Isolation and Screening of Nitrogen Fixers and Phosphate Solubilizing Bacteria

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4. RESULTS AND DISCUSSION

The experimental results derived from the present study are presented anddiscussed here in light of the existing literature under the following sub-headings:

4.1 Isolation and screening of nitrogen fixers and phosphate solubilizing bacteria

4.2 Qualitative assay of phosphate solubilizing activity

4.3 Quantitative assay of phosphate solubilizing activity

4.4 Quantitative assay of Nitrogenase activity

4.5 Detection of Indole acetic acid (IAA) production in nitrogen fixers and phosphatesolubilizing bacteria

4.6 Detection of siderophore production in nitrogen fixing and phosphate solubilizing

bacteria

4.7 Detection of ammonia production

4.8 Characterization, identification and maintenance of isolated microbial strains

4.8.1 Morphological and Biochemical characterization

4.8.2 Molecular characterization of efficient strains

4.9 Development of liquid formulations

4.9.1 Liquid carriers for formulations

4.9.2 Effect of stress conditions on liquid formulation


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4.1 Isolation and screening of nitrogen fixers and phosphate solubilizing

microorganisms

Soil is a complex heterogeneous habitat for a wide variety of organisms, which

include bacteria, fungi, protozoan, nematodes and earthworms that play many functional

roles in the ecosystem in which they exist. Observations have shown that the

concentration of bacteria around the roots of plants is generally much greater than the

surrounding soil, and rhizosphere supports higher microbial growth rates and activities as

compared to the bulk soil (Soderberg and Baath 1998). One of the main reasons for this

is, the increased availability of soluble organic compounds that come from plant rootexudation. However, the composition and quantity of root exudate vary with the species

of plants (Smith 1976) and abiotic stresses such as moisture content and temperature

(Martin and Kemp 1980).

Isolation of nitrogen fixers ( Azotobacter and Azospirillum ) and phosphate

solubilizing bacteria from the 25 soil samples (Table 4.1) of rhizospheric soils of

different crops viz., wheat, maize, potato, brahmi and aloevera grown in Model Organic

Farm of CSK HPKV, Palampur, was carried out on Jensens medium, semisolid NFB

medium and Pikovskayas agar medium (Plate 4.1). A total of 43 Azotobacter , 52

Azospirillum , and 61 phosphate solubilizing bacterial strains were isolated. Nitrogen

fixers ( Azotobacter and Azospirillum ) were screened on the basis of acetylene reduction

assay. It was observed that only 18 Azotobacter and 20 Azospirillum isolates showed

more than 150 nmole C 2H4 h-1 mg -1 protein nitrogenase activity (Table 4.1) and were

selected for further study. Park et al . (2005) used the same criteria for screening of

diazotrophic isolates.

P-solubilizers with a zone of more than 5 mm were selected for further study.

Similar criteria for selecting efficient P-solubilizers were also used by Ostwal and Bhide

(1972) and Illmer and Schinner (1992) to screen their efficient phosphate solubilizing

bacterial isolates.

The efficient isolates of nitrogen fixers ( Azotobacter and Azospirillum ) and

phosphate solubilizers were segregated as depicted in Table 4.2.


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IN THE PRESENT STUDY, IT WAS OBSERVED THAT MAXIMUM

NUMBER OF AZOTOBACTER , AZOSPIRILLUM AND PHOSPHATE SOLUBILIZING

BACTERIA WERE ISOLATED FROM WHEAT AND POTATO CROPS FOLLOWED

BY MAIZE, WHEREAS ALOEVERA AND BRAHMI HAD LOWEST OF THESE

ISOLATES (TABLE 4.1). DIFFERENCES IN BOTH NUMBER AND COMPOSITION

OF MICROORGANISMS IN RHIZOSPHERE OF VARIOUS PLANT SPECIES AND

EVEN VARIETIES WITHIN SPECIES HAVE BEEN REPORTED BY VARIOUS

WORKERS (ELKAN, 1962; LILJEROTH AND BAATH 1988).


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Table 4.1 Isolation of nitrogen fixers and phosphate solubilizing microorganisms from the rhizosphere of different cropand medicinal plants

Plant No. of soilsamples

Azotobacter Azospirillum P-solubilizers

No. ofisolates

obtained

No. of efficientisolates (ARA>150 nmole

C 2H 4 h-1 mg -1

protein)

No. ofisolatesobtained

No. of efficientisolates (ARA>150 nmole

C 2H 4 h-1 mg -1

protein)

No. ofisolatesobtained

No. of efficientisolates (>5 mmzone of

solubilization)

Wheat ( Triticumaestivum)

5 12 4 18 5 16 5

Maize ( Zea mays) 5 8 3 13 6 11 4

Potato ( Solanumtuberosum)

5 10 5 15 5 19 7

Aloevera ( Aloebarbadensis) 5 7 3 7 2 8 4

Brahmi (Bacopamonnieri)

5 6 3 9 2 7 4

Total 25 43 18 52 20 61 24

Table 4.2 Segregation of efficient nitrogen fixers and phosphate solubilizing bacterial isolates obtained from therhizosphere of different plants


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Plant Codes assigned to Azotobacter Codes assigned to Azospiri ll um Codes assigned to P-solubilizingbacteria

Wheat ( Triticumaestivum)

WT-A1*, WT-A2, WT-A3,

WT-A4

WT-AS1*, WT-AS2, WT-AS3,WT-AS4, WT-AS5

WT-P1*, WT-P2, WT-P3, WT-P4,WT-P5

Maize ( Zea mays) MZ-A1, MZ-A2, MZ-A3 MZ-AS1, MZ-AS2, MZ-AS3, MZ-AS4, MZ-AS5, MZ-AS6

MZ-P1, MZ-P2, MZ-P3, MZ-P4

Potato ( Solanumtuberosum)

PT-A1, PT-A2, PT-A3, PT-A4, PT-A5

PT-AS1, PT-AS2, PT-AS3,

PT-AS4, PT-AS5

PT-P1, PT-P2, PT-P3, PT-P4, PT-P5, PT-P6, PT-P7

Aloevera ( Aloebarbadensis)

AV-A1, AV-A2, AV-A3 AV-AS1, AV-AS2 AV-P1, AV-P2, AV-P3, AV-P4

Brahmi (Bacopamonnieri)

BM-A1, BM-A2, BM-A3 BM-AS1, BM-AS2 BM-P1. BM-P2, BM-P3, BM-P4

*W T-A1 represent Azotobacter no. 1 isolated from soil sample of Triticum aestivum , * W T-AS1 represent Azospirillum no. 1isolated from soil sample of Triticum aestivum * WT-P1 represents P-solubilizing bacteria no.1 isolated from soil sample of Triticumaestivum.


