
49
The intensity of mass peaks assigned as surfactins, iturins and fengycins in ICMS
of B. subtilis K1 was significantly higher in comparison to the intensity of
corresponding peaks in ICMS of other six isolates, which again correlates well
with its higher potency as well as the spectrum of antifungal activity. B. subtilis
K1 was found to inhibit almost all test fungi used in this study. Thus, we selected
B. subtilis K1 for further studies on purification and characterization of
antifungal compounds secreted by it in environment.
Table 2.3 (b) Assignment of mass peaks belong to surfactins from ICMS spectra of Banyan endophytic bacilli cells
Identification of
cyclic lipopeptide
Mass
peak
(m/z)
Banyan endophytic bacilli
K1 A2 A4 A11 A12 A13 A32
C11 Surfactin [M+H+] 979.6 - + - - + - +
C12 Surfactin [M+H+] 995.5 + - - + + +
C13 Surfactin [M+H+] 1008.6 - - - + + + +
C14 Surfactin [M+H+] 1022.9 - - - + - + +
C15 Surfactin [M+H+] 1036.7 - - - + - + +
C20 Surfactin [M+H+] 1106.6 + + + - - - -
C 11 Surfactin [M+Na+] 1002.5 - - + - - + -
C 12 Surfactin [M+Na+] 1017.6 - + + - - + -
C 13 Surfactin [M+Na+] 1030.5 - - + + + + -
C 14 Surfactin [M+Na+] 1044.9 - - + + - + -
C 15 Surfactin [M+Na+] 1059.0 - - - + + + -
C18 Surfactin [M+Na+] 1102.9 - - - + + - -
C14 Surfactin [M+K+] 1060.6 - - + - - - +
C15 Surfactin [M+K+] 1074.9 - - + + - + +
Chapter-2
Characterization of Banyan endophytic Bacilli
50
Table 2.3 (c) Assignment of mass peaks belong to fengycins from ICMS spectra of Banyan endophytic bacilli cells Identification of
cyclic lipopeptide
Mass
peak
(m/z)
Banyan endophytic bacilli
K1 A2 A4 A11 A12 A13 A32
Fengycin [M+H+] 1422.2 - - - - + - -
Fengycin [M+H+] 1436.1 + + - - - - -
Fengycin [M+H+] 1450.1 + + + + + - +
Fengycin [M+H+] 1464.1 + + + + + + +
Fengycin [M+H+] 1478.2 + + + + + + +
Fengycin [M+H+] 1492.2 + + - + + + +
Fengycin [M+H+] 1506.2 + + - + + + +
Fengycin [M+Na+] 1472.1 - + + - - - -
Fengycin [M+Na+] 1500.1 - - - + + + -
Fengycin [M+Na+] 1514.1 - - - + - + -
Fengycin [M+Na+] 1528.6 + - - + - - -
Fengycin [M+K+] 1488.0 - - + + - - -
Fengycin [M+K+] 1502.6 + + + - - - -
Fengycin [M+K+] 1516.1 + + + - + - -
Fengycin [M+K +] 1530.2 - + + + + - +
Fengycin [M+K +] 1544.4 - - + - + + +
Chapter-2
Characterization of Banyan endophytic Bacilli
51
Figure 2.7(a) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis K1
Chapter-2
Characterization of Banyan endophytic Bacilli
52
Figure 2.7 (b) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A2
Chapter-2
Characterization of Banyan endophytic Bacilli
53
Figure 2.7 (c) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A4
Chapter-2
Characterization of Banyan endophytic Bacilli
54
Figure 2.7 (d) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. amyloliqeufaciens A11
Chapter-2
Characterization of Banyan endophytic Bacilli
55
Figure 2.7 (e) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A12
Chapter-2
Characterization of Banyan endophytic Bacilli
56
Figure 2.7 (f): Intact cell MALDI-TOF mass spectrometry (ICMS) of B. amyloliquefaciens A13
Chapter-2
Characterization of Banyan endophytic Bacilli
57
Figure 2.7 (g) : Intact cell MALDI-TOF mass spectrometry (ICMS) of Bacillus sp. A32
Chapter-2
Characterization of Banyan endophytic Bacilli
27
2.1 Introduction
Endophytic bacteria colonize internal plant tissue without causing substantial
harm to the host plant and may either benefit the host or benefits may be
reciprocal (Bacon et al., 2002). The symbiotic association of host plants with
bacterial endophytes offers shelter and protection to the microbes against
physiological and environmental conditions while in return microbes provide
several benefits to the host plant such as protection against disease causing
pathogens, fitness by producing functional metabolites, improving soil quality,
increasing plant mineral uptake, inducing plant defense mechanisms, improve the
plant’s ability to withstand environmental stress (e.g. drought) or enhance N2
fixation (Kloepper, 1989; Sturz and Nowak, 2000; Malinowski et al., 2000;
Ciccillo, et al., 2002; Strobel and Daisy, 2003; Kloepper et al., 2004; Melnick et
al., 2008; Li et al., 2008). Recently, endophytes have also been investigated for
their potential application in biodegradation of pollutants in soil and
phytoremediation (Kuiper et al., 2004; Berg et al., 2005; Newman and Reynold,
2005). Bacterial endophytes colonize an ecological niche similar to that of
phytopathogenes, which makes them ideal candidates as biocontrol agents
(Hallmann et al., 1998; Azevedo et al., 2000; Coombs et al., 2004; Kloepper et
al., 2004; Cavaglieri et al., 2004; Berg et al., 2005; Senthilkumar et al., 2007;
Melnick et al., 2008). Endophytes belonging to several bacterial genera such as
Bacillus (Melnick, et al., 2008), Pantoea, Acinetobacter, Serratia (Li, et al.,
2008), Burkholderia (Compant, et al., 2005), Pseudomonas (Geramaine et al.,
2004), Phomopsis, Streptomyces, Enterobacter, Staphylococcus, Azospirillum,
Clavibacter, Herbasprillum (Ryan et al., 2008) have been reported and
investigated for their role in plant protection, plant growth promotion,
phytoremediation and production of novel bioactive compounds. The bioactive
compounds with diverse applications as antibiotics, insecticides,
immunosuppressants, antioxidants and antitumor agents of endophytic origin
have been reported. (Tan and Zou, 2001; Castillo et al., 2002; Strobel and Daisy,
2003; Strobel et al., 2004; Zang et al., 2006).
The plants growing in unique environmental settings with ethnobotanical value
and longevity or endemic location are likely to be a source of rarely occurring
novel microbial endophytes (Strobel, 2003). The novel microbial flora or flora
Chapter-2
Characterization of Banyan endophytic Bacilli
28
from unique plants generally offers the bioactive and chemically novel
metabolites with huge medicinal and agricultural potential (Strobel and Daisy,
2003).
The matrix assisted laser desorption ionization time of flight (MALDI-TOF)
mass spectrometry has been used as an efficient method for rapid investigation of
complex mixture such as a cellular extract or culture filtrates originating from
microorganisms without purification (Saenz et al., 1999; Hitzeroth et al., 2005).
Thus, it permits the direct analysis of biomolecules from intact cells, tissues and
organelles (Vater et al., 2002; Hitzeroth et al., 2005). The intact cell MALDI
mass spectrometry (ICMS) technique has been developed for determining cell
surface associated as well as intracellular molecules. The most significant
application of ICMS has been demonstrated in mass spectrometric finger printing
and metabolic profiling of microorganisms, forming a novel chemotaxonomic
tool for rapid identification of bacteria and differentiation of closely related
strains. It has successfully been used for the rapid identification and taxonomic
characterization of intact bacterial cells based on their specific secondary
metaoblites, proteins, cell wall constituents (Krishnamurthy and Ross, 1996;
Leenders, et al., 1999; Evason et al., 2000; Lay, 2001; Pabel et al., 2003; Keys et
al., 2004; Price et al., 2007). Moreover, this method has been successfully used
for the rapid typing of vegetative cells and spores of bacilli (Leenders et al.,
1999; Williams et al., 2002).
