Distribution Pattern Analysis of Epiphytic Bacteria On

Journal of Microscopy and Ultrastructure 2 (2014) 34–40

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Journal of Microscopy and Ultrastructure

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Original Article

Distribution pattern analysis of epiphytic bacteria on

ethnomedicinal plant surfaces: A micrographical and

molecular approach

∗

Fenella Mary War Nongkhlaw, S.R. Joshi

Microbiology Laboratory, Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong 793022, India

a r a

t i c l e i n f o b s t r a c t

Article history: The epiphytic bacterial community prevalent on ethnomedicinal plant surfaces were stud-

Received 29 November 2013

ied for their diversity, niche localization and colonization using the micrographical and

Received in revised form 4 February 2014

molecular approaches. Scanning electron microscopy (SEM) revealed the presence of large

Accepted 10 February 2014

aggregates of bacterial communities. The bacterial localization was observed in the grooves

Available online 5 March 2014

along the veins, stomata and near the trichomes of leaves and along the root hairs. A total of

20 cultivable epiphytes were characterized which were analyzed for richness, evenness and

Keywords: diversity indices. Species belonging to the genera Bacillus and Pseudomonas were the most

Plant surfaces abundant. Bacillus thuringiensis was the most prevalent epiphyte with the ability to form

Epiphytic bacteria bioﬁlm, as a mode of adaptation to environmental stresses. Bioﬁlm formation explains the

Microscopy potential importance of cooperative interactions of epiphytes among both homogeneous

Bioﬁlm

and heterogeneous populations observed under SEM and inﬂuencing the development of

microbial communities. The study has revealed a deﬁnite pattern in the diversity of cultur-

able epiphytic bacteria, host-dependent colonization, microhabitat localization and bioﬁlm

formation which play a signiﬁcant role in plant–microbe interaction.

1. Introduction moisture, humidity, temperature, wind speed, radiation

and rainfall may inﬂuence epiphyte diversity [33]. Exploita-

Plants maintain a complex system where bacterial tion of ethnomedicinal plants for their natural products has

communities interact continuously supporting the devel- resulted in loss of epiphytic diversity harboured on novel

opment of large microbial population. Epiphytic bacteria ethnomedicinal plants. The purpose of this investigation is

are capable of living (i.e., multiplying) on plant surfaces [1] to determine the sites and extent of growth of epiphytic

and their colonization is controlled by biological factors microﬂora on selected ethnomedicinal plants on the leaf

such as the host plant growth and the life cycle of epi- and root surfaces and to characterize the diversity of cultur-

phytes [2,3]. Epiphytes occupy a narrow ecological niche able epiphytic bacteria associated with these plants using

because of their existence at the interface of vegetation and microscopic (SEM) and molecular techniques.

atmosphere, a variation in climatic conditions including Epiphytic bacterial aggregates on plant surfaces may

vary in size and composition depending on nutrient

availability at a given site [4,5]. These aggregates have

∗ characteristics similar to those cells in bioﬁlms that are

Corresponding author. Tel.: +91 943 610 2171; fax: +91 3642550076.

described in aquatic and medical environments [6]. Bioﬁlm

E-mail addresses: [email protected], [email protected]

(S.R. Joshi). consists of multilayered cell clusters embedded in a

http://dx.doi.org/10.1016/j.jmau.2014.02.003

F.M.W. Nongkhlaw, S.R. Joshi / Journal of Microscopy and Ultrastructure 2 (2014) 34–40 35

complex matrix comprising of a variety of extracellular and ﬁxed by immersion in 2% glutaraldehyde in 0.1 M

polymeric substances (EPS) [7] which facilitates the adher- cacodylate buffer (pH 7.2) for at least 1 h. The samples

ence of epiphytes to other microbial cells and to plant were drained and placed in three consecutive 1 h washes

surfaces. Plants incorporate epiphytes in the strategy to of 0.1 M cacodylate buffer. Samples were then stored in

