chapter 2 - Shodhganga

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CHAPTER 2 Isolation and characterization of biofilm-forming bacteria from Halodule pinifolia (Miki) Hartog and Syringodium isoetifolium (Asch.) Dandy blades

2.1. Introduction

Biofilms are formed on abiotic surfaces and also on living organisms such as aquatic plants and algae (Stanley and Lazazzera, 2004). Plant-associated bacteria interact with host tissue surfaces during pathogenesis, symbiosis and in commensal relationships. The surface properties of the plant tissue, nutrient and water availability and the proclivities of the colonizing bacteria strongly influence the resulting biofilm structure (Ramey et al., 2004). Biofilms on plants may have beneficial or detrimental effects to their hosts sometime they reduce the light availability of the plant by promoting biofouling especially if algae or other organisms embedded on plants, and they may sometime contain pathogens that evade plant cells. On the other hand, the biofilm may reduce grazing or reduce biofouling by producing secondary metabolites Some previous reports also showed that many members of the genus Pseudoalteromonas, on alga Ulva spp., produce multiple extracellular inhibitory compounds that target different classes of marine fouling organisms (Holmstrom et al., 2002). In the past many aquatic bacterial communities have been described on the basis of new methods in various habitats, giving evidence that most aquatic

53

bacteria occur through out the world in various abundances. Aquatic biofilm communities have been described on and in various substrates such as sediments, chlorophytes, diatoms, sponges, lakes, marine snows, stones, steel foils, ceramic tiles or propylene sheets in marine and fresh water. The studies describing biofilm communities are scarce especially on seagrasses. One pair of studies revealed broad microbial diversity associated with the seagrass Halophila stipulacea in the northern Gulf of Elat (Wang et al., 2007; Weidner et al., 1996), and another study identified epiphytic bacterial communities on three seagrass species from the East African coast (Uku et al., 2007). One recent study identified bacteria attached to roots of Zostera marina (Jensen et al., 2007), and another identified methanotrophic bacteria associated with aquatic plants growing in methanogenic sediments (Sorrell et al., 2002). The invention and introduction of molecular methods to microbial ecology has increased our knowledge of bacterial communities immensely over the last two decades (Head et al., 1998). A few studies using molecular techniques, describe the diversity of bacteria in plant-colonized sediments (Bagwell et al., 1998; Cifuentes et al., 2000, 2003), but very few studies describe the diversity of organisms attached to aquatic plant leaf surfaces. Therefore an investigation was made on the biofilm-forming bacteria on two seagrasses blades, Halodule pinifolia (Miki) Hartog and Syringodium isoetifolium (Asch) Dandy. The predominant bacterial colonies obtained from the biofilm were isolated and characterised by 16s rDNA sequence.

54

The two plant species of same habitat exhibited distinct morphological characteristic features. H. pinifolia have creeping, branched moniliferous pale brown roots upto 12 cm long. Shoots upto 40 cm long erect bearing 2–3 leaves at each branch, stems with short internodes. Scales linear–oblong, entire along margins, with many vertical discontinuous tannin dashes. Leaf sheath linear, convolute, entire along margins. Transparent, with numerous reddish-brown tannin streaks, persisting longer than leaf-blades. Lamina flat, linear, narrowed at base, 10–25 cm long 0.75–1.5 mm broad, entire along margins, midrib prominent, widened, lateral teeth minute, or absent. It is the most commonly distributed seagrass that grow luxuriantly in backwaters, estuaries bays and gulfs in sheltered localities. Syringodium isoetifolium have creeping, less branched, pale yellow rhizome and roots up to 8 cm long. Shoots upto 60 cm long bearing erect 2–3 leaves at each branch Internodes up to 7.5 cm long. Scales linear, oblong, entire along margins with discontinuous tannin dashes. Leaf sheath tubular, narrowed at base with numerous tannin dashes. Lamina terrate up to 40 cm long, fleshy, brittle green. It is the pure marine form and is not seen in backwaters and estuaries. Both these

seagrasses

belongs

to

the

(Ramamurthy et al., 1992).