Banyan tree (Ficus bengalensis) is ethanobotanically important Asian endemic
plant with a very long life span. Various parts of the Banyan tree like aerial roots,
latex, stem, bark, leaves, and fruits have been used in preparation of traditional
medicine for the treatment of various ailments like toothache, diarrhea, dysentery,
female sterility, leucorrhoea, rheumatism, skin disorders. The medicinal
properties of Banyan tree led us to work on endophytic flora of this plant.
The present study describes,
i) Isolation, identification and preliminary characterization of
endophytic bacilli from aerial roots of Banyan tree.
ii) In plantae visualization of endophytes in aerial roots of Banyan tree.
Chapter-2
Characterization of Banyan endophytic Bacilli
29
iii) Isolation, purification and identification of antifungal compounds
produced by potential fungal antagonist endophytic bacterial strain, B.
subtilis K1
iv) Fingerprinting of all the endophytic bacilli isolates on the basis of
diversity of cyclic lipopeptides using intact cell MALDI TOF mass
spectrometry (ICMS).
2.2 Materials and methods:
2.2.1 Isolation of endophytes from hanging roots of Banyan tree:
The young aerial roots of Banyan tree were collected from Anand, Gujarat, India
and processed freshly within an hour for the isolation of endophytes. Bacterial
endophytes were isolated from surface sterilized budding aerial roots of Banyan
tree as described below. The aerial roots were dipped into solution of 15%
Savlon™ (v/v) for 15minutes followed by treatment with 70% ethanol for
2minutes. The ethanol treated roots were further immersed in 0.1% HgCl2 (w/v)
for 30 seconds followed by repeated (at least 6-8times) washes with sterile
distilled water to remove excess of HgCl2 from the surface of explants (Khyati et
al., 2009). The surface sterilized root tissues were cut into 8mm pieces and were
placed on PDA and Luria Agar (LA) plates (Himedia limited, Mumbai) for
growth of endophytes. The bacterial growth near surface sterilized aerial root
explants on plates was subjected to further isolation of pure cultures by streak
plate method. The pure cultures were repeatedly sub-cultured to check for purity
and then maintained on LA slopes at 4 ˚C as well as in form of glycerol stocks
stored at -20 °C.
2.2.2 Identification of Banyan endophytes:
The identification of bacterial endophytic isolates was done using MicroLog 1
(bacterial identification system) using GP2 plates procured from BioLog Inc.,
USA. The endophytes were grown on BioLog universal agar (BUG) media for 10
h at 30°C. The Biolog GP2 plates (gram positive2) were inoculated with 150 μL
bacterial suspension of 29% turbidity and incubated at 30 °C. The plates were
read after 18 h for utilization of carbon sources and identification was done on
the basis of carbon source utilization pattern from the gram positive database
using BioLog Microlog software (version 4.2; Biolog, Inc. USA). On the basis of
Chapter-2
Characterization of Banyan endophytic Bacilli
30
carbon source utilization pattern in BioLog GP2 microplates (Gram positive 2
microplates), the similarity coefficients among the endophytic isolates were
determined to construct dendogram using the NTSYS PC Version 2.0 software.
The identification of potential antagonist, endophyte K1, which was selected for
further investigation, was further confirmed by 16S rDNA gene sequence
analysis.
2.2.3 In planta localization/ visualization of endophytes:
2.2.3.1 Vital staining:
The surface sterilized hanging roots of Banyan tree were incubated in sterile 2, 3,
5-Triphenyl tetrazolium chloride (TTC) salts solution (for preparation see
appendix 2) under aseptic condition for 18 h at 30 ± 2 °C . The longitudinal or
cross sections of TTC treated roots were taken using Cryotome (Leica™) for thin
sectioning. The sections were taken on the glass microscopic slides and prepared
for the wet mount using glycerol phosphate buffer. The glycerol was used to
prevent drying of sections. The sections were then examined under light
microscope at a 100 X magnification.
2.2.3.2 Transmission electron microscopy (TEM):
The root tissues were fixed with Karnovsky’s (glutaraldehyde + formaldehyde)
fixative for 2-5 hours at room temperature under vacuum. The roots were then
removed from the fixative and washed thrice with cold solution of 0.1 M sodium
phosphate buffer (pH-6.8) each for 10 min. The roots after fixation were then
transferred into 2% (w/v) Osmium tetroxide (OSO4) and further incubated at
room temperature for 18 h. After the treatment with OSO4 the roots were washed
with buffer solution, twice followed by cold deionized water wash for three
times. The OSO4 treated roots were then stained by soaking the roots into the
cold solution of 1 % (w/v) uranyl acetate for 30 minutes. Once the roots were
fixed, the dehydration step was carried out. To remove the water content,
dehydration was carried out by using acetone water series. Roots were treated
with a series of acetone water solution starting from 10% (v/v) acetone to 100 %
(v/v) acetone and each treatment was carried out for 20 min.
The dehydrated roots were infiltrated with Spurr™ resin. The infiltration step was
carried out by treating the roots with increasing Spurr: acetone ratio for different
Chapter-2
Characterization of Banyan endophytic Bacilli
31
time intervals. The first treatment was carried out by using 3.1 Spurr: acetone
ratio for 2 h followed by treatment with equal proportion (2:2) Spurr: acetone for
12 h. Then the roots were incubated into 3:1 Spurr: acetone solution for 24 h. The
final step was carried out by using pure Spurr and samples were incubated for
24h. All the steps were carried out at room temperature. The infiltrated root
tissues were embedded into spurr resin by putting each infiltrated root piece into
the slot of rubber casting tray along with spurr resin. The spur resin with
embedded root tissues was allowed to polymerize at 70˚C in oven for 12-48 h till
the polymerized spurr became sufficiently hard. The block containing root tissues
were trimmed for ultra thin sectioning. The 1 μm and nm sections were taken
using ultra microtome (Leica™). The ultrathin sections were mounted on formvar
coated grids and with Sato’s lead solution. The coated grids containing root
tissues were then examined under electron microscope.
2.2.4 Characterization of endophytes:
2.2.4.1 In vitro fungal antagonism:
The antifungal activity of banyan endophytic isolates was investigated against
following fungal cultures: Aspergillus niger 40211, A. niger 16404, A. niger 181,
A. flavus, Chrysosporium indicum, Mucor indicus, Fusarium oxysporum f.sp.
lycopersicii, F. oxysporum f.sp. gingiberi, F. oxysporum 1072, Candida
albicans, Alternaria brunsii (1), A. brunsii (2), Cladosporium herbarum 1112,
Sclerotia rolfsii and Lasiodiplodia thoebromae ABFK1. The pure cultures of four
endophytic isolates were spot inoculated in four sectors on sterile potato dextrose
agar plate (3cm away from the center of the petri dish) and incubated at 30 ˚C for
48 h. After 48hrs of incubation, 9mm mycelial plug of each fungal pathogen
mentioned above was placed on the centre of agar medium on the Petri-plate and
further incubation was continued for 5-7 days.
2.2.4.2 Production of extracellular enzymes:
The pure cultures of Banyan endophytic isolates were screened for xylanase,
cellulose, lipase and chitinase. The cultures were spot inoculated on the xylan
agar medium (Luria agar amended with 2.5g/L Birchwood xylan), CMC agar
medium (Luria agar amended with 10g/L carboxy methyl cellulose), chitin agar
medium (4g/L colloidal chitin in Luria agar) and tributyrin agar medium (10g/L
trybutyrin emulsion of Luria agar) for screening of xylanase, cellulase, chitinase
Chapter-2
Characterization of Banyan endophytic Bacilli
32
and lipase production, respectively. All the plates were incubated at 30 ˚C for 48-
72 h. The production of xylanase and cellulase were determined by appearance of
clear zone around colonies upon staining the medium with Congo red followed
by destaining with 2M NaCl. The ability of the cultures to produce chitinase was
determined by presence of dark zones around colonies against fluorescent
background upon treatment with calcofluor white, when observed under UV
light. The ability of the cultures to produce lipase was determined by presence of
clear zones of tributyrin hydrolysis around colonies against the opaque
background of tributyrin emulsion.
2.2.4.3 Hemolytic activity:
Endophytic bacteria were cultivated on sterile blood (5 % v/v) agar medium. The
plates were incubated at 30°C for 24-48 h. The plates were then observed for
zone of haemolysis upon incubation around colonies of bacterial isolates.