survive under stressed conditions [8] and thus, it is more fresh cold cacodylate buffer for transport to the elec-

likely that the phytoepiphytes play an eminent role in plant tron microscopy laboratory. Samples were dehydrated in

defence and other environmental stresses. The knowledge a series of acetone–water washes for 15 min each and

of plant associated bacteria is important for the study of the critical-point dried with liquid CO2. Finally, samples were

effect of pathogens, symbionts and commensals on their sputter coated with a thin layer of gold–palladium and the

hosts. distribution of bacteria on the plants and their morphologi-

cal differentiation were observed at several magniﬁcations

under the scanning electron microscope.

2. Materials and methods

2.1. Plant sample collection 2.3. Isolation of epiphytic bacteria

Healthy plants used by different traditional medic- To analyze the epiphytic microbiota, plant samples were

inal practitioners (TMPs) were collected from different washed thoroughly with tap water followed by sterile

◦ ◦

parts of Meghalaya (N – 25 26.737 , E – 091 44.737 ; N – double distilled water [10]. Each plant sample was cut asep-

◦ ◦

25 36.904 , E – 091 54.121 ) based on their ethnomedicinal tically into 1 cm long segments using a sterile blade under

usages. Rubia cordifolia, Centella asiatica, Potentilla fulgens, the laminar ﬂow hood and allowed to dry. The cut surfaces

Acmella oleracea and Houttuynia cordata are used by the of plant segments were placed on Petri plates contain-

tribal people of Meghalaya, India as folk remedies for treat- ing Nutrient Agar (NA) media (Himedia, India). Each plant

ing a variety of ailments [9]. The taxonomic identity of the segment was inoculated in triplicate. Plates were then incu-

◦

plants was conﬁrmed with the help of Herbarium Curator bated at 32 C for 48 h. Colonies with different morphology

of the parent University. All samples were collected in ster- and pigmentation were randomly selected from each plate

ile polythene bags and brought to the laboratory and used and streaked on fresh NA plates as described above. Simul-

for isolation within 24 h of collection. taneously, the pure isolates were preserved in 20% glycerol

◦

at −20 C for further studies.

2.2. Sample preparation for scanning electron

microscopy 2.4. Molecular characterization of the isolates

The occurrence of bacteria on the surface of plants was Total genomic DNA was extracted using HiPurA bac-

observed using a scanning electron microscope (JSM-6360, terial and yeast genomic DNA Isolation Kits (Himedia,

Jeol). The plant material for observation was prepared India). PCR ampliﬁcation and sequencing of 16S rRNA

according to a modiﬁed version of Baker’s method. Fresh gene was carried out in a 25 ␮l reaction mixture. Using

plant fragments were cut into approximately 1 cm pieces general primers 27F 5 -AGAGTTTGATCCTGGCTGAG-3 and

Table 1

Epiphytic bacteria isolated from ethnomedicinal plants of Northeast India showing bioﬁlm production.

Host plants Epiphytes isolated Isolate name GenBank Accession no. % Similarity Bioﬁlm formation