55

same

family

Potamogetonaceae

2.2. Materials and Methods 2.2.1 Isolation and characterization of biofilm-forming bacteria from Halodule pinifolia and Syringodium isoetifolium blades The isolation and characterization of bacterial biofilm-forming bacteria present on both seagrasses blades which relies on basic microbiological techniques and molecular characterization of pure isolated strains. The bacterial strains were identified using colony characterization and predominant bacteria were identified and analysed phylogenetically by 16s rDNA method to know the phylogenetic position. 2.2.1.1 Seagrass collection Seagrass blades of Halodule pinifolia and Syringodium isoetifolium were collected from extensive shallow seagrass beds from the coast of Kanyakumari. To isolate the biofilm-forming bacteria, five replicate seagrass samples from each species consisting of 10–15 healthy seagrass blades were collected underwater in sterile 50 ml screw cap centrifuge tubes. The seagrass blades were mature green without obvious epiphytes and with no visible sign of infection or injury. These samples were immediately brought to the laboratory. To study and compare the biofilm-forming bacteria, healthy blades were rinsed with 25 mL of filtered seawater (pore size 0.2 µm) and cut into pieces onto Zobell marine agar medium. Replicates were maintained. All the plates were kept in an incubator.

56

After incubation at 37°C for 24 h, the colony forming units (CFU) were counted. The biofilm-forming bacteria were noticed as they grew away from the seagrass leaf tissue and out onto the agar surface. Representative colonies were isolated by repeated transfer on Zobell marine agar medium and examined (Jensen et al., 1998). The isolated bacterial colonies were characterized by morphological, and biochemical methods. The predominant bacteria found were antibiotically tested and further characterized by 16s rDNA sequence method.

2.2.1.2 Morphological characterization Morphological characterization of the colonies were documented using the standard microbiological approaches and based on morphologial appearance such as colony colour, motility, Gram staining, elevation, colony shape, cell shape, margin, etc. 2.2.1.3. Biochemical characterization The standard biochemical tests were carried out by the method of Collins et al. (2004), to identify the bacterial strain upto genus level. For characterization of bacteria, a loop full of isolated bacterial culture was inoculated into Zobell marine broth and incubated overnight and the test like catalase, methyl red (MR) test, Vogues Proskauer (VP) test, amylase, starch hydrolysis, oxidase, gelatinase, casaenase, acid/gas, indole, urease and growth of the bacterial isolate at different salt concentrations were tested. Bacterial isolates were identified using Bergey’s Manual of Systematic Bacteriology (2001 edition).

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2.2.1.4. Antibiotic analysis Various antibiotic discs were placed on Zobell marine agar containing the predominant bacterial strains. The inhibition zones were noticed after 24 h at 37°C. 2.2.1.5. Molecular characterization After morphological, microscopical, and biochemical characterization, the predominant strains were further characterized using molecular tools because it is difficult to identify the bacteria in laboratory by biochemical characterization alone. Hence, a molecular approach was carried out for the confirmation of the strain identity. The 16s rDNA gene of the isolates were sequenced (ABI 3100 sequencer and genotyper; Genei) after the DNA isolation and amplification. DNA sequence was identified with the available sequence in GenBank with the MEGA-1 program to identify their closest relatives based on sequence similarities. The steps involved to find the isolate was first the genomic DNA was isolated from the pure culture pellet. Using consensus primers, the 1.5-kb 16s rDNA fragment was amplified using Taq DNA polymerase. The PCR product was bi-directionally sequenced using the forward reverse and an internal primer. Sequence data were aligned and analysed for finding the closest homologues for the microbe. The sequences were then subjected to distance matrix based on nucleotide sequence homology using the Kimura-2 parameter. The last step for identification is the Phylogenetic Tree based on Nearest Neighbor joining method (MEGA-3.1), which identified the homology of the

58

organism. Phylogenetic analysis was realised by an alignment of sequence consensus of the 16s rDNA genes collected in an international database (GenBank). The results were then expressed in percentage of homology between the submitted sequence and the sequences resulting from the database.

59

2.3. Results 2.3.1. Characterization of biofilm-forming bacteria from Halodule pinifolia All biofilm-forming bacterial

samples were

morphologically and

biochemically observed. A total of four morphologically different bacterial isolates were obtained from H. pinifolia blades namely Staphylococcus, Pseudomonas, E. coli and Bacillus (Table 2.1). 2.3.1.1 Morphological characteristics The colonies isolated from H. pinifolia blades exhibited less diversity when compared to S. isoetifolium blades. Interestingly the sampling sites had a predominant

circular

colony

which

exhibited

very

aggressive

growth.

