chapter-4shodhganga.inflibnet.ac.in/bitstream/10603/9191/9/09_chapter 4.pdfthe purity of the...

Chapter-4

Studies on certain seaweed - bacterial interaction from Saurashtra coast 56

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4.1. Introduction

Among the Rhodophyta, the genus Gracilaria (Gracilariales) is commercially an important

agarophyte and accounts for >70% of the raw materials’ supply to the world agar industry

(McHugh, 2002). There are > 150 different species of Gracilaria that have been reported from

different parts of the world (Byrne et al., 2006). The red alga Gracilaria dura is one of the

economically important macroalgae found on the Indian coast and occurs predominantly in tide

pools in the mid-littoral zone in selective locations. This alga has been used for producing high-

end phycocolloid agarose directly with an improvised process developed by CSMCRI

(Siddhanta et al., 2005; Meena et al., 2007). The thallus is relatively bushy, branched, red to

dark brown in color and is distinguished by abrupt constrictions at the base of the lateral

branches, forming a slender stripe (Jha et al., 2009).

Bacteria are common inhabitants of both the surfaces and the internal tissues of most

plants and play an important role in the development and growth of the host (Matsuo et al.,

2003). Plant-associated bacteria isolated from surfaces are known as epiphytes (Andrews &

Harris, 2000), whereas those isolated from the interior of tissues are called endophytes (Petrini

et al., 1989), with some bacterial populations fluctuating between epiphytic and endophytic

colonization (Hallmann et al., 1997). Both types of bacterial isolates can contribute to the

growth, health and development of seaweeds by the direct production of the plant growth

regulators (PGRs) and nitrogen fixation (Sturz et al., 2000). PGRs have been reported to play a

significant role in the morphogenesis of callus from Rhodophyta members (Yokoya, 2000;

Reddy et al., 2003). Some marine bacteria were reported to produce plant auxins regulating the

morphogenesis pattern and growth in Ulvaceae (Maruyama et al., 1986; 1988). The plant

growth-promoting nature of bacterial isolates associated with Laminaria japonica was reported

by Dimitrieva et al. (2006). The first phytohormone to be identified was IAA, which regulates

the cell elongation, division and differentiation and tryptophan is considered as its precursor

(Park et al., 2005). The study of seaweed-associated bacteria is important not only for

understanding the ecological role of such bacteria in their interaction with seaweeds but also for

the biotechnological application of these bacteria to areas such as the seaweed growth

promotion (Marshall et al., 2006). However, studies on the effect of PGRs are required in order

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to understand the developmental process and growth of Gracilaria spp. There are numerous

reports where bacterial isolates have been shown to influence the life cycle of the Chlorophyta.

The members of Ulvaceae lose their typical foliose thallus morphology when cultured

aseptically in defined synthetic media (Provasoli & Pintner, 1980). This aberrant morphology

has successfully been restored to the foliose thallus following the inoculation of appropriate

morphogenesis inducing bacterial isolates to the culture medium as described in the chapter 3.

On the contrary, there is limited knowledge on bacteria promoting growth and nitrogen fixation

in the Rhodophyta.

The present chapter describes the isolation and screening of epi- and endophytic bacteria

and their role in bud induction as well as growth of commercially important red alga G. dura.

The roles of bacterial isolates, their total fatty acid, protein (total and fractionated protein) and

nitrogen fixation were examined for their effect on this alga. The effect of temperature and

salinity on G. dura–bacteria interaction was also investigated. To the best of our knowledge, this

is the first time that the effect of bacterial protein and nitrogen fixation on the bud induction and

growth of Gracilaria has been investigated.

4.2. Materials and methods

4.2.1. Collection of algal sample and preparation of axenic tissue

Healthy vegetative thalli of Gracilaria dura, G. corticata and G. salicornia were collected from

the intertidal area of Veraval (20º154.870’N, 70º120.830’E) during low tide from August 2008

to June 2010. The fronds were immediately brought to the laboratory under cool conditions and

were cleaned thoroughly to remove adhering debris and other epiphytic contamination. The

fronds were cut into small segments to establish the algal stock culture. The fronds were further

treated with 2% liquid detergent for 10 min, 1% betadine for 2 min and 1% antibiotic mixture

for 48 h at 25±1 ºC (As described in chapter 2). The axenic cultures were maintained by

following methods described in the chapter 2. The axenicity of the algal cultures was tested by

incubating a few algal fragments on Marine Agar 2216, Tryptone Soya Agar, Casein Soya Agar

and Bacillus Medium (HiMedia, India) at 30±1 ºC for 2–15 days.