Rubia cordifolia Citrobacter youngae ME5 JX390623 99.2 +

Bacillus thuringiensis MEA7 JN418875 100 +

Raoultella ornithinolytica ME11 JX390624 99.8 −

Enterobacter soli MEA10 JN680692 99.8 −

Centella asiatica Bacillus tequilensis CEN3E JN628288 99.9 −

Bacillus aryabhattai CEN5E JN628290 100 −

Bacillus thuringiensis CEN6E JN628291 100 +

Pantoea eucalypti CEN7E JN628292 99.3 +

Potentilla fulgens Bacillus thuringiensis POT1 JQ281538 99 +

Pseudomonas palleroniana POT2 JQ281539 99 +

Serratia nematodiphila POT3 JQ281540 99 +

Stenotrophomonas maltophilia POT5 JQ281541 99 +

Acmella oleracea Pseudomonas mosselii Y9 JQ446443 100 +

Pantoea eucalypti Y5 JQ446440 99.6 +

Pseudomonas putida Y6 JQ446441 99.5 +

Bacillus thuringiensis Y7 JQ446442 99.9 +

Houttuynia cordata Lysinibacillus xylanilyticus F1 JN418870 100 −

Bacillus thuringiensis F41 JX390622 99.9 +

Enterobacter asburiae F7 JN418868 100 −

Acinetobacter johnsonii F8 JN418869 100 −

+, Capable of bioﬁlm formation; −, incapable of bioﬁlm formation.

36 F.M.W. Nongkhlaw, S.R. Joshi / Journal of Microscopy and Ultrastructure 2 (2014) 34–40

Table 2

Statistical analysis of epiphytic bacterial diversity of studied ethnomedicinal plants.

Diversity indices Rubia cordifolia Centella asiatica Potentilla fulgens Acmella oleracea Houttuynia cordata

Simpson’s dominance (D) 0.25 0.625 0.25 0.375 0.25

Simpson’s diversity index 0.75 0.375 0.75 0.625 0.75

Shannon-Wiener index (H ) 1.386 0.5623 1.386 1.04 1.386

Species richness (S) 2 1 2 1.5 2

Evenness (E) 1 0.8774 1 0.9428 1

Fisher alpha (F␣) 0 1.592 0 5.453 0

1541R 5 -AAGGAGGTGATCCAGCCGCA-3 with the follow- Applied Biosystems, USA) with Big Dye (3.1) terminator

◦

ing conditions, template DNA denaturation at 94 C for protocol. Sequencing was performed using 20 ␮l reaction

◦

5 min followed by 30 cycles at 94 C for 1 min, anneal- mixture containing about 50 ng DNA template (PCR prod-

◦ ◦

ing at 55 C for 1 min, extension at 72 C for 2 min, ﬁnal uct), 1 pmol of sequencing primer, 3.5 ␮l (5×) big dye buffer

◦ ◦

step was carried out at 72 C for 5 min and then 4 C till and 1 ␮l big dye. This was carried out by denaturation

◦ ◦

inﬁnity using PCR Gene Amp 9700 (Applied Biosystems, step at 96 C for 5 min, followed by 30 cycles at 96 C for

◦ ◦

USA). DNA template replaced with sterile water was used 10 s, 55 C for 10 s and ﬁnal step at 96 C for 5 min. Post

as negative control. The ampliﬁed (approx. 1000 bp) 16S reaction cleanup was carried out to remove unwanted mat-

rRNA gene was then puriﬁed using QIAquick Gel Extraction ters including unincorporated dye terminators by using

Spin Kit (QIAGEN, Germany). The puriﬁed PCR products 125 mM EDTA, 3 M sodium acetate and 70% ethanol and

were bi-directionally sequenced using both forward and then air dried at room temperature for 45 min before

◦

reverse primers in a sequencer Genetic Analyzer (ABI 3130 keeping at −20 C. 10 ␮l formamide was added and then

Fig. 1. Rooted Neighbour-Joining Tree of epiphytic bacteria based on 16S rRNA gene sequences (the numbers at the nodes are bootstrap values based on

1000 replications).

F.M.W. Nongkhlaw, S.R. Joshi / Journal of Microscopy and Ultrastructure 2 (2014) 34–40 37

Fig. 2. Micrograph showing bacterial epiphyte colonization on the ventral side of the leaf surface of Houttuynia cordata. Arrows indicate epiphytes around

the stoma.