Microscopically rod shaped cells were most abundant in H. pinifolia blades. All the isolated colonies were motile forms. Gram staining revealed that rod shaped cells were both Gram-negative E. coli and Pseudomonas and Gram-positive Bacillus. The cocci forms were only Gram-positive Staphylococccus and they were noted in less numbers on H. pinifolia blades. The Gram-positive rod shaped bacteria were abundantly present. The morphological characters of the isolates are shown in Table.2.2. 2.3.1.2 Biochemical characteristics Various tests were made to reveal the genus of the bacterial strains. All the isolates showed catalase positive. Amylase tests were positive only for Bacillus. All isolates showed negative result for gelatinase test. Except Pseudomonas casaenase showed negative for all the isolates. Acid/gas tests were positive for all 60

isolates except Pseudomonas. Most of the Gram-positive rod bacterial isolates showed tolerance to salt concentration up to 5% (Table 2.3). The Bacillus colony was predominantly seen on Halodule pinifolia blades (Plate 2.1) and hence the bacteria Bacillus was further analysed by antibiotic test and 16s rDNA method. The bacteria Bacillus was tested for antibiotic susceptibility and found that it was highly susceptible for various antibiotics (Table 2.8). 2.3.1.3. Molecular characteristics Nucleotide homology and phylogenetic analysis revealed that the predominant strain associated with seagrass H. pinifolia blades belongs to Bacillus pumilus (Accession No HM006706). This strain was closely related to the genus Streptomyces species. (Accession No EU384279). The nearest homolog species was found to be Bacillus stratosphericus (Accession No AJ831841). The identified B. pumilus showed 96% homology with other B. pumilus strains such as St.PRE14, St.NM1C3 and St.CT3 (Table 2.4). The phylogenetic tree made in MEGA-3.1 software using Neighbor Joining method showed the closest relative of B. pumilus (Fig. 2.1). The distance matrix based on Nucleotide Sequence Homology (Using Kimura-2 Parameter) indicates the nucleotide similarity and distance identities between the studied B. pumilus and ten other closest homologous microbes (Table 2.5). The aligned sequence data of B. pumilus are also shown in Data 2.1.

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Table 2.1 The biofilm-forming bacteria isolated and characterized from seagrass blades

Biofilm-forming bacteria from H. pinifolia blades

Biofilm-forming bacteria from S. isoetifolium blades

Staphylococcus

Micrococcus

Pseudomonas

Staphylococcus

E. coli

Vibrio

Bacillus

Pseudomonas –

Bacillus

62

Morphology

Cocci

Rod

Rod

Rod

Gram stain

+

_

_

+

Staphylococcus

Pseudomonas

E. coli

Bacillus

Bacteria

+

+

+

_

Motility

Cream

Slightly convex

63

Cream

Cream

Cream

Colour

Convex

Convex

Flat

Elevation

Circular

Circular

Circular

Irregular

Colony shape

Name of the morphological tests

Entire

Uniform bacillary

Long rods

Undulate

Regular

Undulate

Round in tetrads

Short rod

Margin

Cell shape

Table 2.2. Morphological characterization of biofilm-forming bacteria on Halodule pinifolia seagrass blades

MR _ _ + +

Catalase + + + +

Staphylococcus

Pseudomonas

E. coli

Bacillus

Name of the bacteria

+

_

_

_

VP

+

_

_

_

64

_

_

_

_

Amylase Gelatinase

+

_

+

_

Oxidase

_

_

+

_

+

+

_

+

Casaenase Acid/Gas

Name of the biochemical tests

_

+

_

_

Indole

Table 2.3. Biochemical characterization of biofilm-forming bacteria on Halodule pinifolia blades

_

_

_

_

Urease

2%

2%

5%

6%

NaCl

Plate 2.1a Characteristics of Bacillus pumilus isolated from H. pinifolia in agar plate

Plate 2.1b Microscopic view of Bacillus pumilus isolated from H. pinifolia

Binocular microscope model CH20i (Olympus) at 40x magnification microimage projection system model MIPS (2mpx)

65

Table 2.4 Alignment view of B. pumilus (H4)

Fig 2.1 Phylogenetic tree of B. pumilus (H4)

66

Table 2.5 Nucleotide similarity and distance identities between B. pumilus (H4) and ten other closest homologous microbes

