AP PLI CA TION OF PCR FIN GER PRINT ING MO LEC U LAR TECH NIQUES FOR DIS CRIM I -

NA TION OF AZOTOBACTER ISO LATES ISO LATED FROM VAR I OUS AGRO CLI MA TIC

ZONES OF TAMILNADU

Pasupuleti Reddy Priya*, Ganesan Gopalaswamy and Dananjeyan BalachandarDe part ment of Ag ri cul tural Mi cro bi ol ogy, Tamil Nadu Ag ri cul tural Uni ver sity, Coimbatore 641003, In dia.

* Cor re spond ing au thor

P. Reddypriya, De part ment of Ag ri cul tural Mi cro bi ol ogy, Tamil Nadu Ag ri cul tural Uni ver sity, Coimbatore 641003, In dia.

E-mail ad dress: reddypriyap@ya hoo.com

ABSTRACT

A comparison of different molecular typing methods viz., ERIC PCR, BOX PCR and ARDRA was carried out to analyzetheir discriminatory power and suitability for assessing diversity of selected Azotobacter isolates. Among the threemethods, ERIC PCR showed high polymorphism of about 94.11% followed by BOX PCR and ARDRA i.e., 88.2% and64.7% respectively. Clustering of isolates based on the ERIC, BOX and ARDRA pattern clearly showed the superiority of the former two methods to reveal the intra generic and intra specific diversity. This study clearly shows that typingmethods exploiting the repetitive elements distributed over the genome are more useful for assessing genetic diversity.

Azotobacter spp. are Gram-negative, soil-dwelling, free

living aerobic, polymorphic bacteria, i.e., they may be of

different sizes and shapes. Old populations contain

encapsulated forms that are resistant to heat, desiccation

and other adverse conditions. Their cysts germinate under

favorable conditions to give vegetative cells. One of the

most interesting feature of Azotobacter spp. is that they

have a beneficial effect on the growth of many plant

species due to their ability to fix atmospheric nitrogen by

converting it to ammonia, producing plant growth

promoting substances such as gibberelins, auxins and

cytokinins, vitamins or siderophores. They are capable of

fixing an average of 20 kg N/ha/year and were shown to be

antagonistic to plant pathogens. These properties make

Azotobacter to be interesting research object and

important part of the soil bacteria community, as they are

broadly dispersed in different environments, such as soil,

water and sediments (1).

16S rDNA sequencing technology with the

availability of huge number of reference sequences in

public domain databases (like NCBI, DDBJ, EMBL, RDP

etc.) have provided an excellent opportunity for

identification and characterization of microorganisms at

species and subspecies levels (2). Restriction patterns of

16S ribosomal DNA have also been exploited as a rapid

identification tool for different bacteria. PCR based

techniques using repetitive elementslike BOX, REP,

(GTG)5 and ERIC (Enterobacterial Repetitive Intergenic

Consensus) have recently been used extensively for

genetic characterization of both gram_positive and

gram_negative bacteria and population genetic studies.

The repetitive extragenic palindromic (REP) elements are

palindromic units containing a variable loop in stem_loop

structure (3). ERIC sequences are characterized by

central, conserved palindromic structures (4) while BOX

elements consist of differentially conserved subunits,

namely boxA, boxB, and boxC (5) of which only the

boxA_like subunit sequences are highly conserved

among diverse bacteria (6). Methods based on such

repetitive elements have also been used for studying the

diversity in the ecosystem, presenting the phylogenetic

relationship between strains and discriminating between

microorganisms those are genetically close to each other

(7).

In this study, we compared three different typing

methods based on ERIC, BOX element, restriction

profiling of 16S rDNA to assess the genetic diversity of

seventeen strains of different Azotobacter species and to

evaluate their discriminatory power for the analysis of

diversity.