◦

denaturation step was carried out at 96 C for 2 min then published prokaryotic names at the EzTaxon 2.1 server

immediately kept in ice before loading into the sequencer [11]. Molecular Evolutionary Genetics Analysis software

plate. Sequencing was performed using Genetic Analyzer (MEGA version 4.0) [12] was used for phylogenetic anal-

ABI 3130XL (Applied Biosystems, California, USA). yses. The sequences of identiﬁed phylogenetic neighbours

were aligned with the sequences of representative strains,

2.5. Phylogenetic analyses using Clustal W inbuilt with MEGA 4. Neighbour-Joining

method was employed to construct the phylogenetic tree

Sequence similarities were determined by the BLAST with 1000 bootstrap replications to assess nodal support in

program against the database of type strains with validly the tree.

Fig. 3. Bacterial distribution on the epidermal cell grooves along the veins of Acmella oleracea. Arrows indicate epiphyte colonization.

38 F.M.W. Nongkhlaw, S.R. Joshi / Journal of Microscopy and Ultrastructure 2 (2014) 34–40

Fig. 4. Bioﬁlm formation on leaf surface of Centella asiatica.

2.6. Detection of bioﬁlm formation using Congo red Agar different epiphytes were characterized from ﬁve different

method (CRA) ethnomedicinal plants with Bacillus thuringiensis common

to all tested plants (Table 1). The 16S rDNA nucleotide par-

Bioﬁlm formation was screened using solid medium of tial sequences were submitted to GenBank and accessions

brain heart infusion (BHI) supplemented with 5% sucrose were obtained (Table 1). In comparison with diverse micro-

and Congo red [13]. The medium was composed of BHI bial environments such as soil habitats, the phyllosphere

−1 −1 −1

(37 g L ), sucrose (50 g L ), agar no. 1 (10 g L ) and and rhizosphere represents an environment of reduced

−1

Congo red stain (0.8 g L ). Congo red was prepared as bacterial complexity [19]. The inability of most bacte-

◦

concentrated aqueous solution and autoclaved at 121 C ria to grow in laboratory conditions [20] has caused few

for 15 min, separately from other medium constituents bacterial phyla to deﬁne the phylogenetic structure of epi-

◦

and was then added when the agar had cooled to 55 C. phytic community (Fig. 1). Phylogenetic analysis revealed

Plates were inoculated and incubated aerobically for 24 Bacillus spp. as the most dominant epiphyte followed by

◦

to 48 h at 37 C. Strong bioﬁlm producers are indicated Pseudomonas spp. (Fig. 1). Statistical analysis of epiphytic

by black colonies with dry crystalline consistency. Moder- bacterial diversity of studied ethnomedicinal plants has

ate result is indicated by pink colonies, though occasional revealed similarity in diversity of epiphytes associated with

darkening at the centre, whereas, white colonies indicate the plants Rubia cordifolia, Potentilla fulgens and Houttuynia

negative result. The experiment was performed in tripli- cordata (Table 2). This may be attributed to the different

cate. genera of all the isolates obtained from these plants, unlike

the epiphytes associated with Centella asiatica and Acmella

2.7. Culturable bacterial diversity data analysis oleracea where, several Bacillus spp. and Pseudomonas spp.

were isolated (Table 1).

The diversity of epiphytic bacterial community on each Micrographical observations revealed epiphytic bac-

ethnomedicinal plant under study was evaluated using terial interaction with the host plant through surface

Simpson’s index of dominance (D) [14], Simpson’s diver- colonization, in which the microbes were seen adhered to

sity index (1-D), Shannon–Wiener’s (H ) method [15]. the external plant tissue as individual cells or clusters. SEM

Bacterial alpha diversity was estimated with Fisher’s recovered higher density of epiphytic bacteria from the

alpha (F␣) [16]. Species richness (S) and evenness (E) ventral surface of the leaf (Fig. 2) compared to the dorsal

of species was evaluated using the formula of Pielou surface which corroborates to reports of Lalke-Porczyk and