Data 2.1 Aligned sequence data of B. pumilus

67

2.3.2 Isolation of biofilm-forming bacteria from Syringodium isoetifolium blades A total of five morphologically different samples were detected around seagrass blades. They are Micrococcus, Staphylococcus, Vibrio, Pseudomonas and Bacillus (Table 2.1). 2.3.2.1 Morphological characteristics Morphological characteristic of the colonies showed a circular and irregular colony morphology and most of them are motile forms. Microscopically rod shaped cells were most abundant, followed by cocci shaped cells. Gram staining revealed that cocci form bacteria were Gram-positive Micrococcus and Staphylococcus and the rod form were Gram-negative Vibrio and Pseudomonas. The Gram-positive rod-shaped bacteria was found to be Bacillus. Micrococcus, Pseudomonas, Bacillus and Vibrio showed convex elevation. Staphylococcus showed flat elevation. All the isolates showed cream colour colonies (Table 2.6). 2.3.2.2. Biochemical characteristics The isolated colonies from S. isoetifolium blades were all catalase positive. Except Staphylococcus and Pseudomonas, all were MR positive and except Vibrio and Bacillus all were VP negative. The isolates Vibrio and Bacillus showed amylase positive. Vibrio and Pseudomonas showed casaenase positive. All isolates are gelatinase negative. Except Micrococcus and Pseudomonas all isolates gave positive results for acid/gas production. The Bacillus isolate showed growth only at very low

68

concentration of salt. Other isolated strains were highly resistant to salt concentration (Table 2.7). The predominant strains such as Vibrio and Bacillus (Plates 2.3 and 2.4) were antibioticallly tested. The Vibrio showed high resistance to Amoxillin, Ampicillin, Methicillin and Cefazolin (Table 2.8). 2.3.2.3. Molecular characteristics The predominant strains were further analysed by 16s rDNA sequence. The nucleotides homology and phylogenetic analysis of predominant bacteria was found to be Vibrio alginolyticus (Accession No HM045516). The nearest homologous species was found to be Vibrio parahaemolyticus St.CM11 (Accession No EU660325) which showed 97% homology (Table 2.9). The information about other close homologs for this bacterium was V. natriegens St.01/097, V. natriegens St. CM3 which showed 97% homology. The identified strain showed 98% and 97% homology with other V. alginolyticus strains such as St.YJ0666 and St.YJ06167B respectively (Table 2.9). The phylogenetic tree shows the closest relatives of V. alginolyticus (Fig 2.2). The aligned sequence data of V. alginolyticus is also shown in Data.2.2. The distance matrix based on Nucleotide Sequence Homology (Using Kimura-2 Parameter) indicates the nucleotide similarity and distance identities between the studied V. alginolyticus and ten other closest homologs microbe (Table 2.10). The nucleotides homology and phylogenetic analysis of other predominant bacteria was detected as Bacillus pumilus. The nearest homologous species was found to be Bacillus stratosphericus (Accession No. AJ831841).

69

The identified strain showed 96% homology with other B. pumilus strains such as St. CICCHLJQ74, St.J, St. PRE14 (Table 2. 11). The phylogenetic tree also shows the closest relatives of B. pumilus (Fig.2.3). The aligned sequence data of B. pumilus are also shown in Data 2.3. The distance matrix based on Nucleotide Sequence Homology (Using Kimura-2 Parameter) indicates the nucleotide similarity and distance identities between the studied B. pumilus and ten other closest homologous microbes (Table 2.12).

70

Morphology Cocci

Cocci

Rod

Rod

Rod

Gram stain +

+

_

_

+

Micrococcus

Staphylococcus

Vibrio

Pseudomonas

Bacillus

Bacteria

+

+

+

_

_

Motility

Cream

Slightly convex

71

Cream

Cream

Cream

Cream

Colour

Convex

Convex

Flat

Convex

Elevation

Circular

Circular

Circular

Irregular

Circular

Colony shape

Name of the morphological tests

Undulate

Round in tetrads

Long rods

Long rods

Undulate

Entire

Undulate

Entire

Round in tetrads

Curved rods

Margin

Cell shape

Table 2. 6 Morphological characterization of biofilm-forming bacteria on Syringodium isoetifolium seagrass blades

MR + _ +

_

+

Catalase

+

+

+

+

+

Micrococcus

Staphylococcus

Vibrio

Pseudomonas

Bacillus

Name of the bacteria

+

_

+

_

_

VP

+

_

+

_

_

Amylase

_

_

_

_

_

72

Gelatinase

+

+

+

_

+

Oxidase

_

+

+

_

_

Casaenase

Name of the biochemical tests

+

_

+

+

_

Acid/Gas

__

_

+

_

_

Indole

Table 2. 7 Biochemical characterization of biofilm-forming bacteria on Syringodium isoetifolium blades



_

_

_

_

Urease

2%

5%

9%

6%

4%

NaCl

Table 2.8 Antibiotic susceptibility of predominant bacteria of H. pinifolia and S. isoetifolium