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4.2.2. Isolation of epi- and endophytic bacteria

A number of bacterial isolates were isolated from the fresh thallus of Gracilaria spp.

demonstrated in Table 4.1.

Table 4.1. Distribution of active seaweed associated bacteria collected at Veraval (August

2009).

Species Abbreviation Number of isolates

Gracilaria dura GD 25

G. corticata GC 10

G. salicornia GS 4

The algal material was collected from Veraval coast during low tide. The samples were

immediately brought to laboratory under cool conditions and the tissue was several times

washed gently in autoclaved seawater in the laminar air flow bench and placed on the Marine

Agar 2216, Tryptone Soya Agar, Casein Soya Agar and Bacillus Medium (HiMedia, India) for

isolation of associated bacteria at 30±1°C for 2-15 days (longer incubation period given to

screen for slow growing bacteria). After testing the axenicity of the G. dura with the above

mentioned media, fronds were ground to a fine particle with a mortar and pestle. Thereafter, fine

slurry were dissolved in the 10 ml of autoclaved seawater, vortexed and spread on the same

bacterial media and left for 2-15 days for the endophytic bacterial growth at 30±1°C. Thereafter,

isolation of single colony and maintained of bacterial culture were carried out according to

describe in the chapter 3.

4.2.3. Extraction of fatty acid

The whole cell fatty acids analysis for the bacterial strains was accomplished according to the

method of Dionisi et al. (1999). In brief, 100 mg of dried bacterial samples with internal

standard was transesterified using 1 ml of methanolic HCl (1.5 M) at 80±1ºC for 10 min.

Thereafter, 2 ml of water was added, vortex, centrifuged at 4000 rpm to extract upper organic

layer and concentrated for gas chromatography-mass spectroscopy (GC-MS) analysis. Fatty acid

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Dionisi%20F%22%5BAuthor%5D

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methyl esters were identified by comparing their retention time with those of standard. Zobell

broth was used for growth of all the three putative bud-inducing bacterial isolates.

4.2.4. Genomic DNA isolation and amplification of partial 16S rRNA gene sequences

Bacterial colonies were first incubated with (10 mg ml-1

) lysozyme and the genomic DNA was

extracted according to previous chapter 3. The purity of the isolated genomic DNA, PCR

amplification, conditions and consensus sequences of DNA made according to previous chapter

3. Partial consensus sequence of 16S rRNA gene was generated from forward and reverse

sequences data using Aligner software and submitted to the NCBI Genbank.

4.2.5. Isolation and purification of bacterial extracellular protein

Bacterial isolates were inoculated in 100 ml of Zobell broth and incubated at 30±1ºC in an

orbital shaker for 4 days. The medium was centrifuged at 12,000 rpm for 30 min at 4±1ºC. The

proteins were concentrated from the culture filtrate by salting out with ammonium sulfate (80%

saturation). The precipitated proteins were re-suspended in 25 ml of 0.2 M phosphate-citric acid

buffer (PCA, pH 6.5) and dialyzed against the same buffer. The dialyzed samples were applied

to DEAE–Cellulose column (1.6×20 cm, Sigma) and equilibrated with 400 ml of 0.2 M PCA

buffer. The elution was performed with a linear gradient of 0.0 to 1 M NaCl in PCA buffer with

a flow rate of 0.5 ml min-1

at 4±1°C. All the fractions were pooled and concentrated. The

concentrated fractions were further fractionated using Sephadex G-50 column (Sigma-Aldrich,

Germany) and elution was performed by 0.2 M PCA buffer at a flow rate of 0.5 ml min-1

.

Protein content was determined by the Bradford (1976) method with bovine serum albumin as a

standard.

4.2.6. Assessing the effect of bacteria, their protein (total and fractioned) and fatty acid on

bud induction

Small fronds of 1-2 cm length were excised from the healthy vegetative thalli maintained in the

culture. Care was taken to obtain all the fronds from a single thallus for each set of experiment

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and preferentially not from the basal portion. Fronds of 1-2 cm length were added to each well

containing 5 ml of MP 1 medium in 12 well plates and culture conditions were maintained as

previously described (chapter 3). Each bacterial isolate was added to a separate multi well plate

to assess the effect of individual bacterial isolate on the bud induction in G. dura. The control

was maintained without addition of bacterial isolates. The cultures were grown for 60 days and

the medium was replenished every alternate day. The experiment was carried out in triplicate to

confirm the results. The three putative bud-inducing bacterial isolates were identified at the end

of this preliminary experiment for further study. Further, the total fatty acid and total protein

obtained from the individual putative bud-inducing bacterial isolates as well as fractioned

protein (after second column) was separately incubated in the multi well plate to study its effect

on the bud induction. The control was maintained without addition of any fatty acid and protein.