MATERIALS AND METHODS

Sample collection and isolation of Azotobacter

isolates : The soil samples were collected from different

rice growing places such as Paramakudi, Madurai,

Ramnad and different regions of Kanyakumari and Nilgiri

districts for the Azotobacter isolates by the dilution plating

method on Waksman No 77 medium. Selected isolates

were purified on Waksman No 77 medium by streak plate

method and allowed to grow at 35°C for 48 h. Stock

cultures were made in Waksman No 77 broth containing

60% (w/v) glycerol and stored at -20°C.

Growth and maintenance : Cultures were revived by

streaking on Waksman No 77 medium plates and

incubating at 35 ± °C temperature for 48 hours. Bacterial

cultures were subcultured on Waksman No 77 medium

slants for further studies. For DNA extraction and

biochemical property tests, inoculum was prepared by

Progressive Research – An International Journal Society for Scientific Development Print ISSN : 0973-6417, Online ISSN : 2454-6003 in Agriculture and Technology Volume 10 (Special-II) : 823-827, (2015) Meerut (U.P.) INDIA

growing the cultures in 5 ml Waksman No 77 broth in

screw capped tubes and incubating over for 48 h at 35 ±

2°C .

Biochemical property tests : Gram staining was

performed on the Azotobacter isolates as a preliminary

testing. Several biochemical tests were performed

including the Catalase test, Motility test, Starch hydrolysis

test, Hydrogen sulphide production, Indole formation test

and Oxidasetest.

DNA extraction and quantification : Total genomic DNA

was extracted from 15 Bacillus isolates which was

confirmed as they belong to Azotobacter genus by

biochemical property tests and also from two standard

strains viz., Azotobacter chrococcum (Ac1), a commercial

bioinoculant strains from Tamilnadu Agriculture university

(TNAU). Genomic DNA extraction were performed using

the standard protocol of hexadecyl- trimethyl ammonium

bromide (CTAB) method given by (8). Quantification and

purity check of DNA samples were done spectro-

photometrically by Nano drop instrument. The extracted

pure DNA was stored at -200C for further use .

Amplified ribosomal DNA restriction analysis

(ARDRA) : (a) Amplification of 16S rRNA gene: universal

primer pairs viz., FD1 (5’-AGAGTTTGATCCTGGCT

CAG-3’) and RP2 (5’-ACGGCTACCTTGTTACCACTT-3’)

reported by (9) were used to amplify the 16S rRNA gene.

The 30 µl PCR reaction mixture contains DNA template

50 ng, 10x Taq buffer, 2.5mM of each of deoxyribo-

nucleotide triphosphate (dNTP) mixture, 2.5 mM of

MgCl2, 20 picomole of each primer, and 1 U of Taq DNA

polymerase (all from Bangalore Genei, India). 10 µL of

amplified products along with molecular weight marker

(Step Up 1 kb ladder, Bangalore Genei, India) were

electropohresed on 1.5% agarose gel (Sigma) at 80 volts

for 1.5 hours. Then gel was stained with ethidium bromide

(0.5 µg/ml) solution for 1 min and destained in water for 30

min. Amplified products were visualized under UV light

and gel photograph was documented using Alpha Imager

documentation and analysis system .

(b) Restriction digestion of amplified 16S rRNA gene

products: restriction enzymes HaeIII were procured from

Promega (USA). Restriction digestions were performed in

25 µl reactions following the manufacturer’s instructions.

10 µl of digested products along with molecular weight

marker (Step Up 100 bp ladder, Bangalore Genei, India)

were run on 2.0% agarose gel (Sigma). Gel staining with

ethidium bromide, visualization of bands and

documentation were carried out as described in the

previous sections.

BOX PCR : BOX A1R primer (5’-CTACGGCAAGGCG

ACGCCTGACG-3’) was used for this purpose (10).

Amplification reactions were performed in a reaction

volume of 30.0 µl containing 3.0 µl 10X PCR buffer with

MgCl2, 1.5 µl, 25 mM dNTP mixture, 1.2 µl of BOX A1R

primer (10 pM), 1 units Taq DNA polymerase, 2 µl

template DNA and 17.65 µl sterile distilled deionised

water. Thermal cycling was achieved in Eppendorf

Mastercycler, Germany, thermal cycler with the conditions

824 Pasupuleti Reddy Priya et al.,

Table-1 : List of Azotobacter isolates used for the study.