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Plate 4.1 Isolation of native isolates from the rhizospheric soils: (A) Azospirillum insemisolid NFb medium, (B) PSB on Pikovskayas agar medium, and (C) Azotobacter onJensens medium.

4.2 Qualitative assay of phosphate solubilizing activity

Solubilizing efficiency of different bacterial isolates was compared on

Pikovskayas medium containing TCP as it was reported to be a better source of insoluble

phosphate under laboratory conditions, in comparison to other sources of rock phosphate

(Gaur et al. 1973; Dave and Patel 1999; Chambial 1998).

A B

C


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The diameter of zone of solubilization and colony were recorded on each day upto

10th day of incubation to find out solubilization efficiency (Table 4.3) which varied from

33.3 to 188.8 per cent with highest efficiency shown by PT-P2 and lowest by BM-P2 .

Out of 24 phosphate solubilizing bacterial isolates, 16 isolates showed more than 50 per

cent solubilization efficiency (Table 4.3). Solubilization efficiency of isolates PT-P2

(188.8 %), MZ-P4 (140.0 %), WT-P1 (118.7 %), and WT-P3 (100.0%) was higher than

that shown by standard strain of P.striata (90.9%) thereby indicating the superiority of

these native isolates over the index strain. The solubilization of the phosphate and the

clarity of the zone is primarily dependent upon the nature of the phosphatic compounds

and organisms used (Kapoor et al. 1989). Srivastav et al. (2004) reported P-solubilizationefficiency in the range of 9.0 to 75.0 per cent for bacterial isolates on solid medium.

4.3 Quantitative assay of phosphate solubilizing activity

Quantitative estimation of P-solubilizing activity was done in NBRIP broth

containing 1000 g insoluble P/ml in the form of TCP at pH 6.8. This broth is consistent

in demonstrating higher efficiency as compared to Pikovskayas medium ( Nautiyal 1999)

and has been used by various other workers (Johri et al. 1999; Chatli et al. 2005) also.

After inoculation with P-solubilizing bacteria, the insoluble phosphate was

solubilized and measured as soluble P. As evident from the Table 4.4, thirteen isolates

showed maximum solubilization on the 5 th day of incubation and their maximum values

were varied from 205.42 to 635.60 g P/ml. Eleven isolates showed maximum

solubilization on 7 th day of incubation and their maximum values were from 240.38 to

685.67 g P/ml. After reaching maximum value of solubilization, in most of the isolates

(irrespective of the day of maximum solubilization), the solubilization decreased

Table 4.3 Solubilization efficiency of different P-solubilizing bacterial isolateson Pikovskayas agar medium on 10 th day of incubation

S. No. Isolate Zone (mm) Colonydiameter

(mm)

%S.E

1 WT-P1 17.5 8.0 118.7


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2 WT-P2 16.5 9.5 73.6

3 WT-P3 20.0 10.0 100.0

4 WT-P4 22.0 15.0 46.6

5 WT-P5 15.0 9.5 57.8

6 MZ-P1 15.5 10.5 47.6

7 MZ-P2 12.0 7.0 71.4

8 MZ-P3 24.0 10.0 140.0

9 MZ-P4 19.0 11.0 72.2

10 PT-P1 11.0 6.0 83.3

11 PT -P2 26.0 9.0 188.8

12 PT -P3 17.0 12.0 41.6

13 PT-P4 21.0 11.5 82.6

14 PT-P5 15.0 10.0 50.0

15 PT-P6 21.0 12.0 75.0

16 PT-P7 19.0 12.5 52.0

17 AV-P1 17.0 12.5 36.0

18 AV-P2 17.0 11.0 54.5

19 AV-P3 14.0 9.0 55.520 AV-P4 19.0 13.0 46.1

21 BM-P1 16.0 11.0 45.4

22 BM-P2 24.0 18.0 33.3

23 BM-P3 10.0 5.5 81.8

24 BM-P4 21.0 13.0 61.5

25 P. striata 21.0 11.0 90.9


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Table 4.4 Quantitative assay of phosphate solubilization and pH changes exhibited by different bacterial isolates inNBRIP broth

Phosphate solubilization (g/ml) pH of mediumDays of incubation Days of incubation

Isolate 3 5 7 11 Mean 3 5 7 11 Mean

WT-P1 145.61 360.44 415.46 375.64 324.29 5.00 5.43 5.80 5.33 5.39WT-P2 105.40 285.30 380.33 290.66 265.42 5.10 4.77 4.91 5.28 5.01WT-P3 240.45 575.57 520.43 505.39 460.46 5.53 4.58 4.81 4.81 4.93WT-P4 220.64 490.49 425.56 380.37 379.26 5.03 4.81 4.08 4.60 4.63WT-P5 90.65 195.33 240.50 185.74 178.05 4.93 4.60 5.32 5.57 5.11MZ-P1 90.46 160.52 290.46 265.34 201.69 6.03 5.58 4.33 4.58 5.13MZ-P2 180.57 395.44 325.65 280.45 295.52 6.03 5.04 5.31 5.75 5.54MZ-P3 130.68 370.60 585.53 410.74 374.39 5.70 5.18 4.81 5.20 5.22MZ-P4 320.57 635.60 615.70 580.49 538.09 5.33 5.68 5.05 5.41 5.51PT-P1 110.65 180.53 240.38 220.58 188.04 5.63 4.92 4.44 5.10 5.02PT -P2 375.66 660.46 685.67 620.39 585.54 5.91 5.18 4.78 5.32 5.15PT -P3 150.64 295.67 265.46 210.51 230.57 5.31 4.90 4.60 4.91 4.93PT-P4 105.71 320.55 385.62 210.47 255.58 5.90 5.31 4.91 5.10 5.30PT-P5 120.13 205.42 180.53 140.50 161.64 6.15 5.33 5.72 5.91 5.78PT-P6 145.56 250.83 225.66 170.64 198.17 5.72 4.41 4.70 5.12 4.99PT-P7 125.53 310.49 260.37 205.68 225.52 5.54 4.83 4.42 4.45 4.81AV-P1 120.79 230.48 270.50 240.71 215.62 5.92 4.91 5.04 5.65 5.38