2.2.4.4 Profile of growth, antifungal activity as well as emulsifying activity of
Banyan endophytic isolates:
For inoculum preparation, cells from a single colony of a bacterial isolate was
inoculated into 50 mL of sterile Luria broth (LB) in 250 mL Erlenmeyer flask
and incubated at 30°C for 12 h (O.D. 1.9-2.0) on orbital shaker (150 rpm). For
investigation of antifungal as well as emulsifying activity, the inoculum was
added to 100 mL of sterile LB in 250 mL Erlenmeyer flasks to obtain an initial
O.D.600 nm ∼0.05. The flasks were incubated on orbital shaker (150 rpm) at 30°C
for 96 h and at regular interval of 24 h, one flask of each culture was removed,
cells were separated by centrifugation (10,062 X g for 20 min.) and supernatant
was collected separately. The cell pellet was used for measurement of growth by
gravimetric method while supernatant was subjected to analysis of antifungal and
emulsifying activity. The antifungal activity was assayed by agar cup diffusion
method as described below. The test plates were prepared by seeding 100 μL of
spore suspension (1 x 107spores/ml) of Aspergillus niger 40211 into 4.5ml of
molten soft agar (1% agar, w/v) and over-layered on sterile PDA plates and
allowed to solidify. The plate was divided into four sectors and four wells were
bored, one each in centre of each sector using sterile cup borer. To each well 100
μL aliquot of methanolic antifungal extract obtained from different cultures was
added and allowed to diffuse in medium. The plates were incubated for 48 h at
Chapter-2
Characterization of Banyan endophytic Bacilli
33
30˚C. Upon incubation, the diameter of zone of inhibition was measured and
arbitrary antifungal activity units (AAU) were determined. One arbitrary
antifungal activity unit corresponds to the amount of antifungal active
metabolites which yielded 13 mm zone of inhibition on PDA plates seeded with
A. niger 40211. The emulsifying activity (E.A) was determined by using
modified emulsification assay described by Navon-Venezia et al., 1995. The 1mL
aliquot of culture supernatant was added to 6.5 mL of 20mM TM buffer (20 mM
Tris-HCl buffer [pH-7], 10 mM MgSO4) followed by addition of 0.1mL of 1:1
(v/v) mixture of 2-methyl naphthalene and hexadecane. The samples were
vigorously mixed for 2 min. and allowed to stand for 1 h at 30°C before
measuring turbidity at 600nm. One unit of emulsifying activity was defined as
amount of emulsifier that yielded an A600 nm of 0.1 in the assay mixture.
2.2.5 Isolation and Characterization of antifungal metabolite/s produced by
potential fungal antagonist:
2.2.5.1 Isolation of antifungal metabolites from Banyan endophyte, B. subtilis
K1:
The antifungal metabolites from culture supernatant were precipitated by
lowering the pH of broth to 2 using 6N HCL. The precipitates were harvested by
centrifugation of acidified broth at 10,062 X g for 20 min. The supernatant was
discarded while the pellet was solubilized in pure methanol. The methanolic
extract was then centrifuged to remove undissolved fraction, while supernatant
was collected and subjected to drying by rotary vacuum evaporation (Buchi,
Switzerland) at 30˚C. The yellowish brown sticky substance thus obtained was
dissolved into small volume of methanol for further analysis.
2.2.5.2 Effect of antifungal metabolites produced by B. subtilis K1 on
germination of conidia of A. niger 40211:
The conidia (1x103 spores/mL of potato dextrose broth) were incubated with
various concentrations of crude culture supernatant and methanolic antifungal
extract, for 10 h at 30°C. Upon incubation, the conidiospores were stained with
1% Lacto phenol blue, and observed under light microscope under oil immersion
lens (100 X) (Lawrence & Mayo, Kolkata). Each experiment was performed in
Chapter-2
Characterization of Banyan endophytic Bacilli
34
triplicate. A conidium was considered as germinated if the germ tube was more
than half of the diameter of conidium (Chitarra et al., 2003).
2.2.5.3 Stability studies of antifungal metabolites produced by B. subtilis K1
The cell free culture (CFC) supernatant was adjusted to various alkaline and
acidic pH using 1N NaOH or 1N HCL and incubated at 30˚C for 30 min. The pH
of each treated sample was re-adjusted to pH 7.0. For thermal stability
determination, CFC was incubated at various temperatures varying from 25 to
121˚C for 30 min. The residual antifungal activity upon each treatment was
measured against A. niger as a test culture using agar cup diffusion method.
2.2.5.4 Thin layer chromatography methanolic antifungal extract from B.
subtilis K1
The methanolic extract was spotted to pre coated silica G 60 F254 TLC plates
(Merck Darmstadt Germany) and developed in chloroform: methanol: water:: 65:
30: 5, v/v/v. The separated bands on TLC plate were developed by spraying 1 %
(w/v) Ninhydrin or Pauly’s reagent (Koppel et al. 1973). The antibiogram of
spots resolved on TLC plate was performed by over-layering it with molten soft
PDA agar seeded with 104 spores of A. niger. The over-layered TLC plate was
then incubated at 30˚C in moist chamber for 48 h.
2.2.6 Purification of antifungal metabolites from B. subtilis K1:
The cyclic lipopeptides in the extract were further separated by reverse phase
high performance liquid chromatography (RP-HPLC) using semi-preparative
Phenomenex (Torraance, CA, USA) C18 column (4.6 mm x 250 mm, 10m
particle size, 90 pore size) and MeOH/ H2O/ 0.1% TFA (tri-fluoro acetic acid) as
a mobile phase. The flow rate was maintained at 1mL/min with gradient of 60
min (80-95 %, v/v MeOH in 50 min; 95%, v/v MeOH for 5 min and 95 to 80 %,
v/v MeOH in 5minutes). The elution of metabolites was monitored using UV
detector at 226 and 280 nm. The metabolites eluted under individual peaks were
separately collected in different vials and concentrated using rotary vacuum
evaporator (Buchi, Switzerland) at 30˚C and lyophilized. The concentrated HPLC
peaks, thus obtained were used for the antifungal activity and sequence analysis.
Chapter-2
Characterization of Banyan endophytic Bacilli
35
2.2.7 Antifungal activity of purified HPLC fractions:
A stock solutions (1mg/mL) of metabolites eluted under major HPLC peaks P3,
P4, P5, P14, P15, P16, P17 were prepared by dissolving lyophilized fractions in
MeOH and analyzed for activity against A. niger 40211, A. flavus, A. parasiticus,
F. oxysporum1072, Chrysosporium indicum, Candida albicans, Trichosporon
sp.1110, Alternaria brunsii (2), Cladosporium herbarum1112, Helmethosporium
graminum1126, Lasiodiplodia theobromae ABK1, by paper disc method. The
5mm sterile paper (Whatmann filter paper no. 1) discs were dipped in
aforementioned stock solutions prepared from HPLC fractions and discs were
allowed to air dry under aseptic condition. The discs were then placed on the
PDA plates seeded with 100 μL (1 X 106 spores/mL) spore suspension of each
fungal culture.
Fungal spore suspension was prepared by harvesting spores into sterile distilled
water and the spore counts were determined using haemocytometer. In case of
Candida albicans and Trichosporon 1110, culture suspension was prepared by
growing the yeast cultures in 50 mL of potato dextrose broth under agitated
condition (150 rpm) at 30˚C for 10-12 h. The cell numbers were determined
using a hemocytomer and adjusted to 1 x 106 cells/mL by appropriate dilution.
The MIC and IC50 of HPLC fractions were determined by double dilution
technique against susceptible fungal cultures in sterile 96-well microtiter plates
with each well containing 100 μL of potato dextrose broth. After dilution ∼102
spores of test fungus were inoculated into each well. To control wells,
corresponding aliquot of MeOH instead of sample was added. The plates were
incubated for 24-48 h at 30˚C and MIC values for each fraction were determined
against susceptible test fungi on the basis of highest dilution showing no growth.