The visual interpretation of images taken at weekly intervals with a digital camera on a zoom

stereo microscope (Olympus SZ X 16 microscope, Japan) was adopted as a nondestructive

method to estimate algal growth. The protein expressed by G. dura in the presence and absence

of putative bud inducing bacterial isolates were analyzed by 10% sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970). Protein bands

were developed by silver staining.

4.2.7. Screening for IAA producing and nitrogen fixing activity

Indole-3-acetic acid (IAA) production was analysed using a modification of the qualitative

method developed by Bric et al., (1991). In brief, putative bud-inducing bacterial isolates were

plated onto Marine Agar 2216 amended with 5 mM of L-tryptophan, overlaid with a

nitrocellulose membrane and incubated at 30±1ºC for 24 h. After bacterial growth had occurred

the membrane was removed from the plates and treated with Salkowski reagent (2% (w/v) 0.5

M FeCl3 in 35% perchloric acid) for 15 min at 30±1ºC. IAA producing bacterial isolates were

identified by the presence of a red corona on the membrane corresponding to the position of the

IAA-producing colony. For extraction of IAA, the overnight bacterial culture filtrates of three

putative bacterial isolates were adjusted to pH 2.5 using 5 N HCl and an equal volume of ethyl

acetate was added. The organic solvent was evaporated and the residue was dissolved in 1 ml of

25% methanol and 1% acetic acid (Chung et al., 2003). The IAA standard and organic extract of

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the putative bud-inducing bacterial culture filtrates was spotted on the TLC plates (Kieselgel 60,

Whatman plates of dimension 20 X 20 cm with layer thickness of 0.25 mm). The TLC plates

were run with the following solvent compositions: isopropanol / ammonium hydroxide / water

(8:1:1 v/v) for IAA. The spots were visualized after the plate was sprayed with Ehrlich’s reagent

(this reagent gives red color with IAA). The corresponding spots were scooped out and eluted in

methanol for IAA estimation. The eluted fractions were used for the time of flight liquid

chromatography electrospray ionization- mass spectrometry (TOF LC-ESI-MS) and High-

performance liquid chromatography (HP-LC) analysis.

LC-ESI-MS were performed using a waters micromass Q Tof micro TM

mass

spectrometer connected to Waters alliance HPLC and equipped with electrospray ionization

source. Samples were dissolved in 1 ml of 25% methanol and 1% acetic acid and run at 25ºC for

20 min in TOF-LC-MS for characterization of IAA. The HP-LC used was a Waters instrument

(Model- 2695, Separation Module) comprising a auto injector, a 150 mm X 4.6 mm Nucleosil

C18 stainless steel column (5 μm particle size, 300 A° pore size) (Phenomenex, USA), 6AD

pumps, an SPD-10A UV-vis detector, and an LC-10 chromatography manager. Standards and

organic extracts of putative bud-inducing bacterial culture filtrates were injected with a 20 μL

syringe (Hamilton, Reno, NV). Isocratic elutions were performed at 30ºC with MeOH / H2O

(4:6 v/v) mobile phase containing 1% acetic acid at a constant flow rate of 0.8 ml min-1

. UV

detection was carried out at 254 nm. Standard solutions were prepared with pure IAA (Sigma)

for identification and quantification of IAA produced by the bacterial isolates.

For screening of nitrogen fixing epi- and endophytic putative bud-inducing bacterial

isolation, the isolates were enriched by repeated transfer on nitrogen deficient semisolid

mannitol (NDSM) medium containing the following per litre (pH 8) in deionized water: 5.4 g of

K2HPO4, 4.23 g of KH2PO4, 0.2 g of MgSO4· 7H2O, 4.0 g of NaCl, 0.02 g of CaCl2· 2H2O, 0.01

g of FeCl3, 0.0005 g of Na2 MoO4 ·2H2O, 3 g of mannitol, 0.1 g of yeast extract, 5.0 g of

glucose, 1.5 g of NaOH and 1.75 g of agar (Himedia, India). Thereafter three putative bud-

inducing bacterial isolates were enriched without agar in the aforementioned medium and

production of ammonia (mM) was assayed for nitrogen fixation. The amount of ammonia in the

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medium was measured with the addition of Nessler`s reagent and determined by measuring the

optical density at 410 nm (Emtiazi et al., 2007).