AV-P2 100.44 190.48 265.40 230.25 196.64 5.71 5.03 4.82 5.34 5.22AV-P3 210.35 280.46 200.38 120.49 202.92 5.71 4.83 5.23 5.53 5.32AV-P4 145.57 240.56 325.16 255.73 241.76 5.62 4.58 4.61 4.82 4.91BM-P1 120.34 245.37 210.55 185.44 190.42 5.62 5.18 4.54 4.56 4.97BM-P2 190.43 495.58 385.74 290.28 340.51 5.92 4.40 4.82 5.22 5.09BM-P3 95.32 240.50 180.41 240.35 189.14 5.60 5.31 5.56 5.83 5.58BM-P4 105.48 225.40 195.62 155.60 170.52 6.21 6.03 5.73 5.93 5.97

P. striata 300.46 475.41 560.61 505.42 460.48 6.02 5.54 4.33 4.54 5.11Mean 161.92 332.70 345.51 291.11 5.65 5.05 4.91 5.19

Variant SEm CD (P 0.01) SEm CD (P 0.01)Isolate 0.279 1.027 0.070 0.258

Day 0.112 0.411 0.028 0.103Interaction 0.559 2.055 0.140 0.517


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thereafter, which continued upto 11 th day of incubation. Such an increasing and

decreasing trend in phosphate solubilization was reported by earlier workers also (Gaur

1990; Yadav and Singh 1991; Goenadi et al. 2000). The reason for this trend may beattributed to the fact that when the rate of uptake is higher than that of solubilization, a

decrease in P concentration in the medium could be observed. On the contrary, when the

uptake rate decreases, the level of P in the medium increases (Rodriguez and Fraga

1999). The decrease in soluble phosphorus at later incubation period might be due to

decreased solubilizing activity of microorganisms or increased P-absorption.

Out of 24 bacterial isolates (irrespective of the day of maximum solubilization) it

was observed that only four isolates solubilized more P as compared to the standard,

P.striata (560.61 g P/ml). These isolates were WT-P3 (575.57 g P/ml), MZ-P3 (585.53g P/ml), MZ-P4 (635.60 g P/ml) and PT-P2 (685.67 g P/ml).

The pH of the growth medium changed during the process of solubilization from

its initial value of 6.8 to 4.3 - 5.0 in majority of the isolates. In case of isolate WT-P3,

WT-P4, MZ-P3, MZ-P4, PT-P2, and BM-P2, the pH fell from initial 6.8 to a minimum of

4.58, 4.08, 4.81, 5.05, 4.78, and 4.40, respectively, on the day of maximum of

solubilization. A similar change in the pH of the growth medium was noticed by many

workers (Vora and Shelat 1996; Sujatha et al. 2004). A fall in pH of the liquid culture

during solubilization of inorganic phosphatic compounds has also been reported by

various other workers (Gerretsen 1948; Ahmad and Jha 1968 and Pandey et al. 2006).

Pandey et al. (2006) have reported that a bacterial strain (B0) solubilized 247 g mL 1

TCP under in vitro conditions and the maximum phosphate solubilizing activity

coincided with the concomitant decrease in pH of the medium. The elevation of pH of the

medium on prolonged incubation as also noticed in the present study could be either due

to the death and lysis of microorganisms (Illmer and Schinner 1992) or due to the

consumption of organic acids by the organisms (Dave and Patel 1999).

The trend of pH changes in context with the phosphate solubilizing kinetic as

exhibited by four most efficient bacterial isolates is shown in Figures 4.1. The isolate PT-

P2 obtained from maize showed a maximum solubilization of 685.67 g P/ml at a

minimum pH of 4.78 on 7 th day of incubation. In case of other isolates WT-P3, MZ-P3

and MZ-P4, maximum solubilization of 575.57 g P/ml, 585.53 g P/ml and 635.60 g

P/ml, was observed at pH 4.58, 4.81 and 5.68, respectively.


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Figure 4.1 Trend of pH changes in context with phosphate solubilizing kinetics as exhibited by efficient isolates (----- represent pHchanges and represent P-solubilization)


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In the present study no relationship could be ascertained with the quantity of P-

solubilized and value of pH. These results are in concurrence with those of various other

workers who also could not establish any correlation between the quantity of phosphate

solubilized and decrease in pH (Dave and Patel 1999; Narsian et al. 2000; Sujatha et al.

2004). Thus the pH does not seem to be the sole factor responsible for P-solubilization.

4.4 Quantitative assay of Nitrogenase activity

Nitrogen is an essential nutrient for all forms of life on earth (Sylvia et al . 1999).

In nitrogen cycle, biological nitrogen fixation takes the role of biological conversion of

atmospheric nitrogen (N 2) to available form for plant and microbial growth by a variety

of prokaryotic microbes.

The nitrogenase enzyme catalyzes the reductive breakage of the very strong triple

bond of N 2 to generate NH 3 (Rubio and Ludden 2005). Nitrogenase is able to reduce a

wide range of substrates besides atmospheric nitrogen (Burns and Hardy 1975). The

reduction of acetylene to ethylene (ARA) is proposed as an indirect method to assay for

nitrogenase activity. The ARA is the most common method for measuring N 2 fixation

and is based on the assumption that 3 4 mol acetylene are reduced to ethylene for every

mole of N 2 fixed by nitrogenase enzyme (Stewart et al . 1967; Jensen and Cox 1983).

The comparison of nitrogenase activity of 18 Azotobacter isolates obtained from

different medicinal and crop plants with standard A. chroococcum is depicted in Table

4.5. Six Azotobacter isolates showed significantly higher nitrogenase activity as

compared to standard strain of A. chroococcum (372.85 nmole C 2H4 h-1 mg -1 protein).

These six isolates were WT-A1 (441.58 nmole C 2H4 h-1 mg -1 protein), WT-A2 (451.45

nmole C 2H4 h-1 mg -1 protein), MZ-A2 (440.91 nmole C 2H4 h

-1 mg -1 protein), PT-A1

(444.02 nmole C 2H4 h-1 mg -1 protein), PT-A3 (383.64 nmole C 2H4 h-1 mg -1 protein), and

BM-A3 (374.44 nmole C 2H4 h-1 mg -1 protein). As evident from the results in Table 4.5,

the most efficient Azotobacter which showed highest nitrogenase activity (451.45 nmole

C2H4 h-1 mg -1 protein) was WT-A2, an isolate obtained from wheat crop rhizosphere.