2.2.8 Mass spectrometry (MS) :
The HPLC fractions were subjected to MALDI-TOF MS analysis. The data were
acquired on Ultraflex TOF/TOF spectrometer (Bruker Daltonics, Germany)
equipped with 50 Hz pulsed N2 laser (337nm) operated in positive ion reflectron
mode using 90 ns delay time and 25 kV accelerating voltage. Samples were
prepared by mixing equal volume of purified HPLC fractions with α-cyano-4-
hydroxy-cinnamic acid or 2, 5 dihydroxy benzoic acid saturated in acetonitrile:
Chapter-2
Characterization of Banyan endophytic Bacilli
36
water (1:1, v/v) with 0.1% (v/v) trifluoroacetic acid and applied on the MALDI
sample plate. The sample spots on MALDI plate were allowed to air dry before
analysis.
2.2.8.1 Intact cell MALDI mass spectrometry (ICMS) :
Cells from a single colony of each bacterial isolate grown on Luria agar plate was
transferred into 100 μL of methanol: Water (1:1) and from that 1 μL was used for
mass spectrometric (MS) analysis using MALDI-TOF mass spectrometer as
mentioned above.
2.3 Results and Discussion:
Most of the studies on bacterial endophytes has been focused on agriculturally
important plants (Cavaglieri et al., 2004; Compant et al., 2005; Naik et al., 2006;
Melnick et al., 2008) while literature on bacterial endophytes from woody trees is
sparse (Wang et al., 2006). The young aerial roots of Ficus benghalensis (Banyan
tree) originates near the crown of the tree and grows down towards soil through
the air which appears to be an ideal plant organ to study endophytic flora
(Suryanarayan et al., 2001). Hence, we selected aerial roots tips of Ficus
benghalensis for the isolation of endophytic bacteria.
2.3.1 Isolation, identification, in plantae localization and characterization of
Banyan bacterial endophytes:
The surface sterilized tender aerial root tips of Banyan tree were placed on LA
and PDA plates and incubated at 30°C up to 10 days. On the fourth day of
incubation, bacterial growth was observed at the edges of surface sterilized
Banyan aerial root pieces. The bacterial growth thus obtained was then sub-
cultured on fresh sterile LA plates for isolation of pure cultures. Seven different
morphotypes could be isolated in pure form, which were designated as K1, A2,
A4, A11, A12, A13, and A32. All the seven isolates were found to be motile,
gram positive, spore forming bacilli.
All bacterial isolates except A32 could be identified on the basis of carbon source
utilization profile using GP2 plates of Biolog. The isolates designated as K1, A2,
A4 and A12 were identified as Bacillus subtilis with similarity coefficients of
0.86, 0.68, 0.77 and 0.65, respectively. The isolates A11 and A13 were identified
as Bacillus amyloliquefaciens with 0.78 and 0.74 similarity coefficients,
Chapter-2
Characterization of Banyan endophytic Bacilli
37
respectively. The isolate A32 produced highly mucoid colonies preventing the
preparation of dense homogenous cell suspension, which is a pre-requisite for
identification using Biolog. The GP2 plate contains 95 different carbon
substrates, amongst which 48 substrates were not utilized by all the six isolates;
however these isolates exhibited significant variation in utilization of remaining
47 carbon sources (Table 2.1). The dendogram on the basis of carbon source
utilization profile for above six cultures grouped these cultures into two clusters
sharing more than 80% similarity. One of the cluster consisted of all four B.
subtilis isolates, amongst which K1 and A2 exhibited about 90% similar carbon
utilization profile. The other cluster grouped the two B. amyloliquefaciens
isolates, A11 and A13 which also exhibited high degree of similarity (90%)
(Figure 2.1). On the basis of carbon source utilization profile, it was confirmed
that all the seven isolates were different from each other.
Table 2.1: Utilization profile of 47 carbon sources by Banyan endophytes
No. Carbon source K1 A2 A4 A11 A12 A13 1 Dextrin + + + + + + 2 N Aacetyl ß-D glucosamine + + + + + + 3 Amygdalin + + + + + + 4 Arbutin + + + - + V 5 D-Cellobiose + + + + + + 6 D-Fructose + + + - + V 7 D-Galactose - - + - - - 8 Gentiobiose + + + - + + 9 D-Gluconic acid - - + V V - 10 Α-D-Glucose + + + - + V 11 m-Inositol - - - V - - 12 Maltose + + + + + + 13 Maltotriose V + + V V V 14 D-Mannitol + + + V + - 15 D-Mannose + + + + + B 16 3-Methyl glucose V V + V V + 17 Α-Methyl D-glucoside + + + + + + 18 ß-Methyl D-glucoside + + + + + + 19 Palatinose + + + + + - 20 D-psicose + + + V V + 21 Salicin + + + + + - 22 D-Sorbitol V + + + + - 23 Stachyose - - - - V - 24 Sucrose + + + + + - 25 D-Trehalose + + + + + + 26 Turanose + + + + + + 27 ß-Hydroxy butyric acid - - - - + -
Chapter-2
Characterization of Banyan endophytic Bacilli
38
28 Γ-Hydroxy butyric acid + - - - - - 29 P-Hydroxy phenylacetic acid - - - - + - 30 Α-Ketovaleric acid - V V - + - 31 L-Lactic acid - - + - - - 32 L-Malic acid + V + - + - 33 Pyruvatic acid methyl ester + V + V + V 34 Pyruvic acid + V + V + V 35 N-Acetyl L-Glutamic acid - - - - + - 36 L-Alanine V + + - - V 37 L-Aspargine V V + - + V 38 L-Glutamic acid V + + - + V 39 Glycerol V + V + + + 40 Adenosine + + + - + V 41 2' Deoxyadenosine + + V - - - 42 Inosine + + + - + V 43 Thymidine + + + V + + 44 Uridine + + + V + + 45 Thymidine 5’-
monophosphate V - + - - - 46 D Glucose 6phosphate V V V V - - 47 DL α Glycerol phosphate - - - V - V
+, utilization of substrate as carbon source; -, substrate not metabolized; V, borderline reaction or weak positive.
Figure 2.1: Dendogram based on similarity coefficients calculated from carbon source utilization profile of six endophytic bacilli. The carbon source utilization profile was determined by employing GP2 plates of Biolog.
Chapter-2
Characterization of Banyan endophytic Bacilli
39
The transverse sections (T.S.) of hanging roots of Banyan tree upon staining with
tetrazolium dye revealed the presence of pink to purple colour stained bacteria
and fungi in the parenchyamtous cells of cortex and pith area. They appeared to
be localized in intercellular spaces as well as in paranchymatous cells of cortex as
well as around xylem vessels (figure 2.2a). The presence of rod shaped bacteria
was demonstrated in roots of Brassica sp. by Shefali et al., (1987) using
tetrazolium reducing dye. The presence of rod shaped bacterial cells could also be
demonstrated by transmission electron microscopy (figure 2.2b). Thus, light as
well as transmission electron microscopy supports the occurrence of bacterial
cells as endophytes in vascular as well as parenchymatous cells of Banyan aerial
roots. Furthermore, endophytes are known to produce extracellular hydrolyases
like cellulases, pectinases, xylanases in order to penetrate the host tissues for
colonization or as a resistance mechanism to overcome attack by host against
pathogenic invasion and/or to obtain nutrients from the host cells (Tan and Zou,
2001). All our endophytic isolates were found to hydrolyze xylan and cellulose
on solid media indicating their ability to produce xylanase/s and cellulose/s,
which are plant cell wall degrading enzymes. The ability to produce xylanase and
cellulase further supports their endophytic nature. None of the cultures exhibited
chitin hydrolysis while all the isolates hydrolyzed tributyrin. Thus, the
microscopic observations as well as their ability to produce hydrolyases, supports
the endophytic nature of our bacterial isolates.
Figure 2.2: Light micrograph (X 100) of T.S. cortical cells of TTC treated Banyan aerial roots (a); Electron micrograph of T.S. of Banyan aerial root (b).
Chapter-2
Characterization of Banyan endophytic Bacilli
40
The endophytic isolates were then investigated for antifungal activity against 14
different fungal cultures. It was surprising to observe that all the endophytic
isolates exhibited antifungal activity (Table 2.2 and Fig. 2.3). The bacterial
cultures K1, A13, A2 and A4 were found to inhibit the growth of all the 14 test
fungal cultures, while A32 inhibited only 5 fungal cultures amongst 14 tested.