4.2.8. Optimization of salinity and temperature on interaction

Fronds of 1-2 cm length of G. dura were used for investigating the role of total protein of B.

pumilus in bud induction. A total of 100 fronds were used during the entire experiment.

Different combinations of salinity and temperature were investigated to determine the optimum

conditions for the interaction. The sterilized PES medium of different salinities viz. 40, 35, 30,

25, 20 and 15‰ was prepared. A total of 2 fronds were incubated in 5 ml of medium in

sterilized multi wells plates. The plates representing all the six salinities were incubated under

different temperature regimes separately at 40, 35, 30, 25, 20 and 15°C in a Multi Thermo

Incubator (MTI-202, Eyela, Japan). The fronds of thallus were observed under the inverted

microscope (IX 70, Olympus, Japan) to record the induction of new buds. Each treatment was

tested in triplicate. Two-way ANOVA analysis was used for finding the effect of salinity and

temperature on the interaction for bud induction and significant differences were determined at

p ≤ 0.05. The optimum salinity and temperature that resulted in maximum bud induction was

maintained for all the further experiments.

4.3. Results

4.3.1. Selection of the bacterial isolation and identification

The present chapter reports three putative bud-inducing bacterial isolates (one epi- and two

endophytes) showing IAA production and nitrogen fixation which regenerated new buds from

G. dura fronds These putative bud-inducing bacterial isolates were identified using biochemical,

fatty acid analysis and partial 16S rRNA gene sequences. The growth conditions of the putative

bud-inducing bacterial isolates showed that these isolates can survive under high pH (9) and

tolerate high NaCl (10%) concentration. The physical properties, carbohydrate utilization and

antibiotic sensitivities were also tested for the three putative bud-inducing bacterial isolates

(Table 4.2).

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Table 4.2. Morphological and biochemical test for identification of bacterial isolates.

Test B. licheniformis B. pumilus E. homiense

Colony morphology

Configuration Circular Circular Umbonate

Margin Entire Entire Irregular

Elevation Convex Convex Flat

Surface Smooth Smooth Rough

Pigment Milky White off White off, dry

Opacity opaque Translucent Translucent

Gram’s reaction + + +

Cell shape Rod Rod Rod

Arrangements Single Single Single

Biochemical test

Indole utilization - - -

Methyl red utilization + + +

Voges-Proskauer

utilization

- + -

Citrate utilization + - -

Lipolysis ++ - ++

Esculin hydrolysis + - ++++

Gelatin hydrolysis -

Starch hydrolysis +++ + +

Urea hydrolysis -

Catalase + + +

Oxidase ++ + +

Nitrate reductase +++ - ++

Ornithine decarboxylase - - +

Lysine decarboxylase + + +

Carbohydrate utilization

Arabinose + - +

Adonitol - - -

Cellobiose + - +

Dextrose + - +

Dulcitol - - -

Fructose + - +

Galctose - + -

Glucose + + +

Inositol - - -

Inulin - - -

Lactose

- - -

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Table 4.2. Continued.


Maltose - - +

Mannitol - + -

Mannose + - +

Melobise - - -

Raffinose - ++ -

Rhamnose - - -

Salicin + - +

Sorbitol - - -

Sucrose + + +

Xylose + + -

Trehalose + + +

Antibacterial effect

Inhibitory zone

(mm)

Inhibitory zone

(mm)

Inhibitory zone

(mm)

Chloramphenicol (30 µg) 22 21 21

Ampicillin (10 µg) 17 16 17

Tetracycline (30 µg) 21 30 20

Gentamycin (10 µg) 15 22 16

Streptomycin (10 µg) 15 14 15

Kanamycin (30 µg) 12 14 10

Co-Trimoxazole (25 µg) resistance 30 Resistance

Amikacin (30 µg) 14 20 13

Physical condition

pH 2 - - -

pH 4 - - -

pH 6 + - -

pH 7 ++ ++ +

pH 8 +++ ++ +++

pH 9 ++++ ++++ +++

pH 10 ++ ++ ++

pH 12 + + +

pH 13 - - -

4 ºC - - -

15 ºC - - -

20 ºC + + +

25 ºC ++ ++ ++

30 ºC ++++ ++++ ++++

35 ºC ++- +++ ++

40 ºC + + +

45 ºC + + -

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Table 4.2. Continued.