[17]. Donderski [21]. This may be due to comparatively lower

environmental stress such as, reduced radiation, rainfall

3. Results and discussion and higher stomatal openings on the ventral side of the

leaves. Homogeneous as well as heterogeneous popula-

Culturable technique has revealed the diversity and tions were observed using SEM among the phyllobacteria

host dependence of epiphytic bacteria [18] that are capa- and rhizobacteria of ethnomedicinal plants (Fig. 3). Micro-

ble of bioﬁlm formation (Table 1). Epiphytic bacterial scopic technique revealed the patchy recovery of bacteria

population differed largely among plant species, ﬁfteen from leaves in leaf imprint studies which suggests that

F.M.W. Nongkhlaw, S.R. Joshi / Journal of Microscopy and Ultrastructure 2 (2014) 34–40 39

Fig. 5. Bioﬁlm formation on the root of Potentilla fulgens.

bacteria do not occur in a uniform pattern across leaf sur- 4. Conclusion

faces but tend to be localized in particular sites [22–24].

The most frequent sites of bacterial colonization were the Epiphytic bacteria physically interact with plants

stomata (Fig. 2), epidermal cell grooves along the veins through surface colonization, in which they adhere to

(Fig. 3), at the base of trichomes and root hairs [25,26]. external plant surfaces as individual cells, in clusters or

These observations suggest a relatively scattered coloniza- bioﬁlm. In the present study, culturable and molecular

tion and variation in population sizes, embedded in an technique has characterized 15 different epiphytic bacte-

exopolymeric matrix as reported on leaf surfaces of dif- rial isolates from selected ethnomedicinal plants out of

ferent plant species [27]. which Bacillus spp. was recovered as the most dominant

Epiphytes colonized plant surfaces to form dense epiphyte. Remarkable differences in bacterial coloniza-

biofilm (Figs. 4 and 5). CRA method used for testing biofilm tion and sites of growth of the epiphytic microflora on

formation revealed moderate bioﬁlm formation for some ethnomedicinal plants were revealed using SEM. Bioﬁlm

epiphytes, with the formation of pink colonies (SFig. 1). formation on plant surfaces were observed as a mode of

Out of twenty characterized culturable epiphytic bacteria, adaptation of epiphytes to environmental stresses. The

thirteen isolates showed the ability for bioﬁlm formation present study of plant-associated epiphytes is important

(Table 1). Micrographical and molecular approach recov- not only for understanding the ecological role of such

ered B. thuringiensis as the most prevalent epiphyte capable bacteria in their interaction with plants but also for the

of forming bioﬁlm on plant surfaces which has also been biotechnological application of these bacteria to areas such

reported as a biocontrol agent [28]. Leaf and root micro- as the plant growth promotion. Future efforts could be

biota are reported to provide plant growth-promoting and considered on how environmental stress affects epiphyte

protection against pathogens [29]. Microbial cells within diversity on plant surfaces. Micrographical imaging may

bioﬁlms have better chances of adaptation and survival due provide evidence on changes in colonization pattern, if any,

to the protection from the bioﬁlm matrix [30]. Considerable resulted by the environmental changes.

evidence has indicated bacteria to modify their environ-

ment, such as by increasing nutrient concentrations to

enhance their colonization on plant surfaces [2,31]. This Conﬂict of interest

habitat modiﬁcation may be augmented by cooperative

interactions among bacteria which may occur among both None declared.

homogeneous and heterogeneous populations. Bacterial

interaction is mediated by quorum-sensing in which bacte-

ria sense the presence of neighbouring cells by detecting an Acknowledgements

increase in concentration of constitutively produced extra-

cellular molecules, N-acyl homoserine lactones (HSL) [32]. We acknowledge UGC, Govt of India for ﬁnancial assis-

Supplementary material related to this article can tance in the form of fellowship and DBT, Govt of India for

be found, in the online version, at doi:10.1016/j.jmau. the research project. We thank SAIF, NEHU for assistance

2014.02.003. with scanning electron microscopy.

40 F.M.W. Nongkhlaw, S.R. Joshi / Journal of Microscopy and Ultrastructure 2 (2014) 34–40

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