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Table 4.5 Nitrogenase activity of native isolates of nitrogen fixers isolated fromdifferent crop plants

S.No. Isolate(Azotobacter ) Nitrogenaseactivity* Isolate(Azospirillum ) Nitrogenaseactivity*

1 WT-A1 441.58 a WT-AS1 202.50 a

2 WT-A2 451.45 b WT-AS2 304.58 b

3 WT-A3 225.48 c WT-AS3 458.33 c

4 WT-A4 256.29 d WT-AS4 219.68 d

5 MZ-A1 287.52 e WT-AS5 414.83 e

6 MZ-A2 440.91 a MZ-AS1 157.53 f

7 MZ-A3 194.37f

MZ-AS2 462.33g

8 PT-A1 444.02 g MZ-AS3 229.61 h

9 PT -A2 183.23 h MZ-AS4 358.47 i

10 PT -A3 383.64 i MZ-AS5 175.57 j

11 PT-A4 155.40 j MZ-AS6 327.73 k

12 PT-A5 237.63 k PT-AS1 398.46 l

13 AV-A1 241.28 l PT -AS2 153.23 m

14 AV-A2 168.49 m PT -AS3 405.55 n

15 AV-A3 291.60 n PT-AS4 346.32 o 16 BM-A1 151.51 o PT-AS5 428.46 p

17 BM-A2 207.41 p AV-AS1 177.79 q

18 BM-A3 374.44 q AV-AS2 421.38 r

19 A. chroococcum 372.85 r BM-AS1 312.29 s

20 BM-AS2 264.72 t

21 Azospirillumbrasilense

394.48 u

Each value represents mean of three replicates. In the same column, significantdifferences according to LSD at P 0.01 levels are indicated by different letters. Sameletters represent that their values are at par.*nmol C 2H4 released h -1 mg -1 protein.

In case of Azospirillum isolates, seven isolates showed significantly higher

nitrogenase activity (Table 4.5) as compared to standard strain of A. brasilense (394.48


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nmole C 2H4 h -1 mg -1 protein). These isolates were WT-AS3 (458.33 nmole C 2H4 h -1 mg -1

protein), WT-AS5 (414.83 nmole C 2H4 h -1 mg -1 protein), MZ-AS2 (462.33 nmole C 2H4 h -

1 mg -1 protein), PT-AS1 (398.46 nmole C 2H4 h-1 mg -1 protein), PT-AS3 (405.55 nmoleC2H4 h

-1 mg -1 protein), PT-AS5 (428.46 nmole C 2H4 h-1 mg -1 protein), and AV-AS2

(421.38 nmole C 2H4 h-1 mg -1 protein). The most efficient Azospirillum isolate was MZ-

AS2 (462.33 nmole C 2H4 h-1 mg -1 protein) obtained from the rhizosphere of maize crop.

In the present study the nitrogenase activity was quantified in nitrogen free

medium as it is evidenced that nitrogen fixation is depressed in presence of nitrogen in

the medium (Mishustin and Shilnikova 1969; Laane et al . 1980).

4.5

Detection of indole acetic acid (IAA) production in nitrogen fixing andphosphate solubilizing microorganisms

The most common auxin produced by microorganisms showing plant growth

promoting traits is indole acetic acid (IAA) which facilitates the development of shoots.

Bacterial IAA can directly promote plant growth by stimulating plant cell elongation,

proliferation, and development of lateral and adventitious roots. Rapid establishment of

roots, whether by elongation of primary roots or by proliferation of lateral and

adventitious roots, is advantageous for young seedlings as it increases their ability to

anchor themselves to the soil and to obtain water and nutrients from their environment,

thus enhancing their chances of survival.

Out of eighteen Azotobacter strains isolated from the rhizosphere of different

crops and medicinal plants, thirteen isolates were found to produce IAA (Table 4.6; Plate

4.2) . The maximum production of IAA was shown by AV-A1 (20.35 g/ml), followed by

MZ- A1 (18.14 g/ml). The standard strain A. chroococcum produced 15.51 g/ml of

IAA. Among all the Azotobacter isolates studied for IAA production, WT-A1 showed

least IAA production (8.65 g/ml). Five isolates of Azotobacter among 18 studied

isolates, exhibited no IAA production. WT-A2, the most efficient isolate with respect tonitrogenase activity, showed 17.45 g/ml of IAA production which was significantly

higher than the standard strain .


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Table 4.6 Indole Acetic Acid (IAA) Production by Phosphate solubilizing and nitrogen fixing native bacteria

S.No. Isolate(PSB)

IAA Conc.(g/ml)

Isolate(Azotobacter )

IAA Conc.(g/ml)

Isolate(Azospirillum )

IAA Conc.(g/ml)

1 WT-P1 14.52 a WT-A1 8.65 a WT-AS1 -2 WT-P2 - WT-A2 17.45 WT-AS2 9.32 a

3 WT-P3 17.30 WT-A3 10.66c

WT-AS3 13.424 WT-P4 - WT-A4 - WT-AS4 -5 WT-P5 - MZ-A1 18.14 WT-AS5 12.73 c 6 MZ-P1 - MZ-A2 10.96 e MZ-AS1 -7 MZ-P2 - MZ-A3 - MZ-AS2 18.278 MZ-P3 19.15 c PT-A1 - MZ-AS3 -9 MZ-P4 15.91 PT -A2 12.51 MZ-AS4 -

10 PT-P1 - PT -A3 15.14 g MZ-AS5 14.16 e 11 PT -P2 16.68 PT-A4 16.06 MZ-AS6 7.3912 PT -P3 13.28 e PT-A5 12.05 PT-AS1 15.87 g 13 PT-P4 - AV-A1 20.35 PT -AS2 -14 PT-P5 15.29 AV-A2 12.82 PT -AS3 12.26 c 15 PT-P6 - AV-A3 16.37 PT-AS4 14.78 e 16 PT-P7 - BM-A1 - PT-AS5 -17 AV-P1 - BM-A2 11.89 AV-AS1 10.5618 AV-P2 - BM-A3 - AV-AS2 8.95 a 19 AV-P3 12.51 e A. chroococcum 15.51 g BM-AS1 -20 AV-P4 - BM-AS2 20.0621 BM-P1 - Azospirillum brasilense 14.36 e 22 BM-P2 11.89 g 23 BM-P3 -24 BM-P4 -25 P. striata 14.40 a

Each value represents mean of three replicates. In the same column, significant differences according to LSD at P 0.01 leve ls areindicated by different letters. Same letters represent that their values are at par.