NaCl (%)

2.5 - - -

4 + + ++

5 ++++ ++++ ++++

7 +++ +++ ++

10 + + +

15 - - -

+, presence of activity; -, absence of activity.

The partial consensus sequence of 16S rRNA gene sequences of three putative bud-

inducing bacterial isolates were obtained and compared with the NCBI Genbank data through

BLAST search. Bacillus licheniformis (accession number GU723480) showed 97% homology

with the B. licheniformis strain JS-17 (accession number GQ280087), B. pumilus (accession

number HQ318731) showed 98% homology with the B. pumilus strain U4 (accession number

GU297606) and Exiguobacterium homiense (accession number HQ318732) showed 97%

homology with the E. homiense strain EQH16 (accession number FJ999945). The saturated

fatty acids were the major constituents (86 to 95%) of all the three bud-inducing bacterial

isolates. 13-methyltetradecanoic acid (22.65% and 24.20%), 12-methyltetradecanoicacid

(27.91% and 18.62%), hexadecanoic acid (14.63% and 16.17%) and branched-chain fatty acids

were observed in B. pumilus and B. licheniformis while 13-methyltetradecanoic acid (16.84%)

and 12-methyltetradecanoic acids (5.3%) were present in E. homiense. The polyunsaturated fatty

acid was only observed in B. licheniformis (Table 4.3). Hydroxyl (4.06%) and trans (0.3%) fatty

acid were characteristic of E. homiense. Cis and cyclic fatty acids were presented in E. homiense

and B. pumillus (Table 4.3). Branched chain fatty acids were more constituent in Bacillus sp.

than E. homiense. One putative bud-inducing epiphyte was identified as E. homiense and the

other two putative bud-inducing endophytes were B. pumilus and B. licheniformis.

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Table 4.3. Fatty acid composition of putative bud-inducing bacterial isolates.

Fatty acids

B. pumillus B.

licheniformis

E.

homiense

Dodecanoic acid (C12:0) ND 0.41 0.28

2-hydroxydodecanoic acid (2C12:OH) ND ND 1.45

3-hydroxydodecanoic acid (3C12:OH) ND ND 2.05

Tridecanoic acid (C13:0) 3.69 2.68 6.43

Tetradecanoic acid (C14:0) 2.31 3.54 24.25

2-hydroxytetradecanoic acid (2C14:OH) ND ND 0.33

3-hydroxytetradecanoic acid (2C14:OH) ND ND 0.2

Pentadecanoic acid (C15:0) 1.03 1.07 0.5

13-Methyltetradecanoic acid (Iso-C15:0) 22.65 24.20 16.84

12-Methyltetradecanoic acid (anteiso C15:0) 27.91 18.62 5.3

Hexadacenoic acid (C16:0) 14.63 16.17 13.9

14-Methylpentadecanoic acid (iso C16:0) 3.85 4.46 ND

7- Hexadecenoic acid C16:1n-7 1.15 0.39 3

15-Methylhexadecanoic acid (iso C16:0) ND ND 3.87

Methylenehexadecanoic acid (Cyc. C17) ND ND 6.56

Heptadacenoic acid (C17:0) 0.55 0.73 0.33

15-Methyl hexadecanoic acid (iso C17:0) 4.35 7.82 0.31

14-Methylhexadecanoic acid (anteiso C17:0) 6.33 6.56 ND

Octadecanoic acid (C18:0) 8.80 6.65 8.28

Cis-9- Octadecenoic acid (cis-C18:1n-9) 0.62 2.03 5.78

Trans-9- Octadecenoic acid (trans-C18:1n-9) ND 0.84 0.32

Cis-11- Octadecenoic acid (C18:1n-11) ND ND ND

15-Methylheptadecanoic acid (anteiso C18:0) 0.66 0.81 ND

Nonadecanoic acid (C19:0) 0.43 0.25 ND

Propaneoctanoic acid (Cyc. C19) 0.43 ND ND

Eicosanoic acid (C20:0) 0.64 0.39 0.3

8,11,14 -Eicosatrienoic acid (C20:3 n-6) ND 0.31 ND

5,8,11,14-Eicosatetraenoic

acid (C20:4 n-6)

ND

1.81

ND

Docosanoic acid (C22:0) ND 0.25 ND

SFA 93.25 94.61 86.92

MUFA 1.77 3.28 9.03

PUFA 0 2.11 0

Br FA 40.74 52.47 26.09

cyc FA 0.43 0 6.57

FA-OH 0 0 4.06

cis FA 1.77 0 8.73

trans FA 0 0 0.30

ND, not detected; PUFA, polyunsaturated fatty acid. Organisms of this group were grown on Zobell Marine Agar.