No

Antibiotics

Bacillus

Vibrio

Bacillus

1

Amoxicillin

36mm

Nil

39mm

2

Ampicillin

30mm

Nil

28mm

3

Chloramphenicol

29mm

23mm

25mm

4

Neomycin

20mm

12mm

19mm

5

Gentamycin

21mm

11mm

17mm

6

Novobiocin

24mm

14mm

25mm

7

Methicillin

33mm

Nil

30mm

8

Oxytetracycline

20mm

9mm

15mm

9

Streptomycin

25mm

16mm

22mm

10

Bacitracin

11mm

1mm

8mm

11

Lincomycin

27mm

14mm

33mm

12

Cefozolin

33mm

Nil

30mm

73

Plate 2.2a Characteristics of Vibrio alginolyticus isolated from S. isoetifolium

Plate 2.2b Microscopic view of Vibrio alginolyticus isolated from S. isoetifolium

Binocular microscope model CH20i (Olympus) at 40x magnification microimage projection system model MIPS (2mpx)

74

Table 2.9 Alignment view of V. alginolyticus (S3)

Fig 2.2 Phylogenetic tree of V. alginolyticus (S3)

75

Table 2.10 Nucleotide similarity and distance identities between V. alginolyticus (S3) and ten other closest homologous microbes

Data 2.2 Aligned sequence data of V. alginolyticus

76

Plate 2.3a Characteristics of Bacillus pumilus isolated from S. isoetifolium

Plate 2.3b Microscopic view of Bacillus pumilus isolated from S. isoetifolium

Binocular microscope model CH20i (Olympus) at 40x magnification microimage projection system model MIPS (2mpx)

77

Table 2.11 Alignment view of B. pumilus (S5)

Fig 2.3 Phylogenetic tree of B. pumilus (S5)

78

Table 2.12 Nucleotide similarity and distance identities between B. pumilus (S5) and ten other closest homologous microbes

Data 2.3 Aligned sequence data of B. pumilus

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2.4. Discussion Plants and marine animals are constantly exposed to high concentration of microbes. These microbes in nature exist as epibionts on living organisms and non living surfaces. Plant leaves host complex assemblages of bacteria (Wigand and Stevenson, 1997), especially leaves of aquatic plants support very active bacterial communities that are thought to be influenced by plant primary production (Haglund et al., 2002; Tornblom and Sondergaard, 1999). Biofilms on plants may have beneficial effects for the plants, they can attract zoospores, enhance and restore growth form on plantlets and produce metabolically beneficial for the plant. (Joint et al., 2000; Marshall et al., 2006) or they may be phytopathogens. The bacteria that are known phytosymbionts or phytopathogens are classified into only four bacterial phyla, viz., the cyanobacteria, proteobacteria, firmicutes and actinobacteria. Various morphological biochemical and antibiotic test of the present study revealed that the biofilm-forming bacteria found on Halodule pinifolia were Staphylococcus, Pseudomonas, E. coli and Bacillus. The bacteria present in Syringodium isoetifolium were Micrococcus, Staphylococcus, Pseudomonas, Vibrio and Bacillus. The predominant bacteria present in the seagrass H. pinifolia was Bacillus pumilus. The predominant bacteria present in S. isoetifolium were Bacillus and Vibrio. The diversity of the bacterial biofilm on the two plant species was more or less similar and limited. The studied two seagrass samples were taken from the same site moreover both the plants grow side by side as continuous beds. The earlier studies revealed that the terrestrial plant associated microbial diversity

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was influenced more by soil type (Singh et al., 2007). Plot typology and local vegetation profile (Nunon et al., 2005). The similarily might also be because, the bacterial communities on the surface of aquatic plants might be influenced by the host plant and environmental factors (Hempel et al., 2008). The limited varieties of bacteria might be because the aquatic angiosperms produce antimicrobial agents, like zosteric acid, that may limit bacterial colonization on plant surfaces and allow only certain microbes to become established (Bushmann and Ailstock, 2006; Harrison, 1982; Jensen et al., 1998; Newby et al., 2006) Moreover, the biofilm bacteria itself have antimicrobial properties that defend other bacteria to get established (Holmstrom et al., 2002). The occurrences of bacteria Bacillus were predominant in these two seagrasses. Algam et al. (2005) also reported that Bacillus species are among the most common bacteria found to colonize seagrasses and it is likely that they could play a role in the biocontrol of the vascular plant pathogens. The members of Firmicutes especially Bacillus have been documented as plant-associated bacteria with the capability of producing secondary metabolites, antibiotics, phytohormones and toxins that are of great importance to the health, establishment and proliferation of the host plant (Bargabus et al., 2002; Mcirroy and Kloepper, 1995a; Mcspadden-Gardener, 2004). These bacteria can also contribute to abiotic stress

adaptation

by

production

of

indole

acetic

acid

in

pepper

(Sziderics et al., 2007) and serve as biocontrol agent against phytopathogenic fungi (Cho et al., 2007).