The isolates A11 and A12 were also able to inhibit 13 out of 14 test fungal
cultures; they could not inhibit the growth of Sclerotia rolfsii.
Table 2.2 Spectrum of antifungal activity of cell free fermentation broth of
Banyan endophytes monitored by agar diffusion assay
Sr. No.
Fungal cultures Diameter of zone of inhibition (mm) K1 A11 A12 A13 A2 A4 A32
1 Aspergillus niger 40211
36 36 34 36 22-24 26 -
2 Aseprgillus niger 181
32-34 34 32-34 38 20 22 -
3 Aspergillus niger 16404
32 30 34 36 22 24 -
4 Aspergillus flavus 30 28 31 30 28 28 26 5 Alternaria brunsii (1) 32 22 30 20 24 30 20 6 Alternaria brunsii (2) 28-30 24 30 20 22 34 24 7 Chrysosporium
indicum 34-38 30-36 36-40 32-36 28 30 22-24
8 Fusarium oxysporum(1072)
32 28 24 20 28 24 -
9 Fusarium oxysporum lycopercisi
30 24 26 24 32 28 -
10 Fusarium oxysporum gingiberi
32 26-28 24 24 26 20 -
11 Cladosporium herbarum1112
30 28 25 25-28 28-30 28 25-27
12 Lasiodiplodia theobromae ABK1
32 28 24 20 28 24 -
13 Sclerotia rolfsii 30 - - 15 20 25 - 14 Mucor indicus 24 18 22 18 26 24 -
In some interactions between bacilli and sensitive fungi especially Aspergillus
species, a precipitation line was observed in the inhibitory zone between bacterial
and fungal growth. Similar observations have been documented by Cornea et al.,
(2003) in their in vitro antifungal assays of Bacillus sp. B209 against Sclerotinia
sclerotiorum. Several bacterial endophytes have been known for their fungal
Chapter-2
Characterization of Banyan endophytic Bacilli
41
antagonism. The bacterial endophyte, B. amyloliquefaciens ES-2 isolated from
Scultellaria baicalensis Georgi inhibited the growth of various plant pathogenic
fungi viz., A. niger, A. flvus, A. ficuum, A. oryzae, Mucor wuntungkiao, F.
culmorum, F. oxysporum, Maganporthe grisea and Botryodiplodia theobromae
(Sun et al., 2006). Similarly, Serratia marcescens isolated from Rhyncholacis
penicillata; Paenibacillus polymyxa isolated from wheat and Streptomyces sp.,
isolated from rice have been reported for their fungal antagonism (Beck et al.,
2003; Ezra et al., 2004; Strobel et al., 2004; Naik et al., 2006; Li et al., 2007).
Figure 2.3: Inhibition of growth of different fungal cultures by Banyan
endophytes on potato dextrose agar plates.
Furthermore all our endophytic isolates exhibited prominent haemolysis on sheep
blood agar plates. The haemolytic activity of Bacillus sp. has been correlated to
their ability to produce and secrete surface active metabolites which act on red
blood corpuscles (Thimon et al., 1992; Vanittanakom et al., 1986; Pabel et al.,
2008). The fungal antagonistic action and hemolytic activity exhibited by our
Chapter-2
Characterization of Banyan endophytic Bacilli
42
isolates suggest their ability to produce antifungal compounds that may also have
surface active properties. The antifungal compounds produced by some Bacilli
species have been reported for their surface active properties as well as their
antifungal activity (Vanittanakom et al., 1986; Peypoux et al., 1999; Vater et al.,
2002; Puja and Cameotra, 2004; Stein , 2005; Sanket et al., 2008; Tendulkar et
al., 2007).
Amongst all the Banyan endophytic isolates, B. subtilis K1 was found to be most
potent fungal antagonist based on in vitro inhibition assay and thus was selected
for further characterization. The B. subtilis K1 was also identified on the basis of
full length nucleotide sequence of 16 S rDNA (accession number EU056571).
Figure 2.4 shows the phylogenetic relatedness of B. subtilis K1 with other Bacilli
on the basis of 16 S rDNA sequence analysis using neighbor joining method.
Figure 2.4 Phylogenetic tree showing relatedness of Bacillus subtilis K1 with other Bacilli spp. on the basis of 16S r DNA analysis
Chapter-2
Characterization of Banyan endophytic Bacilli
43
2.3.2 Profile of growth and extracellular antifungal as well as emulsifying
activity of B. subtilis K1.
The extracellular emulsifying activity in fermentation broth of B. subtilis was
found to increase with growth, reaching maximum in mid log growth phase at
about 33 h of incubation and then onwards it started decreasing upto 50 h of
incubation (Fig. 2.5). This emulsifying activity again started slowly increasing
with further incubation upto 84 h before reaching plateau when further increase in
biomass ceased. In contrast to emulsifying activity, the extracellular antifungal
activity in the fermentation broth of B. subtilis K1 could not be detected upto 31
h of incubation. The antifungal activity started appearing after 33 h of incubation
i.e. approximately in the mid logarithmic growth phase and increased upto 51
hours of incubation (i.e. late logarithmic growth phase). This antifungal activity
then sharply decreased and then remained constant till further incubation upto 96
h of incubation (Fig. 2.5). This suggests that emulsifying and antifungal activities
of B. subtilis K1 are independent of each other and may be attributed to different
metabolites with varying production profiles.
0
1
2
3
4
5
6
7
8
9
0 10 20 30 40 50 60 70 80 90 100
Time (h)
Gro
wth
(Abs
orba
nce
660
nm)
0
2
4
6
8
10
12
14
16
18
20
Emul
sify
ing
activ
ity (U
/mL)
A
ntifu
ngal
act
ivity
(AA
U/m
L )
Growth Antifungal activity Emulsifying activity
Figure 2.5: Profile of growth and production of extracellular antifungal as well as emulsifying activity by B. subtilis K1.
The haemolytic zone around the colonies of all our endophytic isolates on blood
agar plate may be attributed to their ability to produce compounds that can
Chapter-2
Characterization of Banyan endophytic Bacilli
44
penetrate into cell membrane and causes cell lysis. The fungal antagonistic action
and hemolytic activity exhibited by the isolates suggested that bacterial isolates
might be producing antifungal compounds with surface active properties. Bacilli
are known to produce surface active agents with antifungal and/or haemolytic
activity (Vanittanakom et al., 1986; Peypoux et al., 1999; Vater et al., 2002; Puja
and Cameotra, 2004; Stein, 2005; Tendulkar et al., 2007; Sanket et al., 2008).
2.3.3 Influence of antifungal extract on germinability of A. niger
conidiospores
In order to determine whether extracellular antifungal agents produced by B.
subtilis K1 affects fungal spores and its germination, conidiospores of A. niger
40211 were incubated with different dilutions of cell free crude fermentation
broth made with distilled water. The treatment of conidiospores with 10%, 25%
and 50% (v/v) of cell free culture supernatant of B. subtilis K1 obtained after 51 h
of incubation resulted in inhibition of A. niger conidiospores germination by
80%, 89% and 96%, respectively. Similar effect of methanolic extract from B.
subtilis YM 10-20 on germination of conidiospores of P. roquefortii has been
reported by Chitarra et al., (2003).
2.3.4 pH and Temperature stability of antifungal activity
The antifungal activity of crude extract from B. subtilis K1 was found to be stable
over wide range of pH (2-10) and temperature (30-121˚C). The antifungal
activity remained same upon 30 min. incubation at 121˚C. The stability against
high temperature and wide range of pH have been also been observed in the
antifungal compounds produced by B. licheniformis and B. subtilis (Tendulkar et
al., 2007; Nagorska et al., 2007). This type of pH and thermal stability of
antifungal metabolites have been reported for cyclic lipopeptides, produced
commonly by Bacilli sp.(Winkelmann et al., 1983; Chitarra et al. 2003; Stein,
2005; Tendulkar et al., 2007). Thus, it seemed that antifungal activity of B.
subtilis K1 might be due to its ability to produce and secrete cyclic lipopeptides
in environment.