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4.3.2. Effect of the bacterial isolates on G. dura for the bud induction and growth

Fig. 4.1. Total number of buds regenerated from individual frond with co-culture of different

bacterial isolates (a) and their total protein (b). UT-V- B. pumilus, GC-endo- B. licheniformis,

ENT-4- E. homiense.

This is the first findings to the seaweed-associated putative bud-inducing bacterial

isolates regenerating the new buds and enhancing the growth in G. dura. The highest number of

bud induction was associated with the putative bud-inducing B. pumilus while the lowest with

the E. homiense. The putative bud-inducing B. pumilus was able to regenerate up to 10 numbers

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of buds per frond with varied length of 6, 4, 2 and 1 mm while B. licheniformis was able to

regenerate up to 9 buds per frond with varied length of 3, 2 and 1 mm. The induction of 5 buds

by E. homiense varied in length by 3, 2 and 1 mm (Fig. 4.1a). The total protein of the

endophytes putative bud-inducing B. pumilus and B. licheniformis was found to regenerate most

(10) number of buds with varied length of (6, 5, 3 and 2 mm) and (4, 3 and 2 mm) respectively

(Fig. 4.1b).

Fig. 4.2. Regeneration of new buds from individual fronds in control at 0 day (a) and 45 days

(b) and comparison with fronds treated with total protein of B. licheniformis (c and f), B.

pumilus (d and g) and E. homiense (e and h) at 45 and 60 days, respectively.

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Although the total protein of the epiphytic putative bud-inducing E. homiense was less

effective and was able to regenerate 5 buds with 3, 2 and 1 mm length when compared to the

control in which 1-2 buds were observed (Fig. 4.2). The total fatty acid and the fractionated

protein from DEAE–Cellulose and Sephadex G-50 columns of the putative bud-inducing

bacterial isolates were not able to regenerate new buds in G. dura. Maximum protein expression

was observed with B. pumilus while E. homiense and B. licheniformis showed approximately

equal expression when incubated with G. dura as comparison to control after 60 days of growth.

4.3.3. Qualitative analysis for IAA and nitrogen fixation

The three putative bud-inducing bacterial isolates were found positive for IAA and nitrogen

fixation activity. The TOF LC-MS revealed a peak at 174.05-175 m/z and 176.05-177 m/z for

IAA production (Fig. 4.3). The putative bud-inducing bacterial isolates B. pumilus, B.

licheniformis and E. homiense were produced 445.5, 335 and 184.1 μg ml-1

IAA in the culture

filtrates respectively with 9.094 to 9.172 retention times with HP-LC (Fig. 4.4). The putative

bud-inducing B. pumilus, B. licheniformis and E. homiense produced 12.51, 10.14 and 6.9 mM

ml-1

ammonia respectively in the nitrogen free bacterial culture medium. The production of

ammonia was evidenced by the change in colour of the culture filtrate from yellow to brown

after the addition of Nessler’s reagent.

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Fig. 4.3. LC-MS spectra of IAA for B. pumilus (a), B. licheniformis (b) and E. homiense (c).

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Fig. 4.4. HP-LC chromatogram of IAA for B. pumilus (a), B. licheniformis (b) and E. homiense

(c).

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4.3.4. Effect of salinity and temperature on growth

There was a varied response in bud induction in G. dura when treated with the total protein of

the putative bud-inducing B. pumilus when incubated in different salinities and temperatures

(Fig. 4.5). The association between G. dura and the total protein of the putative bud-inducing B.

pumilus regenerated the formation of 10 numbers of buds when incubated under 25°C and 30‰

while 8 numbers of buds were formed under the 25°C and 25‰ regime. The lowest number of

the buds was regenerated during incubation at 40°C and 40‰. Further, the effect of the different

salinities and temperatures on the interaction was significant at p ≤ 0.05 in two-way ANOVA

(Table 4.4).