S.No.

Isolates Area of sampling (paddy crop)

1 Ac1 Standard strain, Bioinoculant TNAU

Azotobacter chrococcum

2 MTCC

2460

Azotobacter vinelandii, a Standard strain

(MTCC, Chandigarh)

3 Azt1 Paramakudi, Dryland

4 Azt2 Paramakudi, Dryland

5 Azt3 Paramakudi, Dryland

6 Azt4 Madurai Wetland

7 Azt5 Vadugapatti, Wetland

8 Azt6 Madurai, Wetland

9 Azt7 Ramanathapuram, Dryland

10 Azt8 Ramanathapuram, Dryland

11 Azt9 Palliyadi, Kanyakumari District, Wetland

12 Azt10 Puthaeri, Kanyakumari District ,Wetland

13 Azt11 Puthaeri, Kanyakumari District, Wetland

14 Azt12 Parvathipuram, Kanyakumari District,

Wetland

15 Azt13 Thirumogur, Madurai District, Wetland

16 Azt14 Thirumogur, Madurai District, Wetland

17 Azt15 Poothapandi, Kanyakumari District,

Wetland

Table-2 : Biochemical properties of Azotobacter isolates.

Isolate Catalase activity

Starch hydrol

ysis

H2Sproduction

Oxidase

Indole production

Ac1 + + + + -

MTCC2460

+ + + + -

Azt1 + + + + -

Azt2 + + + + -

Azt3 + + + + -

Azt4 + + + + -

Azt5 + + + + -

Azt6 + + + + -

Azt7 + + + + -

Azt8 + + + + -

Azt9 + + + + -

Azt10 + + + + -

Azt11 + + + + -

Azt12 + + + + -

Azt13 + + + + -

Azt14 + + + + -

Azt15 + + + + -

described by Rademaker and De Bruijn (7).PCR

conditions was given in the table.10 µl of amplified

products along with molecular weight marker (Step Up

100 bp ladder, Bangalore Genei, India) were

electropohresed on 2% agarose gel (Sigma) at 80 volts for

1.5 hours. Amplified products were visualized under UV

light and gel photograph was documented using gel

documentation unit as described earlier.

ERIC PCR : ERIC PCR was performed using specific

ERIC primers viz. ERIC 1R (5’-ATTAAGCTCCTGGG

GATTCAC-3’) and ERIC 2 (5’-AAGTAAGTGACTGGGGT

GAGCG-3’) (10). Amplification was performed in a

reaction volume of 30 µl containing 3.0 µl 10X PCR buffer

with MgCl2, 1.5 µl 25 mM dNTP mixture, 0.6 µl of each

primer (10 pM), 0.5 units Taq DNA polymerase, 2 µl

template DNA and 19.3 µl sterile distilled deionised water.

PCR conditions was given in the table. Electrophoresis,

gel staining with ethidium bromide, visualization of bands

and documentation were carried out as described in the

previous section.

Statistical analysis : Pair wise genetic similarities among

the isolates under study were determined using Jaccard’s

coefficient (11). Cluster analyses were carried out on

similarity estimates using the unweighted pair group

method with arithmetic average (UPGMA) using

NTSYSpc, version 2002(12).

RESULTS

A total of twenty-six isolates were obtained. For

preliminary identification, morphological traits (cell and

colony morphology), Gram reaction and pigment

production in the Waksman No 77 medium were

considered. Among them fifteen isolates were selected for

further studies remaining were discarded as they do not

belong the genus Azotobacter. The list of Azotobacter

isolates and its source was given in the Table1. Most

isolates presented whitish (cream color), smooth,

irregular, shining, 3-8mm diameter colonies; nevertheless,

colonies with transparent, glistening, shining, 2-5 mm

diameter also appeared. Two cell type morphologies were

identified: Gram-negative bacilli short and large and

Gram-negative bacilli short and small. Some showed a

brown pigmentation and others presented a dark brown

pigmentation; a single one displayed a yellowish green

pigmentation, which is a characteristic of both A. vinelandii

and A. paspali (13) and all the isolates were positive for

catalase, starch hydrolysis and oxidase test. They were

showed negative for H2S production and indole

production test. These tests confirmed they were all

belong to the genus Azotobacter. Additionally, both

control A. vinelandii MTCC2460 and A.chrococcum (Pb1)

standard culture from TNAU strains identities were also

confirmed with the same tests (Table-2).