Chapter-2
Characterization of Banyan endophytic Bacilli
45
2.3.5 Thin layer chromatography of antifungal compounds:
The methanol soluble antifungal active fraction obtained upon acid precipitation
from fermentation broth of B. subtilis K1 were resolved into 6 bands on silica gel
TLC plates using chloroform: methanol: water :: 65: 30: 5, v/v/v. and made
visible upon exposure to iodine vapors (figure 2.6 a). All the separated bands
could also be stained with ninhydrin reagent and showed positive Pauly’s test,
suggesting that the resolved metabolites consisted of peptides with aromatic
amino acid residues such as tyrosine (Kopple et al., 1973). These bands
fluoresced in UV upon development with Rhodamine, suggesting the presence of
lipid moiety as well in the compounds. In order to determine, which bands on
TLC had antifungal activity, the developed TLC plate was over-layered with
spores of A. niger 40211 seeded in molten 1% (w/v) PDA agar and upon 48 h of
incubation, zone of no growth was observed around bands with Rf values 0.51,
0.31 and 0.15. The complete inhibition of fungal growth was observed around
band at 0.51 Rf value, while only inhibition of sporulation was observed around
bands at 0.31 and 0.15 Rf (figure 2.6 (b)).
Figure 2.6: (a) TLC and (b) anti-biogram of methanolic antifungal extract
(AFK1) against A. niger 40211
Chapter-2
Characterization of Banyan endophytic Bacilli
46
2.3.6 Intact Cell MALDI-TOF mass spectrometry of Banyan endophytic
bacilli.
In this study, MALDI-TOF mass spectrometry technique was applied to
investigate the secondary metabolites produced by all seven endophytic bacilli
using intact cell as a target. The Intact Cell MALDI-TOF mass spectra (ICMS) of
all seven endophytic bacilli shows mass peaks ranging from m/z 551.0 to m/z
2047.3 which were compared with the reported m/z values of compounds
produced by other bacilli strains and from that three groups of mass peaks could
be identified (Figure 2.7, a-g; Table 2.3, a-c). These were putatively assigned
based on literature as surfactins (m/z, 979 to 1096.8), iturins (m/z, 1014.5-1123.5
and fengycins (m/z, 1422.2-1558.2), which represent the well-known families of
cyclic lipopeptides produced by Bacillus sp. (Leenders et al., 1999; Vater et al.,
2002; Yu et al., 2002; Pabel. 2003; Meng gong et al., 2006; Price et al., 2007;
Pyoung et al., 2010). Iturin is a cyclic heptapeptide and known for its strong
antifungal and hemolytic activity, while fengycin is cyclic depipeptide with 10
amino acids which also possess strong antifungal activity specific to filamentous
fungi with very limited hemolytic activity (Winkelmann et al., 1983;
Vanittanakom et al., 1986; Maget-Dana and Peypoux, 1994). Surfactin is a cyclic
heptapeptide which is known for its excellent surface activity and other biological
activities such as, antiviral, antitumor, antimycoplasma, mosquitocidal (Peypoux,
1997; Vollenbroich et al., 1997; Kim et al., 2007; Geeta et al., 2010). On the
basis of mass spectra profile, five isolates viz., B. subtilis K1, B. subtilis A2, B.
subtilis A4, B. amyloliquefaciens A11 and B. subtilis A12 seemed to produce
higher proportion of iturin homologues in comparison to surfactins. All these five
isolates produced fengycin homologues but the intensity of fengycin m/z peaks
were significantly lower in comparison to the intensity of iturin peaks (Fig. 2.7 a-
e). Similarly on the basis on MALDI-TOF M/S data, isolate A32 seemed to
produce higher proportion of fengycins in comparison to surfactins and iturins
(Fig. 2.7 g). In surfactin-iturin cluster of ICMS of Bacillus sp. A32, three peaks
corresponding to iturins and seven mass peaks of surfactins were assigned (Table
2.3 a-c). Furthermore, peaks at m/z 1220.9, 1234.0, 1248.0 observed in ICMS
spectra of isolates B. subtilis A2, B. amyloliquefaciens A13 and Bacillus sp. A32
differed from each other by 14 Da, suggesting that the corresponding metabolite
Chapter-2
Characterization of Banyan endophytic Bacilli
47
belonged to the same family varying from each other in mass by multiples of 14
da. The peak at 1270.0 may be assigned as sodium adduct of m/z 1248.0. There
are no reports in literature on bacilli producing cyclic lipopeptides with m/z
1220.9 to 1270.0. The mass peaks with m/z 1901.3 in ICMS of A4 and m/z
2047.3 in ICMS of A12 could not be assigned. The molecules at m/z 551.0, 614.7
and 660.8 in ICMS of A13 also could not be assigned. These unassigned m/z
peaks may belong to new molecules produced by the strains of endophytic bacilli
but their low intensity makes it difficult to select and fragment them further for
their structural elucidation. On the basis of ICMS profile, the similarity
coefficients among these isolates were determined and used to construct a
dendogram (Figure 2.8). The similarity coefficients of B. subtilis A2, B. subtilis
A4, B. amyloliquefaciens A11, B. subtilis A12, B. amyloliquefaciens A13;
Bacillus sp. A32 with Bacillus subtilis K1 were calculated to be 0.64, 0.55, 0.50,
0.51, 0.64 and 0.54, respectively. Similarity coefficient values of ICMS pattern of
all seven bacilli suggested their variability in production of metabolites as none
of them shared 100% similarity. The isolates B. subtilis K1, B. subtilis A2, B.
subtilis A4, B. amyloliquefaciens A11, B. subtilis A12 and B. amyloliquefaciens
A13 exhibited higher heterogeneity as well as intensity of mass peaks
corresponding to iturins and fengycins, in comparison to isolate A32, which may
be correlated with their spectrum and potency of antifungal activity. The Bacillus
sp. A32, which produced more of surfactins and fengycins, exhibited relatively
weaker antifungal activity with narrow spectrum. According to literature, most
strains of Bacilli, have been reported to produce cyclic lipopeptides of a single
family (Vanittanakom et al., 1986; Winkelmann et al., 1983; Beson et al., 1987;
Sen and Swaminathan, 1997; Yu et al., 2002; Cho et al., 2003; Bais et al., 2004;
Meng-gong et al., 2006; Mizumoto and Shoda, 2007). Nevertheless, there are
reports of Bacilli producing mixture of lipopeptides belonging to two different
families such as surfactins + iturins (Ohno et al., 1995) or iturins + fengycins
(Pryor et al., 2007; Cazorla et al., 2007; Ongena et al., 2007) or fengycins +
surfactins (Sun et al., 2007; Cazorla et al., 2007). However, reports of Bacilli co-
producing lipopeptides of sufactin, Iturin as well as fengycin families, with high
degree of microheterogeneity are sparse (Vater et al., 2002; Toure et al., 2004;
Price et al., 2007; Romero et al., 2007). More significantly such strains have
Chapter-2
Characterization of Banyan endophytic Bacilli
48
been found to exhibit broader range as well as higher potency of antifungal
activity, suggesting synergism between members of different families of cyclic
lipopeptides (Thimon et al., 1992; Ongena et al., 2007; Romero et al., 2007). It is
noteworthy to mention here that all the endophytic Bacilli exhibiting antifungal
activity that could be isolated from Banyan aerial roots were found to be co-
producers of surfactins, iturins and fengycins. This implies that, these organisms
must be playing a definite biological role while residing as endophytes in Banyan
aerial roots, which would be worth investigating.
Table 2.3(a) Assignment of mass peaks belong to iturins from ICMS spectra of Banyan endophytic bacilli cells Assignments of
cyclic lipopeptide
Mass
peak
(m/z)
Banyan endophytic bacilli
K1 A2 A4 A11 A12 A13 A32
C12 Iturin [M+H+] 1014.6 + - - - - - -
C13 Iturin [M+H+] 1028.9 + - - - + - -
C14 Iturin [M+H+] 1043.6 + - - - + + -
C15 Iturin [M+H+] 1057.6 + + - - + + -
C16 Iturin [M+H+] 1071.7 + + - - - - -
C17 Iturin [M+H+] 1084.7 + + - - - - -
C14 Iturin [M+Na+] 1065.6 - - - + - + -
C15 Iturin [M+Na+] 1079.7 + - - - - + -
C17 Iturin [M+Na+] 1107.7 + + + - - - -
C18 Iturin [M+Na+] 1121.7 - + + + - - -
C19 Iturin [M+Na+] 1134.7 - - + - - - -
C20 Iturin [M+Na+] 1150.8 - + + - - - -
C21 Iturin [M+Na+] 1165.9 - - - + + - +
C 15 Iturin [M+K+] 1095.7 - - + + + + +
C 17 Iturin [M+K+] 1123.8 - + + + - - -
Chapter-2
Characterization of Banyan endophytic Bacilli
49
The intensity of mass peaks assigned as surfactins, iturins and fengycins in ICMS
of B. subtilis K1 was significantly higher in comparison to the intensity of
corresponding peaks in ICMS of other six isolates, which again correlates well
with its higher potency as well as the spectrum of antifungal activity. B. subtilis
K1 was found to inhibit almost all test fungi used in this study. Thus, we selected
B. subtilis K1 for further studies on purification and characterization of
antifungal compounds secreted by it in environment.