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Plate 4.2 Pink color development showing indole acetic acid (IAA) production by nativeisolates


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Out of twenty isolates, twelve isolates of Azospirillum were found to be positive

for the IAA production (Table 4.6). The isolate BM-AS2 was found to produce maximum

IAA (20.06 g/ml), followed by MZ -AS2 (18.27 g/ml). The least IAA production was

observed in isolate MZ- AS6 (7.39 g/ml). Eight isolates did not show any IAA

production. MZ-AS2, the most efficient isolate with respect to nitrogenase activity,

showed 18.27 g/ ml of IAA production which was significantly higher than the standard

strain .

Out of 24 efficient phosphate solubilizing bacteria, only nine isolates were found

to produce IAA (Table 4.6). The maximum IAA production was recorded in case of

isolate MZ-P3 (1 9.15 g/ml), whereas the most efficient isolate with respect to

nitrogenase activity PT- P2 showed 16.68 g/ml of IAA production. The standard strain

P. striata produced 14.40 g/ml, IAA which was significantly lower than the isolates

MZ-P3 and PT-P2.

The production of auxins (IAA) depends upon the strain and age of the

microorganism. The promotion and expansion of root growth is one of the major markers

by which the beneficial effect of plant growth promoting bacteria is measured (Glick1995). Supplementation of culture medium with tryptophan helps the microorganisms to

produce IAA from it. It has been reported by various workers that the precursor, L-

tryptophan is necessary for IAA production by microorganisms (Bent et al . 2001; Asghar

et al . 2002; Park et al . 2005; Tsavkelova et al . 2007). In the present study also, the

assessment of IAA was performed in the presence of L-tryptophan. Under natural

conditions, L-tryptophan may be available in root exudates as noticed by Beniziri et al .

(1998) which is inducing the microorganisms to produce IAA in the rhizosphere.

4.6 Detection of siderophore production in nitrogen fixing and phosphatesolubilizing microorganisms

Siderophores are low molecular weight iron chelating ligands synthesized by

microorganisms (Winkelmann 1991). Most bacteria and fungi produce siderophores that

differ according to their functional groups. Siderophore production by 18 Azotobacter

and 20 Azospirillum and 24 phosphate solubilizing bacterial isolates was studied by spot


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inoculation on CAS agar medium (Plate 4.3). It was found that out of 18 Azotobacter

isolates, only 9 isolates (Table 4.7) had shown siderophore production along with

standard strain. These nine isolates were WT-A2, WT-A3, MZ-A1, MZ-A2, PT-A1, PT-A3, AV-A2, AV-A3 and BM-A2.

The Azospirillum isolates were also tested for siderophore production, and out of

twenty isolates, twelve isolates were found to be positive for siderophore (Table 4.7). The

siderophore producing isolates were WT-AS1, WT-AS3, WT-AS5, MZ-AS2, MZ-AS4,

MZ-AS6, PT-AS1, PT-AS3, PT-AS5, AV-AS1, AV-AS2 and BM-AS1. The standard

strain of A. brasilense was also positive for siderophore production.

Out of 24 efficient P-solubilizing bacterial isolates, only 12 isolates showed

siderophore production (orange halo zone formation) on CAS agar plates. These twelve

isolates were WT-P1, WT-P3, WT-P4, MZ-P3, MZ-P4, PT-P1, PT-P2, PT-P3, PT-P5,

AV-P2, AV-P3 and BM-P2 (Table 4.7). The standard strain of P. striata was also

positive for siderophore production.

It is well known that siderophores are beneficial to plants by solubilizing iron

formerly unavailable to the plants (Prabhu et al. 1996). These siderophores have

multifaceted role in plant growth and protection as reported by other investigators

(Schwyn and Neilands 1987; Sujatha et al. 2004; Pandey et al. 2006).

4.7 Detection of ammonia production in nitrogen fixing and phosphatesolubilizing microorganisms

Ammonia is consider one of the plant growth promoting substances produced by

various microbes inhabiting rhizosphere. The isolated nitrogen fixers and phosphate

solubilizers were qualitatively analyzed for ammonia production (Plate 4.4). Out of

eighteen isolates of Azotobacter , eleven isolates were positive for ammonia production

(Table 4.8). These eleven isolates were WT-A1, WT-A2, WT-A3, MZ-A2, PT-A1, PT-

A4, PT-A5, AV-A2, AV-A3, BM-A1 and BM-A3. The standard strain also, found positive for ammonia production.

Out of twenty Azospirillum isolates, thirteen isolates were found positive for

ammonia production. These isolates were WT-AS2, WT-AS3, WT-AS4, MZ-AS1, MZ-

AS2, MZ-AS3, MZ-AS4, PT-AS1, PT-AS4, PT-AS5, AV-AS1, AV-AS2, and BM-AS2


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Table 4.7 Siderophore Production by Phosphate solubilizing and nitrogen fixing native bacteria

S.No. Isolate (PSB) Siderophoreproduction

Isolate(Azotobacter )

Siderophoreproduction

Isolate(Azospirillum )

Siderophoreproduction

1 WT-P1 ++ WT-A1 - WT-AS1 +2 WT-P2 - WT-A2 ++ WT-AS2 -

3 WT-P3 +++ WT-A3 + WT-AS3 ++4 WT-P4 + WT-A4 - WT-AS4 -5 WT-P5 - MZ-A1 + WT-AS5 +6 MZ-P1 - MZ-A2 +++ MZ-AS1 -7 MZ-P2 - MZ-A3 - MZ-AS2 ++8 MZ-P3 + PT-A1 + MZ-AS3 -9 MZ-P4 + PT -A2 - MZ-AS4 +++

10 PT-P1 + PT -A3 + MZ-AS5 -11 PT -P2 ++ PT-A4 - MZ-AS6 +12 PT -P3 +++ PT-A5 - PT-AS1 +13 PT-P4 - AV-A1 - PT -AS2 -14 PT-P5 + AV-A2 + PT -AS3 ++15 PT-P6 - AV-A3 ++ PT-AS4 -16 PT-P7 - BM-A1 - PT-AS5 +17 AV-P1 - BM-A2 ++ AV-AS1 ++18 AV-P2 ++ BM-A3 - AV-AS2 +++19 AV-P3 + A.chroococcum + BM-AS1 +20 AV-P4 - BM-AS2 -21 BM-P1 - Azospirillum

brasilense +

22 BM-P2 ++23 BM-P3 -24 BM-P4 -25 P.striata ++

- : No Siderophore Production+, ++, +++: represents 8-10, 11-20 and >20 mm orange zone respectively