Based on the preliminary identification, fifteen

presumptive Azotobacter isolates designated Azt1 to

Azt15 were selected for DNA extraction and restriction

analysis. A.chrococcum (Pb1) and A. vinelandii MTCC

2460 were included in the molecular analysis. The in silico

restriction analysis with Restriction endonuclease Hae III

generated different restriction patterns for all Azotobacter

sequences and a polymorphism of 64.7%. A total of 4-8

bands and these amplified DNA bands varied from

150-800 bp length (Fig 1). All the bands were scored for

their presence or absence of band in the seventeen

cultures and a similarity matrix was constructed using the

UPGMA program. Cluster analysis carried out based on

the similarity data generated from the 17 cultures. In

ARDRA analysis, 100% similarity for the following isolates

viz., Azt12 and Azt15; Azt 4, Azt 7 and Azt 8; Azt 3, Azt 5

and Azt 6; Azt 13 and Azt 14. The Jaccard’s similarity

coefficient among these isolates ranges from 0.15 to 1.00

(Fig.-2). This shows it has less discriminatory power

among the Azotobacter isolates.

A high level of polymorphism was seen in PCR with

BOXA1R primer and ERIC primer set when compared to

ARDRA which was about 88.2% and 94.11%

respectively(Fig 1). The number of DNA bands generated

Pasupuleti Reddy Priya et al., 825

Fig . 1 0. AR DR A p rof ile of A zo toba cter s tra in s an d i so late s. M - 100 b p D NA m ark er; C – Az otob acter

ch rococcum AC 1; V – A . v in ela nd ii (M TC C 2460) ; 1-15: Nativ e isolates .

A. ARDRA profile of Azotobacter strains and isolates. M-100 bpDNA marker; C-Azotobacter chrococcum AC1; V-A. vinelandii(MTCC2460);1-15:Native isolates

B. ERIC fingerprinting profile of Azotobacter strains and isolates. M- 100 bp DNA marker; AC-Azotobacter chrococcum AC1; AV –A. vinelandii (MTCC); 1-15: Native isolates.

Fig. 11. BOX fingerprinting profile of Azotobacter strains and isolates. M- 100 bp DNA marker; C – Azotobacter chrococcum AC1; V – A. vinelandii (MTCC); 1-15: Native isolates.

C. BOX fingerprinting profile of Azotobacter strains andisolates.M-100 bp DNA marker ;C- AzotobacterchrococcumAC1;V- A.vinelandii(MTCC);1-15:Native isolates.

Fig-1 : Molecular Fingerprints of Azotobacter Isolates A.ARDRA; B. ERIC; C. BOX

by this PCR varied in size from 100 -1000 bp length.

Cluster analysis carried out based on the similarity data

generated from the 17 cultures using the UPGMA

program as previously described. A few of the bacterial

isolates showed unique bands indicating the ability of

Rep-PCR to distinguish many of the isolates. The

dendogram of the ERIC and BOX PCR analysis was

depicted in figure 3 and 4. There was 100 % similarity for

the isolates Azt 13 and Azt14 in all the above cases as

because those isolates were isolated from the same place

and hence they may belong to same species.