Table 2.3 (b) Assignment of mass peaks belong to surfactins from ICMS spectra of Banyan endophytic bacilli cells
Identification of
cyclic lipopeptide
Mass
peak
(m/z)
Banyan endophytic bacilli
K1 A2 A4 A11 A12 A13 A32
C11 Surfactin [M+H+] 979.6 - + - - + - +
C12 Surfactin [M+H+] 995.5 + - - + + +
C13 Surfactin [M+H+] 1008.6 - - - + + + +
C14 Surfactin [M+H+] 1022.9 - - - + - + +
C15 Surfactin [M+H+] 1036.7 - - - + - + +
C20 Surfactin [M+H+] 1106.6 + + + - - - -
C 11 Surfactin [M+Na+] 1002.5 - - + - - + -
C 12 Surfactin [M+Na+] 1017.6 - + + - - + -
C 13 Surfactin [M+Na+] 1030.5 - - + + + + -
C 14 Surfactin [M+Na+] 1044.9 - - + + - + -
C 15 Surfactin [M+Na+] 1059.0 - - - + + + -
C18 Surfactin [M+Na+] 1102.9 - - - + + - -
C14 Surfactin [M+K+] 1060.6 - - + - - - +
C15 Surfactin [M+K+] 1074.9 - - + + - + +
Chapter-2
Characterization of Banyan endophytic Bacilli
50
Table 2.3 (c) Assignment of mass peaks belong to fengycins from ICMS spectra of Banyan endophytic bacilli cells Identification of
cyclic lipopeptide
Mass
peak
(m/z)
Banyan endophytic bacilli
K1 A2 A4 A11 A12 A13 A32
Fengycin [M+H+] 1422.2 - - - - + - -
Fengycin [M+H+] 1436.1 + + - - - - -
Fengycin [M+H+] 1450.1 + + + + + - +
Fengycin [M+H+] 1464.1 + + + + + + +
Fengycin [M+H+] 1478.2 + + + + + + +
Fengycin [M+H+] 1492.2 + + - + + + +
Fengycin [M+H+] 1506.2 + + - + + + +
Fengycin [M+Na+] 1472.1 - + + - - - -
Fengycin [M+Na+] 1500.1 - - - + + + -
Fengycin [M+Na+] 1514.1 - - - + - + -
Fengycin [M+Na+] 1528.6 + - - + - - -
Fengycin [M+K+] 1488.0 - - + + - - -
Fengycin [M+K+] 1502.6 + + + - - - -
Fengycin [M+K+] 1516.1 + + + - + - -
Fengycin [M+K +] 1530.2 - + + + + - +
Fengycin [M+K +] 1544.4 - - + - + + +
Chapter-2
Characterization of Banyan endophytic Bacilli
51
Figure 2.7(a) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis K1
Chapter-2
Characterization of Banyan endophytic Bacilli
52
Figure 2.7 (b) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A2
Chapter-2
Characterization of Banyan endophytic Bacilli
53
Figure 2.7 (c) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A4
Chapter-2
Characterization of Banyan endophytic Bacilli
54
Figure 2.7 (d) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. amyloliqeufaciens A11
Chapter-2
Characterization of Banyan endophytic Bacilli
55
Figure 2.7 (e) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A12
Chapter-2
Characterization of Banyan endophytic Bacilli
56
Figure 2.7 (f): Intact cell MALDI-TOF mass spectrometry (ICMS) of B. amyloliquefaciens A13
Chapter-2
Characterization of Banyan endophytic Bacilli
57
Figure 2.7 (g) : Intact cell MALDI-TOF mass spectrometry (ICMS) of Bacillus sp. A
Chapter2
Characterization of Banyan endophytic Bacilli
59
Figure 2.8: Dendogram based on similarity coefficient of ICMS analysis of seven
endophytic bacilli.
2.3.7 Purification and identification of antifungal compounds:
The crude methanolic antifungal extract from B. subtilis K1 was separated into 23 well
isolated peaks on semi preparative reverse phase C18 column. Figure 2.9 shows elution
profile using gradient of 80-95% MeOH. The separated peaks were collected in different
vials which were then subjected to mass spectrometry analysis. The m/z values of isolated
compounds were compared with the molecular mass ions of reported antifungal
molecules produced by genus Bacillus in order to identify them. The metabolites eluted
in peak no. 2, 3 and 4 corresponding to mass ions at m/z 1028.0, 1042.9, 1057.1, 1065.0,
1079.6, 1071.5 and 1081.1 were assigned as iturins (Winkelmann et al., 1986; Gong et
al., 2007; Vater et al., 2002; Pyoung II et al., 2010). The metabolites eluted in peaks 20
to 23 with corresponding m/z values of 1008.6, 1022.7, 1044.6 and 1058.6 were assigned
as surfactins (Table 2.5) (Kowall et al., 1998; Vater et al., 2002; Pyoung II et al., 2010).
The metabolites eluted in peaks 8 to 19 with m/z in range of 1421.8 to 1521 were
Coefficient0.45 0.47 0.49 0.51 0.53 0.55 0.57 0.59 0.61 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80
K1
A2
A4
A11
A12
A13
A32
Chapter2
Characterization of Banyan endophytic Bacilli
60
putatively assigned as fengycins (Vater et al., 2002; Hue et al., 2007; Bie et al., 2009;
Pyoung II et al., 2010).
Figure 2.9: Elution profile of metabolites separated from crude methanolic extract (obtained from cell free fermentation broth of B. subtilis K1) by semi-preparative reverse phase C18 HPLC column (4.6 mm x 250 mm, 10m particle size, 90 pore size).
2.3.8 Antifungal activity of major HPLC fractions consisting of pure putative iturin
and fengycin homologues
The HPLC peak no. P1, P3, P4, P14, P15, P16 and P17 were tested for their fungal
antagonistic activity using disc diffusion method. The two iturin homologues eluted in P3
(m/z 1042.9 m/z) and P4 (m/z 1057.1) fractions inhibited the growth of A. flavus, A.
parasiticus, Chrysosporium indicum, Fusarium oxysporum 1072, Lasiodiplodia
theobromae ABK1, Alt. brunsii, Candida albicans, Trichosporon 1110 and
Cladosporium herbarum 1112 but were ineffective against Helminthosporium
graminum1126 (Table 2.6, Figure 2.10). The fengycin homologues eluted in P14 to P17
fractions inhibited only sporulation of A. niger 40211 with affecting its vegetative growth
or germination. However, these fengycin homologues inhibited the growth of A. flavus,
A. parasiticus, C. indicum, F. oxysporum 1072, Alt. brunsii, C. herbarum 1112 and
Chapter2
Characterization of Banyan endophytic Bacilli
61
Helminthosporium graminum 1126 but were ineffective against Trichospron sp. 1110
and Candida albicans (Table 2.6 and Figure 2.10). The fengycin homologues have been
reported for their antifungal activity against Pyricularia oryzae, Condiobolus coronatus,
Curvaularia lunata, Fuasrium sp., Fusarium oxysporum, F. moliniformae, Rhizomucor
miehei, A. kikuchiana, R.solani, Colletotrichum gloeosporioides, Podospaera fusca
(Vanittanakom et al., 1986; Hue et al., 2007; Romero et al., 2007; Pyoung II et al.,
2010).
