Chapter 2 Characterization of Banyan endophytic Bacilli …shodhganga.inflibnet.ac.in/bitstream/10603/7223/6/06_chapter 2.pdf · Characterization of Banyan endophytic Bacilli and

Chapter 2

Characterization of Banyan endophytic Bacilli and

identification of their antifungal metabolites

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

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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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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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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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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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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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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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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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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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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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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.

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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).

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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 -


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


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


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


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


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


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


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


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


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


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)


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


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)


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


51

Figure 2.7(a) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis K1

Chapter-2


52

Figure 2.7 (b) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A2

Chapter-2


53

Figure 2.7 (c) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A4

Chapter-2


54

Figure 2.7 (d) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. amyloliqeufaciens A11

Chapter-2


55

Figure 2.7 (e) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A12

Chapter-2


56

Figure 2.7 (f): Intact cell MALDI-TOF mass spectrometry (ICMS) of B. amyloliquefaciens A13

Chapter-2


57

Figure 2.7 (g) : Intact cell MALDI-TOF mass spectrometry (ICMS) of Bacillus sp. A32

Chapter-2


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


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


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


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


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


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


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


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


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


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


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


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


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


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


36 36 34 36 22-24 26 -

2 Aseprgillus niger 181

32-34 34 32-34 38 20 22 -


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


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


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


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


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


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


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


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


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)


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 - + + + - - -

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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)


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 - - + + - + +

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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)


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 - - + - + + +

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51

Figure 2.7(a) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis K1

Chapter-2


52

Figure 2.7 (b) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A2

Chapter-2


53

Figure 2.7 (c) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A4

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54

Figure 2.7 (d) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. amyloliqeufaciens A11

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55

Figure 2.7 (e) : Intact cell MALDI-TOF mass spectrometry (ICMS) of B. subtilis A12

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56

Figure 2.7 (f): Intact cell MALDI-TOF mass spectrometry (ICMS) of B. amyloliquefaciens A13

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57

Figure 2.7 (g) : Intact cell MALDI-TOF mass spectrometry (ICMS) of Bacillus sp. A

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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

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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

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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.

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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

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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.

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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

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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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