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Table 4.8 Ammonia Production by Nitrogen and Phosphate solubilizing native bacteria

S.No. Isolate Ammonia(PSB) productionIsolate Ammonia(Azotobacter ) production

Isolate Ammonia(Azospirillum ) production

1 WT-P1 + WT-A1 + WT-AS1 -2 WT-P2 + WT-A2 + WT-AS2 +

3 WT-P3 + WT-A3 + WT-AS3 +4 WT-P4 - WT-A4 - WT-AS4 +5 WT-P5 + MZ-A1 - WT-AS5 -6 MZ-P1 - MZ-A2 + MZ-AS1 +7 MZ-P2 + MZ-A3 - MZ-AS2 +8 MZ-P3 - PT-A1 + MZ-AS3 +9 MZ-P4 + PT -A2 - MZ-AS4 +

10 PT-P1 + PT -A3 - MZ-AS5 -11 PT -P2 + PT-A4 + MZ-AS6 -12 PT -P3 + PT-A5 + PT-AS1 +13 PT-P4 + AV-A1 - PT -AS2 -14 PT-P5 + AV-A2 + PT -AS3 -15 PT-P6 - AV-A3 + PT-AS4 +16 PT-P7 - BM-A1 + PT-AS5 +17 AV-P1 + BM-A2 - AV-AS1 +18 AV-P2 - BM-A3 + AV-AS2 +19 AV-P3 + A. chroococcum + BM-AS1 -20 AV-P4 + BM-AS2 +21 BM-P1 - Azospirillum brasilense +22 BM-P2 -23 BM-P3 +24 BM-P4 -25 P. striata +

- : Negative for ammonia Production+: Positive for ammonia production


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Plate 4.3 Orange color zone showing siderophore production by native isolates

Plate 4.4 Yellow to brownish coloration indicate ammonia production by native isolates

(Table 4.8). The standard strain was also found positive for ammonia production. There

are indirect evidences of usefulness of free living N 2 fixing bacteria in crop improvement

under tropical and sub-tropical conditions especially with strains excreting a high amount


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of ammonia in addition to a variety of growth promoting factors (Narula and Gupta

1986).

Out of twenty four efficient phosphate solubilizers, fifteen isolates showed

ammonia production (Table 4.8). These isolates were WT-P1, WT-P2, WT-P3, WT-P5,

MZ-P2, MZ-P4, PT-P1, PT-P2, PT-P3, PT-P4, PT-P5, AV-P1, AV-P3, AV-P4, and BM-

P3. The standard strain also showed ammonia production.

There are number of sources of ammonia secretion by rhizospheric

microorganisms. Ammonia and extracellular proteins are the nitrogenous secretions of

nitrogen fixers in nitrogen free or deficient medium (Saribay 2003; Behl et al . 2006 ).Amidases catalyze the hydrolysis of the carboxylic amide bonds to liberate carboxylic

acid and ammonia (Asano and Lubbehusen 2000). One of the major mechanisms utilized

by PGPR to facilitate plant growth and development is lowering of the ethylene levels by

hydrolysis of 1-aminocyclopropane-1- carboxylic acid (ACC), the immediate precursor

of ethylene in plants. The enzyme catalyzing this reaction (ACC deaminase) hydrolyzes

ACC to -ketobutyrate and ammonia (Zahir et al . 2008). Some authors consider the

production of ammonia to be involved in antagonistic interactions that result in diseasecontrol (Saraf et al . 2008), however, meticulous experimentation is required to exactly

pin point the role of ammonia in influencing the growth of plant and suppressing the

diseases.

Some of the indigenous microorganisms obtained in the present study possessing

high nitrogen fixing and P-solubilizing capacities coupled with high IAA production,

siderophore production and ammonia production might provide them better tools to

survive under the local conditions and thus can become good candidate for biofertilizers

production.

4.8 Characterization and identification of phosphate solubilizing and nitrogenfixing bacterial isolates

4.8.1 Morphological and Biochemical characterization


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Twenty four phosphate solubilizing bacterial isolates were identified on the basis

of morphological and biochemical characteristics as depicted in Table 4.9. Only one

isolate was Gram-negative coccus, while others were Gram-negative rods. Only threeisolates (MZ-P2, AV-P2, and AV-P3) were non-motile whereas, others were motile.

Majority of the phosphate solubilizing bacterial isolates were oxidase and catalase

positive. It was found that out of 24 phosphate solubilizing bacterial isolates, 15 were

belong to genus Pseudomonas , 1 to Alcaligenes , 1 to Microccocus, 1 to Flavobacterium

and 2 to Acinetobacter . The most efficient P-solubilizing strain PT-P2 showing highest P

solubilizing activity (685.67 g P/ml) was identified as Pseudomonas (Plate 4.5). Four

isolates could not be identified in the present study due to their unusual characteristics

(Table 4.9). Presence of various genera in the rhizospheric soils shows the extent of

microbial diversity existing in these specialized niches of different plants. Similar

observations were made by Louw (1970) and Thakkar et al. (1993). Results of this study

showed that in the rhizosphere of the different medicinal and crop plants, most of the

bacterial isolates exhibited various traits like phosphate solubilization, IAA production,

ammonia production and siderophore production, and the predominant genus was of

Pseudomonas . Dominance of this genus in the root zones of various crops has also been

reported by other workers (Parmar and Dadarwal 1997; Vazquez et al. 2000; Saxena and

Sharma 2003).

Eighteen Azotobacter and twenty Azospirillum isolates were screened from therhizospheric soils and characterized biochemically. The various morhphological and

biochemical tests performed are depicted in Table 4.10 and Table 4.11. The most

efficient Azotobacter and Azospirillum isolates were WT-A2 and MZ-AS2, respectively

(Plate 4.5). Various workers have isolated these isolates from the rhizosphere of different

crops (Tsavkelova et al . 2007; Khan et al . 2008; Reinhardt et al . 2008; Khan and Doty

2009 ).

Therefore, in the present study the observance of Pseudomonas, Azotobacter and

Azospirillum as the dominant native flora reflects that these organisms are probablyadapted to the agroclimatic conditions of Himachal Pradesh in a better way and thus need

to be exploited for the preparation of bioinoculants.