The minimal inhibitory concentration (MIC) for iturin homologues (HPLC peaks 3 and
4) and fengycin homologues (HPLC peaks 14-17) were determined using double dilution
method in 96-well micro-titer plate (Table.2.7 and 2.8). The MIC values of iturin
homologues against Candida albicans were found to be 10 μg which is in agreement to
observations reported by Winkelmann et al., (1983). The iturins were found to be more
potent against A. niger 40211, C. indicum, Alt. brunsii, Cladosporium herabarum1126,
A. flavus L. theobromae in comparison to Candida albicans, Trichosporon 1110 and F.
oxysporum. Klich et al., (1991) also reported requirement of higher concentration iturin A
to inhibit the growth of A. parasiticus, A. flavus and Fusarium moliniforme. The MIC
values of fengycins in four fractions (HPLC peaks 14-17) against F. oxysporum 1072
were found to be 50μg/mL which was 5-fold higher than reported for Fusarium sp. by
Vanittanakom et al. (1986). However MIC values of fengycins against Alt. brunsii were
in the range of 0.31-2.5μg/mL, which were significantly lower than reported for Alt.
kikuchiana (10μg/mL) (Vanittanakom et al., 1986). In present study, amongst all the test
cultures tested, Fengycins were found to be most potent against Clad. herbarum.
Chapter2
Characterization of Banyan endophytic Bacilli
62
Table 2.5: MALDI-TOF analysis of HPLC fractions collected upon separation of metabolites from crude methanolic extract (obtained from cell free fermentation broth of B. subtilis K1) by semi-preparative reverse phase C18 HPLC column (4.6 mm x 250 mm, 10m particle size, 90 pore size).
HPLC fraction
no.
R.T Molecular mass ions (m/z) Identification of compounds based on
literature P1 15.2 802.0,891.7,972.5,1184.7,1237.8,1372.9 Unidentified P2 19.3 1028.0 Iturin [M+H]+
P3 21.8 1042.9 1065.0 1081.1
Iturin [M+H]+
Iturin [M+Na]+
Iturin [M+K]+
P4 23.8 1057.1 1079.6
Iturin [M+H]+
Iturin [M+Na]+
P5 24.2 1071.5 Iturin [M+H]+
P6 27.2 1071.5 1435.7 1453.7,1467.8,1481.8
Iturin [M+H]+
Fengycin [M+H]+
Linear fengycin [M+H]+
P7 28.7 1071.5 1481.7
Iturin [M+H]+
Linear fengycin [M+H]+
P8 30.9 1072.6 1421.9,1435.8, 1459.8
Iturin [M+H]+
Fengycin [M+H]+
P9 32.9 1435.6,1449.7,1464.81467.9, 1481.9,1495.9
Fengycin [M+H]+
Linear fengycin [M+H]+ P10 &
11 34.6 1449.4,1464.5,1478.7
1481.7,1495.9,1510.9,1525.1 Fengycin [M+H]+
Linear fengycin [M+H]+ P12 37.5 1449.8, 1463.8, 1477.8
1510.9, 1524.9 Fengycin [M+H]+
Linear fengycin [M+H]+ P13 38.2 1449.8,1463.8, 1477.9 Fengycin [M+H]+
P14 39.8 1449.8,1463.8 1471.8,1485.9
Fengycin [M+H]+
Fengycin [M+Na]+
P15 41.9 1463.8, 1477.8, 1491.9, 1505.9 Fengycin [M+H]+
P16 42.6 1477.8, 1491.8, 1506.11485.8,1499.8,1513.8
Fengycin [M+H]+
Fengycin [M+Na]+ P17 44.3 1477.8,1491.9,1505.9
1513.9 Fengycin [M+H]+
Fengycin [M+Na]+ P18 47.6 1449.8,1491.9,1505.9,
1528.0,1544.0 Fengycin [M+H]+
Fengycin [M+Na]+ P19 50.5 1447.8, 1462.8
1469.8,1484.9,1513.9, 1527.9,1544.0 Fengycin [M+H]+
Fengycin [M+Na]+ P20 52.0 1008.6, 1022.7
1030.6, 1044.6 1441.9, 1624.7
Surfactin [M+H]+ Surfactin [M+Na]+
Unidentified P21 & 22 54.6
1008.5, 1022.6 1044.6, 1058.6 1441.9, 1033.6, 1166.7, 1272.7, 1372.7
Surfactin [M+H]+
Surfactin [M+Na]+
Unidentified
P23 57.9 1022.5, 1036.5, 1441.9, 1624.7
Surfactin [M+H]+
Unidentified
Chapter2
Characterization of Banyan endophytic Bacilli
63
Table 2.6 Spectrum of antifungal activity spectrum of crude extract, purified iturin (P3 and P4) and fengycin (P14-P17) homologues from B. subtilis K1
Test pathogen Zone of Inhibition (mm)
crude P-3
P-4
P-14
P-15
P-16
P-17
A. niger 40211 20 18 19 12a 8a 9a 14a
A. flavus 6 6 7 7 5 6 5 A. parasiticus 12 10 7.5 8.5 10 9.3 8.3C. indicum 15 12 13 16 18.6 18 17 F. oxysporum 1072 12 8 9 10 8.6 9 9L. theobromae ABFK1 16 14 16 15 15 15 16 Alternaria brunsii ND 18 19 20 18 21 20Candida albicans 9 8.5 8.6 ND ND ND NDTrichosporon 1110 7 7 8 ND ND ND ND Helminthosporium graminum 1126
9 ND ND 8 9 7 7
a only spore germination was inhibited
Figure 2.10: Photograph showing bioassay of putative purified Iturins (P3, P4) and fengycins (P14-P17) by reverse phase C-18 HPLC using A. niger 40211, C. indicum, Alt. brunsii, F. oxysporum. Control (C) corresponds to only solvent i.e. MeOH.
Chapter2
Characterization of Banyan endophytic Bacilli
64
Table 2.7 Minimum Inhibitory Concentration (MIC) of purified iturin homologues from B. subtilis K1
Test culture MIC and IC50 of Iturin homologues
(μg/ml) Iturin (1042.9m/z)
(MIC) Iturin(1057.0m/z)
(MIC) A. niger 40211 2.5 1.25
A. parasiticus 5.0 5.0 C. indicum 5.0 2.5 F. oxysporum 1072 5.0 5.0 Alternaria brunsii 2.5 1.25 Candida albicans 5.0 10.0 Trichosporon sp. 1110 5.0 10.0 Cladosporium herbarum 1126 5.0 2.5
Table 2.8 Minimum Inhibitory Concentration (MIC) fengycin containing fractions separated from cell free crude extracellular extract of B. subtilis K1 by RP-HPLC.
Test culture MIC of fengycin containing fractions
(μg/ml)P-14
P-15
P-16
P-17
A. parasiticus 50 50 50 50 C. indicum 0.625 1.25 2.5 2.5 F. oxysporum 1072 50 50 50 50 A. brunsii 0.625 1.25 2.5 2.5 C. herbarum 0.15 0.125 0.062 0.062
2.4 Summary and conclusion:
Seven gram positive spore forming rod shaped bacilli exhibiting the broad spectrum
antifungal activity were isolated from the aerial roots of Banyan tree. Light microscopy
and transmission electron microscopy studies showed presence of rod shaped bacteria
located in intercellular spaces as well as within parenchymatous cells of Banyan root
tissue which supported presence of bacterial endophytes in the aerial roots of Banyan
tree. The isolates designated as K1, A2, A4, A12 were identified as B. subtilis whereas
isolates A11 and A13 were identified as B. amyloliquefaciens. All the seven cultures
exhibited haemolytic as well as emulsifying activity. Amongst, all seven cultures, B.
subtilis K1 was found to be most potent fungal antagonist, hence was selected for
Chapter2
Characterization of Banyan endophytic Bacilli
65
purification and characterization of antifungal compounds produced by it. The
metabolites in the cell free culture supernatant of B. subtilis K1 were separated by C-18
reverse phase HPLC and could be putatively identified as homologues of surfactins,
iturins and fengycins. The extracellular metabolites from the Banyan endophytes were
also analyzed using Intact Cell MALDI-TOF Spectrometry (ICMS). The similarity
coefficients of seven isolates determined on the basis of ICMS profile could be used to
differentiate them from each other.
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