Fig. 4.5. Effect of different temperatures and salinities on regeneration of new buds from

individual fronds treated with total protein of B. pumilus.

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Table 4.4. Two-way ANOVA analysis to show effects of different temperature and salinity on

regeneration of new buds from individual fronds with total protein of B. pumilus

Sources Effect on the bud formation per frond

df SS MS F P < .05%

Temperature (T) 5 90.89 18.18 23.1 0.000

Salinity (S) 5 110.22 22.04 28.01 0.000

Interaction (TxS) 25 109.89 4.4 5.58 0.000

Error 72 56.67 0.78

Total 107 367.67

4.4. Discussion

An insight into the structure and species composition of seaweed-associated bacterial

populations is fundamental to understand as how seaweed-associated biological processes are

influenced by environmental factors and consequently has important biotechnological

implications. The present chapter for the first time describes the environmental and

physiological factors involved in G. dura-associated bacteria interaction especially with the bud

regeneration and the process of plant development.

In the present chapter, putative bud-inducing bacterial isolates were identified by partial

16S rRNA gene sequences, biochemical and fatty acid analysis. The biochemical analysis

revealed that bud-inducing bacterial isolates could tolerate high pH and salt concentration. The

predominance of terminal methyl branched iso and anteiso fatty acids having 12 to 17 carbons is

a characteristic feature observed in all the species of Bacillus (Kaneda, 1969). The presence of

methyl branched iso and anteiso fatty acids could be attributed to the high pH tolerance

(Ntougia & Russell, 2000). The 13-methyltetradecanoic and 12-methyltetradecanoic acids are

generally the constituent fatty acids the genus Bacillus (Kaneda, 1977; Ntougia & Russell,

2000). The major fatty acid of E. homiense was found to be tetradecanoic acid with 24.25% and

13-methyltetradecanoic acid with 16.84% (Table 4.3). The presence of hydroxyl (4.06%), trans

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(0.3%) and tetradecanoic acid (24.25%) revealed the characteristic feature of E. homiense. Fatty

acids of similar structure usually differ by two carbons arising from the same precursor

(Kaneda, 1969; 1977).

The B. licheniformis reported in this chapter found to be inducing growth and

morphogenesis in U. fasciata (Described in chapter 3). The members of Ulvaceae lose their

typical foliose thallus morphology when cultured in axenic culture (Provasoli, 1958; Provasoli

& Pintner, 1980). This aberrant morphology could successfully been reversed to the foliose

thallus following the inoculation of appropriate morphogenesis-inducing bacterial isolates to the

culture media (Nakanishi et al., 1996; Tatewaki et al., 1983; Matsuo et al., 2003). Further,

Tatewaki et al. (1983) reported that the addition of culture filtrate of morphogenesis-inducing

bacterial isolates was capable of giving the same effect as that of the addition of bacterial culture

itself in Monostroma oxyspermum. But in the present chapter, there were varied responses for

bud induction from G. dura when the fronds were incubated with the different putative bud-

inducing bacterial isolates and their total fatty acid, total protein and fractionated protein. The

protein of putative bud-inducing B. pumilus was found to regenerate more buds than other two

putative bud-inducing bacterial isolates (B. licheniformis and E. homiense). Only the total

protein of the three putative bud-inducing bacterial isolates showed significant effect on the bud

induction in G. dura. The column purified proteins did not have any significant effect on the

bud induction in G. dura which revealed that more than one protein is required for the bud

induction. Putative bud-inducing bacterial isolates induced the expression of more protein which

may be involved in the bud induction from G. dura (Fig. 4.6).

Several studies have reported that temperature (Pakker & Breeman, 1996) and salinity

(Bird & McLachan, 1986) are the most important factors in determining the growth of

Gracilaria spp. In the present study, G. dura showed various responses with regard to bud

induction according the changing environmental factors like temperature and salinity. The

combination of 25°C and 30‰ showed high bud induction in G. dura. Similarly, these

combinations were known to regenerate more growth in G. verrucosa and G. chorda (Choi et

al., 2006). It is not easy to show the effect of environmental factors like temperature and salinity

with all three putative bud-inducing bacterial isolates along with their total protein, hence the

Chapter-4


total protein of putative bud-inducing B. pumilus was chosen due to its highest activity in

inducing buds than the other two putative bud-inducing bacterial isolates.

Fig. 4.6. The 10% SDS-PAGE protein profile of G. dura in the presence of different bacterial

isolates along with control. M - Markers, Eh - E. homiense, Bp - B. pumilus, Bl - B.

licheniformis, C - control.