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Plate 4.5 Most efficient bacterial isolates of (A) Phosphate solubilizing bacteria (PT-P2)on Pikovskayas agar medium, (B) Azotobacter (WT-A2) on Jensens medium and (C)

Azospirillum (MZ-AS2) on NFb medium

A B

C


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Table 4.9 Morphological and Biochemical characteristics of phosphate solubilizing bacteria

Isolate Gramstaining

Morphology Motility O C I MR VP Ci U H 2S Utilization of PossibleorganismD M S L R A

WT-P1 - rods + + + - - + + - - - + - + - - PseudomonasWT-P2 - rods + + - - - + + - - - - + - - - PseudomonasWT-P3 - rods + - + - - - + - - - - - + - - AcinetobacterWT-P4 - rods + + + - - + - - - + - + - - + PseudomonasWT-P5 - rods + + + - - + + - - - + - - - - PseudomonasMZ-P1 - rods + - + - - + + - - - - + + - - U.IMZ-P2 + cocci - + + - - - - - - - + - - - - M icrococcusMZ-P3 - rods + + - - - + - - - - + - + - - PseudomonasMZ-P4 - rods + + + - - - + - - + + + + - - PseudomonasPT-P1 - rods + + - - - + + - - - - + - - - U.IPT -P2 - rods + + + - - - + - - - + + + + - PseudomonasPT -P3 - rods + + + - - - + - - - - - - - - PseudomonasPT-P4 - rods + + + - - + - - - + + - - - - PseudomonasPT-P5 - rods + + + - - + + - - + - - - - - PseudomonasPT-P6 - rods + + + - - - + - - + - - - - - Al caligenesPT-P7 - rods + + + - - + + - - + - - - - - PseudomonasAV-P1 - rods + + - - - + - - - - - - - - - PseudomonasAV-P2 - rods - - + - - + + - - - - + + - - U.IAV-P3 - rods - + + + + - - - - - - - - - - FlavobacteriumAV-P4 - rods + - + - - - + - - - - - + - - AcinetobacterBM-P1 - rods + + - - - + - - - - - - - - - PseudomonasBM-P2 - rods + + - - - + - - - + + - - - - PseudomonasBM-P3 - rods + - + - - + + - - - - + + - - U.IBM-P4 - rods + + - - - + - - - - - - - - - PseudomonasO- Oxidase, C-Catalase, I-Indole, MR-Methyl red, VP-Voges Proskauer, Ci Citrate utilization, U- Urease, D-Dextrose, M-Mannitol, S-Sucrose, L-Lactose, R-Rhamnose, A-Adonitol

Table 4.10 Morphological and Biochemical characteristics of strains isolated on Jensens medium


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Morphology Motility O C I MR VP Ci U H 2S Utilization ofF T S Du So Ma

WT-A1 - rods + + + - - - - - - + - + - + -

WT-A2 - rods + + + - - - - - - + - + - + +

WT-A3 - rods + + + - - - - - - + - + - + -

WT-A4 - rods - - + - - + - - - + + + - + +

MZ-A1 - rods + + + - - - + - - + - + - - -

MZ-A2 - rods + + + - - - - - - + + + - + -

MZ-A3 - rods + + + - - + - - - + - + - + -

PT-A1 + rods + + + - - + + - - + - + - + -

PT -A2 - rods + + + - - - - - - + - + - + -

PT -A3 - rods - + + - - - + - - + - + - - -

PT-A4 - rods + + + - - - - - - + + + - + -

PT-A5 - rods + - + - - + - - - + + + - + -

AV-A1 - rods + + + - - - - - - + + + - + -

AV-A2 - rods + + + - - - + - - + - + - - -

AV-A3 - rods - + + - - + - - - + + + - + -

BM-A1 - rods + + + - - - + + - + - + - + -

BM-A2 - rods + - + - - - - - - + - + - - -

BM-A3 - rods + + + - - - - - - + + + - + -

O- Oxidase, C-Catalase, I-Indole, MR-Methyl red, VP-Voges Proskauer, Ci Citrate utilization, U- Urease, F-Fructose, T-Trehalose, S-Sucrose, Du- Dulictol, So- Sorbitol, Ma- Maltose


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Table 4.11 Morphological and Biochemical characteristics of strains isolated on NFb medium


Morphology Motility O C I MR VP Ci U H 2S Utilization ofF Me S Du So M

WT-AS1 - rods + + + - - - + + - + + - - - -

WT-AS2 - rods + + + - - + - + - + - - - - +WT-AS3 - rods + + + - - - - + - + - - - - -WT-AS4 - rods + + - - - - + + - + - - - -WT-AS5 - rods + + + - - - - + - + + - - + -MZ-AS1 - rods + + + - - + + + - + - - - - -MZ-AS2 - rods + + + - - - - + - + - - - - -MZ-AS3 - rods + + + - - - + + - + - + - - -

MZ-AS4 - rods + + + - - - - + - + + - - - -MZ-AS5 - rods + + + - - - - + - + - - - - -MZ-AS6 - rods + + + - - - - + - + - - - - -PT-AS1 - rods + + + - - - - + - + - - - - -

PT -AS2 - rods + + + - - + - + - + + - - - -PT -AS3 - rods + + - - + - + + - + - - - - -PT-AS4 - rods + + + - - - + + - + - - - - -PT-AS5 - rods + + + - - - - + - + - + - - -AV-AS1 - rods + + + - - + + + - + - - - + -AV-AS2 - rods + + + - - - + + - + - - - - -BM-AS1 - rods + + + - - + - + - + - - - - +BM-AS2 - rods + + + - - - - + - + - - - - -

O- Oxidase, C-Catalase, I-Indole, MR-Methyl red, VP-Voges Proskauer, Ci Citrate utilization, U- Urease, F-Fructose, Me-Melibiose, S-Sucrose, Du- Dulictol, So- Sorbitol, M- Mannitol


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4.8.2 Molecular characterization of efficient strains

An attempt was made to characterize the efficient bacteria isolated from the

rhizosphere of medicinal and crop plants using 16S rRNA gene sequencing to identify

and decipher their phylogenetic affilation of these bacteria. The 16S rRNA gene sequence

is about 1,500 bp long and is composed of both variable and conserved regions. The Plate

3.2 shows the 1,500 bp PCR amplicons of efficient strains amplified using universal

primers. Universal primers are usually chosen as complementary to the conserved regions

at the beginning of the gene or at either the 540-bp region or at the end of the whole

sequence (about the 1,500-bp region), and the inbetween sequence of the variable region

is used for the comparative taxonomy (Chen et al . 1989; Relman 1999). The gene is large

enough, with sufficient interspecific polymorphisms, to provide distinguishing and

statistically valid measurements. The 16S rRNA gene serve as molecular chronometer,

since it is the most conserved part during evolution (Clarridge 2004). Therefore, 16S

rRNA gene sequencing is used and accepted worldwide for identification and

phylogenetic analysis of the bacterium.