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Some studies revealed the vital role of Bacillus strains for stimulating plant defence mechanisms, and thereby providing enhanced health and fitness to its host plant. Toro and collaborators (1997) showed that Bacillus species can solubilize phosphorus and nitrogen for the plant bearing an extremely important asset to nutrient supply. The genus Bacillus is generally reported to be distributed widely across many terrestrial and aquatic habitats (Ivanova et al., 1999; Siefert et al., 2000) including marine sediments (Miranda et al., 2008). Dhanasekaran et al. (2009) also identified the biofilm-forming Bacillus species in the marine habitat. The sequence analysis of genes encoding 16s rDNA is currently the most promising approach for phylogenetic classification of bacteria. The phylogenetic analysis revealed that B. pumilus isolated from the blades of H. pinifolia and S. isoetifolium were same but belong to different strains. The nearest homologous species of B. pumilus bacteria was found to be Bacillus stratosphericus St.41KF2 (Accession No AJ831841) which showed 96% similarity. The nearest homologous genus was found to be Streptomyces species (Accession No EU384279). B. pumilus also showed 96% similarity with other B. pumilus St.PRE14, St.NMIC3 and St.CT3. The identified predominant Bacillus strain of S. isoetifolium showed 96% similarity with B. pumilus strains such as St.CICCHLJQ74, St.J and St.PRE14. The alignment sequence of B. pumilus strain of H. pinifolia showed 1554 base pair and that of S. isoetifolium showed 1511 bp (Data 2.2 and 2.3). The base pair sequence was also different between these two strains that indicates the difference in their strains. The two seagrass-associated B. pumilus were aerobic, motile, Gram-positive, rod-shaped bacteria. It is reported to be the second most 82

dominant species among aerobic spore-forming bacterium (La Duc et al., 2004), highly resistant to unfavourable conditions such as low or no nutrient availability, extreme dessication, gamma and UV radiation and chemical disinfection (Nicholson et al., 2000). Various reports on B. pumilus in terrestrial habitats were studied by a few researchers. It is reported to protect cotton roots against Fusarium oxysporum (Chen et al., 1995). It is used for alkaline protease production in environmental decontamination of dioxins and in the baking industry. Also it has been found that it has a growth-promoting activity by the secretion of high amount of physiologically active gibberllins (Gutierrez-Manero et al., 2008). It has also been documented as a biocontrol agent in agriculturally important crops such as tomato, reducing significantly whitefly crawlers, nymphs and pupae which threatened the plants (Kloepper et al., 2004). It is reported to be an epiphyte in plants like rice (Cao et al., 2001) and is documented to promote growth in Pinus species (Probanza et al., 2001). It induces systemic disease protection like curcubit wilt a disease in cucumber (Zehnder et al., 2001) late blight in tomato (Yan et al., 2003) and Cercospora leaf spot in sugar beets (Bargabus et al., 2002). This bacterium was known to produce a compound that has fungicidal activity aganst Mucoraceae and Aspergillus species (Bottone and Peluso, 2003). Bacillus pumilus have also been detected in marine environments (Ivanova et al., 1999). According to previous studies by Ivanova and colleagues (1992, 1999) the strain B. pumilus along with other Bacillus strains are the most

83

abundant among those associated with marine sponges, ascidians, soft corals and are present in sea water as well. All the strains studied were able to utilize a wide range of organic compounds were halotolerant and alkali-tolerant which reflects their great metabolic flexibility. On the whole the previously reported works suggest that the various activity of B. pumilus might be vital for the health of seagrasses assuming the roles similar to those reported earlier in terrestrial plants. These biofilm bacterial communities are generally more resistant to antibiotics or detergents than single cells due to the surrounding matrix (Stewart and Costerton, 2001; Burnmolle et al., 2006; Harrison et al., 2007), but the antibiotic tests revealed that Bacillus pumilus were more susceptible to various antibiotics. The current susceptibility tests only use agar media that encourage bacteria to grow more within a planktonic/quasi-sessile state than as a true biofilm phenotype (Clutterbuct et al., 2007) and also recent research has indicated that the results from the disc diffusion test are open to interpretive error and that is only useful as preliminary screening for susceptibility testing (Manoharan et al., 2003) and also the organism that is sensitive in vitro may not be effective in vivo condition. The other bacterium isolated from Syringodium isoetifolium was Vibrio alginolyticus that was not present in H. pinifolia blades. This bacterium possesses a strong adhesive power to both biotic and abiotic inert surfaces. These properties allow persisting in planktonic state or attached to both biotic and abiotic surfaces (Snoussi et al., 2008). The genus Vibrio includes more than 30 species, atleast 12 of which are pathogenic to humans and or have been associated with food borne diseases (Chakraborty et al., 1979). They are the natural inhabitants of aquatic 84