It is well established that auxin produced by some marine bacteria enhances

morphogenesis pattern in green seaweeds (Mooney & Van, 1986). Previous studies showed the

ability of bacteria living on Ulva spp. (formerly Enteromorpha) to convert tryptophan into IAA

(Fries, 1975). The present chapter unveiled the role of IAA and nitrogen fixation in the

physiology of G. dura. The production of phytohormone by plant-associated bacteria is an

important mechanism influencing plant growth and development (Sessitsch et al., 2004). IAA is

the most abundant member of the auxin family of phytohormone (Bartel, 1997). Many

beneficial bacteria synthesize IAA, predominantly by a tryptophan-dependent pathway, which

can stimulate host root cell proliferation and elongation (Patten & Glick, 2002). The first step of

the Trp-dependent indole-3-pyruvate pathway is catalyzed by a tryptophan aminotransferase

(Trp-AAT), which converts Trp into indole-3-pyruvate. Aminotransferases associated with IAA

production have been reported in a variety of plant-associated diazotrophs like Azospirillum,

Chapter-4


Enterobacter, Gluconacetobacter, Pseudomonas and Rhizobium spp. (Kittell et al., 1989; Koga

et al., 1994; Pedraza et al., 2004). Several isoforms of Trp-AATs have been isolated and each is

capable of using several amino acids as substrates and may function in multiple biosynthetic and

catabolic pathways (Kittell et al., 1989; Pedraza et al., 2004). The second step in the Trp-

dependent pathway is the decarboxylation of indole-3-pyruvate to indole-3-acetaldehyde, which

is further oxidized by indole-3-acetaldehyde dehydrogenase to IAA. The decarboxylation of

indole-3-pyruvate by indolepyruvate decarboxylase is the key step in the pathway (Koga, 1995)

and the enzyme indolepyruvate decarboxylase is regenerated in the presence of Trp (Koga et al.,

1994; Patten & Glick 2002). The production of the IAA from putative bud-inducing B. pumilus

has been confirmed with TOF LC-MS peak between 174-177 m/z and quantity produced by the

putative bud-inducing B. pumilus (445.5 μg ml-1

), B. licheniformis (335 μg ml-1

) and E.

homiense (184.1 μg ml-1

) was confirmed with HP-LC. The bacterium belonging to the

Roseobacter group was responsible for the gall formation in the red alga (Prionitis lanceolata)

due to overproduction of IAA (Ashen & Goff, 2000). Tobacco associated bacteria B. pumilus

SE34 was reported for the production of IAA which enhanced the root growth (Kang et al.,

2006). An endosymbiotic bacterium from the Agrobacterium and Rhizobium group, containing

the nifH gene encoding for nitrogenase involved in nitrogen fixation, was isolated from rhizoids

of the green alga, Caulerpa taxifolia (Chisholm et al., 1996). The three putative buds inducing

bacterial isolates were found to be positive for nitrogen fixation that may help the better growth

of G. dura in oligotrophic environment. These associations secure the supply of nitrogen to the

macroalgae and might be one of the reasons for the successful invasion of these noxious

macroalgae (like Caulerpa taxifolia or Codium fragile) into oligotrophic environments

(Chisholm et al., 1996). The nitrogen fixing Paenibacillus with cellulase activity produced 1.3

mM ammonia (Emitiazi et al., 2007) while the present study reports the highest production of

ammonia (12.51 mg ml-1

) by B. pumillus has indicated for more growth for buds of G. dura

(Fig. 4.2). The production of NH3 is very toxic so it is immediately converted into non toxic

form of amino acid which is the building block of protein and supply for other additional

purposes. It is suggested that these putative bud-inducing bacterial isolates are important for

nitrogen supply to the G. dura. The growth of the plankton could be favourable with the

associated nitrogen fixing bacteria because they fixing atmospheric nitrogen and provide

nitrogen supplement sources (Maruyama et al., 1988). In addition to nitrogen fixation, microbes

Chapter-4


play a role in the protection of macroalga against toxic heavy metals (Dimitrieva et al., 2006). In

conclusion, present finding suggested that the bud induction and growth of G. dura are due to

protein conjugated with IAA and nitrogen-fixation.

chapter-4shodhganga.inflibnet.ac.in/bitstream/10603/9191/9/09_chapter 4.pdfthe purity of the...

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