DISCUSSION

Azotobacter species are found in agricultural soils playing

different beneficial roles: atmospheric nitrogen fixation,

production of phytohormones, degradation of toxic

compounds(14) and driving the ecological balance in

agro-ecosystems. For the isolation and molecular

characterization of isolates of the genus Azotobacter soils

samples were collected from paddy crop of different

agroclimatic regions of Tamilnadu. An important

characteristic of Azotobacter is the Gram-negative

bacillary morphology, with cells between 2 ìm and 4 ìm in

diameter. Many isolates presented this morphology, while

few consisted of small Gram-negative bacilli but with a

morphology very similar to that displayed by Azospirillum,

Beijerinckia, Herbaspirillum and Derxia .However, it is

known that Azotobacter is a pleiomorphic microorganism

(13). It has been reported that Azotobacter has the

capacity to produce soluble pigments(15), this can be a

useful tool in the characterization of some Azotobacter

species. As Brown or black pigmentation in the

Waksmann no77 medium produced by A. chroococcum

(16). All the Isolates exhibited morphological traits and

similar pigmentation to those displayed by A.

chroococcum (Pb1). In addition, all Azotobacter species

have the capacity to produce oxidases and catalases for

the protection of their nitrogenase.

Amplified ribosomal DNA restriction analysis is one

of the molecular tools which can be helpful for

differentiating among the bacterial species. But definitely

it does not have the advantage of considering genome

wide diversity and selection of ideal restriction enzyme for

digestion becomes a tricky thing. Exploiting repetitive

genetic elements which are wide spread over the bacterial

genomes, can also be a good and powerful recourse to

identify and discriminate different bacterial species. This

method can be used to generate more accurate

information because it is capable of screening several

parts of the bacterial genome (17).

In the present study, the comparison among the

different molecular typing methods viz.,ARDRA, BOX

PCR and ERIC PCR reveals that the discriminatory power

of the BOX and ERIC PCR is comparatively higher than

the ARDRA which is evident from the number bands

generated and the range of similarity coefficient obtained

after cluster analysis. Differences observed between two

fingerprinting methods used are most probably the result

of their varying sensitivity. This difference in sensitivity

should be particularly visible in bacterial genomes

containing only very infrequent repetitive regions (18).

(19) reported that among different repetitive element

based PCR systems viz., (GTG)5, BOX and ERIC, the

later two were more efficient than the former one and

particularly ERIC PCR resulted in similar clustering of

Bacillus isolates as obtained with 16S rDNA sequence

based phylogenetic analysis. They also reported ERIC

PCR as a powerful tool for examining genetic relationship

among the unknown Bacillus isolates. In congruence with

results reported by (19), our results also showed that PCR

based on repetitive elements have better discriminatory

826 Pasupuleti Reddy Priya et al.,

Fig.-2 : Dendrogram showing the genetic similarity ofAzotobacter isolates, obtained on the basis of ARDRAanalysis.The den drogram was constructed using the UPGMA method based on the Jaccard coefficient.

Fig.-3 : Dendrogram showing the genetic similarity ofAzotobacter isolates, obtained on the basis of ERIC PCRanalysis.The den drogram was constructed using the UPGMA method based on the Jaccard coefficient.

Fig.-4 : Dendrogram showing the genetic similarity ofAzotobacter isolates, obtained on the basis of BOX PCRanalysis.The den drogram was constructed using the UPGMA method based on the Jaccard coefficient.

power for intra generic as well as intra specific diversity of

Azotobacter.

CONCLUSION

The results of the present study clearly indicates that

ERIC PCR is a better tool than BOX PCR or ARDRA in

terms of intra generic and intra specific discrimination.

Even different strains of one species could be

differentiated based on the ERIC and BOX PCR which

was not possible with ARDRA. By converting the ERIC

and BOX fingerprints into SCAR marker it will be possible

to increase the realibility of this method although a number

of such SCAR primers need to be tested for diversity

analysis and also for authentication of specific strain .

ACKNOWLEDGEMENT

The financially support given by Department of

Biotechnology, New Delhi through R & D Project

(Development of SCAR markers for strain authentication

and to improve the quality assessment of bioinoculants,

Sanction No. BT/PR6450/AGR/21/358/2012) is

acknowledged..

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