environments and form symbiotic or pathogenic relationships with eukaryotic hosts. Previously the abundantly seen Vibrio associated with seagrass Posidonia oceanica were reported by Marco-Noales et al. (2006) and they reported some species of Vibrio causes diseases in aquatic angiosperm. Wahbeh and Mahasenah (1984) studied the heterotrophic bacteria associated with leaves, rhizomes and roots of three seagrasses and they found that Vibrio was reported to occur on H. uninervis. Vibrio spp. are more resistant to dessication, predation and toxic chemicals (Ophir and Gutnik, 1994) Recent studies revealed the ability of these organisms to form biofilms depends upon specific structural genes (flagella, pili and exopolysaccharide biosynthesis and regulatory processes (two-component regulatory, quorum sensing and c-di-GMP signalling) (Yildiz and Visick, 2009). Data from previous taxonomic studies on Vibrio have suggested that most strains are saprophytic in marine environment and are associated with such metabolic functions as mineralization of organic compounds. Marine organisms like Vibrio proteolyticus was found to involve in the biofilm process (Paul and Jeffrey, 1985). Adherence of pathogenic Vibrio sp. to biotic surfaces could elucidate the pathogenesis of this bacteria in the host. Some bacteria belonging to Vibrio natriegens which are predominantly present in estuarine and marine environment can able to fix nitrogen (West et al., 1985). Islam et al. (1989) found that toxigenic V. cholerae had a greater tendency to attach to the green alga Rhizoclonium fontanum, aquatic angiosperm

85

Elodea canadensis and found that prolonged survival of this bacteria to R. fontanum that indicate the ability to derive nutrients from the extracellular products released by this species. V. alginolyticus from tin panel-associated biofilm was reported by Bhosle et al. (1995). The adhesion of Vibrio on S. isoetifolium blades alone suggests that the distribution patterm of the bacterial genera may be related partly to inhibiting substance such as flavones, phenolic acids and tannins and partly to difference in quality and quantity of soluble organic exudates released by plants. (Kurilinko et al., 2007). Further Islam et al. (1990) ascertained that two toxigenic strains of Vibrio survived for longer periods when they are attached to freshwater macrophytes Lemna minor than when they were suspended in water. They suggested that aquatic plants could be the environmental reservoirs of these microbes through either a non-specific association or commensal relationship. Borroto (1997) isolated the toxigenic V. cholerae from the roots of Eichhornia crassipes and he observed that adhesion of the microorganism to the plant roots favours its survival. Bagwell et al. (2006) analysed the diazotrophic microbial community from various seagrass beds and he found that seagrass roots yielded a collection of organisms identical to Vibrio diazotropicus. The present identified Vibrio alginolyticus isolated from seagrass blades of S. isoetifolium was Gram-negative curved rods which are able to hydrolyse starch, sugar and indole producer that can withstand high salinity (up to 9%). The Vibrio that associated with Posidonia oceanica showed greater enzymatic potential (Marco-Noales et al., 2006). Various susceptibility tests revealed that

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Vibrio alginolyticus was resistant to methicillin, amoxicillin, ampicillin and cefozoline. Other bacterial species present in both the seagrasses were Pseudomonas, Micrococcus, Staphylococcus and E. coli. Presence of Pseudomonas, Micrococcus and Staphylococcus on seagrass leaves were already reported by various researchers. The bacterial community associated with leaves of marine seagrass Halophila stipulacea in the northern Gulf of Elat recorded Pseudomonas species (Weidner et al., 2000). Presence of Pseudomonas in biofilm on both abiotic and biotic surfaces was reported by Parsek and Fuqua (2004). Pseudomonas species has been extensively researched for their strong relationship with plants like production of phytohormones, aiding in nutrient intake, solubilization of phosphate, production of siderophores and fixing nitrogen (Garbeva et al., 2001; Hinton and Bacon, 1995; Kuklinsley-Sobral et al., 2004). The proteobacteria Pseudomonas aeruginosa was demonstrated to promote seedling emergence, root length, shoot length, dry weight and pod yield of groundnut in the field (Kishore et al., 2005) and has broad-spectrum antifungal activity. Presence of Micrococcus in Halodule uninervis and Staphylococcus in Halophila ovalis were also reported by Wahbeh and Mahasneh (1984). The Micrococcus which belongs to Actinobacteria have been well characterized due to their ability of producing secondary metabolites which are of economic and medical importance, such as antibiotics (Kieser et al., 2000). Some bacteria related to Micrococcus luteus has been previously reported in association to lettuce

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promoting root growth by secretion of indole acetic acid (Barazani and Friedman, 1999). The members of the genus Staphylococcus that comes under Firmicutes are ubiquitous and diverse and have been isolated from soil, plants, animals and humans. This species has been reported as endophytes in carrot (Daucas carota L. var. sativa), sweet corn, cotton and sweet pepper (Mclnroy and Kloepper, 1995b; Surette et al., 2003; Rasche et al., 2006). The presence of bacteria Escherichia coli on H. pinifolia showed the polluted nature of seagrass beds because E. coli are typically

used

as

faecal

pollution

indicator

(Bordallo

et

al.,

2002;

Kinzelman et al., 2003). The seagrass H. pinifolia occurs along the shoreline and the proximity to the shore makes it susceptible to input variety of contaminants and in consequence increases the opportunity of colonizing this bacteria. The presence of E. coli on these seagrass suggests that they can naturally occur in the marine water and related ecosystem. Further studies indicate that in temperate and subtropical coastal areas affected by a large tidal range E. coli can survive and seen to replicate on dessicated and rewetted sediments (Desmarais et al., 2002; Hartel et al., 2004). There are also reports that indicate that this bacterium are capable of surviving on the phyllosphere of fresh water and sun dried algae (Whitman et al., 2003, 2005; Ott et al., 2001; Muller et al., 2001). The exact knowledge of the occurrence of E. coli bacteria on seagrass was not known but in terrestrial habitat E. coli have the ability to colonize corn, bean, and cilantro plants under humid conditions, ability to lower population levels than

88

those of common bacterial epiphytes (Brandl et al., 2001; O'Brien and Lindow, 1989). As recent research on several aquatic angiosperms demonstrated that a number of different microbial metabolic processes are elevated on plant surfaces

including

sulphide

oxidation

(Lee,

1999),

methane

oxidation

(Sorrell et al., 2002), iron reduction (King and Garey, 1999), sulphate reduction and nitrogen fixation (Nielsen et al., 2001) all of which are influenced by plant activity and some of which are arguably beneficial to the plant. Thus most plants form intimate relationships with microorganisms, ranging from mutualistic to parasitic. Some researchers suggest that the success of plant–microbe interactions is significantly influenced by the formation of bacterial attachment mechanisms of the bacteria within plant tissues such as presence of adhesions, secretion of exopolysaccarides and surface proteins with initial contact often mediated by active motility (Danhorn and Fuqua, 2007), some of which produce compounds that promote plant growth (Dey et al., 2004; Hornschuh et al., 2006; Ryu et al., 2006), including the plant growth-regulating auxin, indole-3acetic acid, a compound involved in plant development (Koch and Durako, 1991; Lindow and Brandl, 2003). The diazotrophic bacteria were usually found to grow on both the roots and the leaves of aquatic plants and potentially provide as much as 50% of the nitrogen demand of the plant (Capone, 1983). According to Beattie (2002) most of the plant associated bacteria are commensals that have no detectable effect on plant growth or physiology; these are found primarily on plant surfaces.

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In general, bacteria in biofilm physically interact with plants in diverse ways and these bacteria have characteristic saturation levels, nutrient availabilities, and surface chemistries, season and climatic conditions all of which strongly influence the composition as well as type of bacteria in biofilm. The diversity of biofilm-forming bacterial communities on these two seagrasses has indicated interesting research information about the microbial community. The data obtained for this study will be a baseline data to understand the seagrass associated ecosystem.

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chapter 2 - Shodhganga

CHAPTER 2 Isolation and characterization of biofilm-forming bacteria from Halodule pinifolia (Miki) Hartog and Syringodium isoetifolium (Asch.) Dandy ...

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