Sequence data of 16S rRNA gene of six efficient strains obtained through

automated sequencer using eubacterial universal primers revealed 1366 bp partialsequence in isolates, WT-A2, PT-A1, MZ-AS2, WT-AS3 and MZ-P4. However, the

sequence of PT-P2 isolate could be partially sequenced yielding a 920 bp sequence read

only (Figure 4.2).

4.6.3.1 Nucleotide sequence analysis

Nucleotide sequence analysis of test isolates using clustalW program revealed that

isolate WT-A2 showed maximum homology (99%) with Stenotrophomonas maltophilia

(DQ257429), isolate PT-A1 showed homology (87%) with Bacillus licheniformis

(GU201863), isolate MZ-AS2 showed maximum homology (88%) with Azospirillum

brasilense (AY324110), isolate WT-AS3 showed homology (96%) with Azospirillum

brasilense (GU256438), isolate MZ-P4 showed maximum homology (99%) with

Pseudomonas aeruginosa (GU586139) and isolate PT-P2 showed homology (98%) with

Burkholderia cepacia (GQ383907). The test bacterial isolates clustered with members of

the genera Stenotrophomonas, Bacillus, Azospirillum, Azospirillum, Pseudomonas and


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Table 4.12 Pair wise genetic distance of the six efficient isolates with other selected sequences from the NCBI

ISOLATES 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Stenotrophomonas maltophilia WT-A2 (GU371215)*

Stenotrophomonas maltophilia (DQ257429) 0.000

Stenotrophomonas sp. CK6 (AJ870967) 0.000 0.000

Stenotrophomonas rhizophila (EU977698) 0.000 0.000 0.000

Stenotrophomonas like sp.V4BP15 (AJ244720) 0.000 0.000 0.000 0.000

Stenotrophomonas maltophilia(AJ131117) 0.000 0.000 0.000 0.000 0.000

Bacillus licheniformis PT-A1 (GU371216)* 0.249 0.249 0.249 0.249 0.249 0.249

Bacillus licheniformis (GQ375247) 0.149 0.149 0.149 0.149 0.149 0.149 0.103

Bacillus licheniformis (GQ375245) 0.149 0.149 0.149 0.149 0.149 0.149 0.103 0.000

Bacillus licheniformis (GQ375244) 0.149 0.149 0.149 0.149 0.149 0.149 0.103 0.000 0.000

Bacillus licheniformis (AJ293011) 0.149 0.149 0.149 0.149 0.149 0.149 0.103 0.000 0.000 0.000

Bacillus licheniformis (AB525389) 0.148 0.148 0.148 0.148 0.148 0.148 0.105 0.001 0.001 0.001 0.001

Azospirillum brasilense MZ-AS2 (GU371217)* 0.256 0.256 0.256 0.256 0.256 0.256 0.322 0.250 0.250 0.250 0.250 0.249

Azospirillum brasilense WT-AS3 (GU371218)* 0.217 0.217 0.217 0.217 0.217 0.217 0.274 0.206 0.206 0.206 0.206 0.207 0.163

Azospirillum brasilense (AY324110) 0.150 0.150 0.150 0.150 0.150 0.150 0.255 0.152 0.152 0.152 0.152 0.151 0.095 0.063

Azospirillum brasilense (Z29617) 0.150 0.150 0.150 0.150 0.150 0.150 0.255 0.152 0.152 0.152 0.152 0.151 0.095 0.063 0.000

Azospirillum brasilense (AB480699) 0.150 0.150 0.150 0.150 0.150 0.150 0.255 0.152 0.152 0.152 0.152 0.151 0.095 0.063 0.000 0.000

Azospirillum brasilense (DQ288688) 0.151 0.151 0.151 0.151 0.151 0.151 0.255 0.153 0.153 0.153 0.153 0.152 0.094 0.064 0.001 0.001 0.001

Azospirillum sp. Ptl3 (GQ284588) 0.150 0.150 0.150 0.150 0.150 0.150 0.255 0.152 0.152 0.152 0.152 0.151 0.095 0.063 0.000 0.000 0.000 0.001

Azospirillum sp. 7C (AF411852) 0.150 0.150 0.150 0.150 0.150 0.150 0.255 0.152 0.152 0.152 0.152 0.151 0.095 0.063 0.000 0.000 0.000 0.001 0.000

Azospirillum sp. DA10-2 (AY118225) 0.150 0.150 0.150 0.150 0.150 0.150 0.255 0.152 0.152 0.152 0.152 0.151 0.095 0.063 0.000 0.000 0.000 0.001 0.000 0.000

Pseudomonas aeruginosa MZ-P4 (GU371219)* 0.084 0.084 0.084 0.084 0.084 0.084 0.261 0.154 0.154 0.154 0.154 0.153 0.249 0.204 0.142 0.142 0.142 0.144 0.142 0.142 0.142

Pseudomonas aeruginosa (FJ985806) 0.084 0.084 0.084 0.084 0.084 0.084 0.261 0.154 0.154 0.154 0.154 0.153 0.249 0.204 0.142 0.142 0.142 0.144 0.142 0.142 0.142 0.000

Pseudomonas sp. YKM-M4 (GU272400) 0.084 0.084 0.084 0.084 0.084 0.084 0.261 0.154 0.154 0.154 0.154 0.153 0.249 0.204 0.142 0.142 0.142 0.144 0.142 0.142 0.142 0.000 0.000

Pseudomonas aeruginosa (GU199190) 0.084 0.084 0.084 0.084 0.084 0.084 0.261 0.154 0.154 0.154 0.154 0.153 0.249 0.204 0.142 0.142 0.142 0.144 0.142 0.142 0.142 0.000 0.000 0.000

Pseudomonas aeruginosa (GU181320) 0.084 0.084 0.084 0.084 0.084 0.084 0.261 0.154 0.154 0.154 0.154 0.153 0.249 0.204 0.142 0.142 0.142 0.144 0.142 0.142 0.142 0.000 0.000 0.000 0.000

Burkholderia cepacia_PT-P2 (GU371220)* 0.135 0.135 0.135 0.135 0.135 0.135 0.298 0.194 0.194 0.194 0.194 0.192 0.292 0.243 0.189 0.189 0.189 0.190 0.189 0.189 0.189 0.143 0.143 0.143 0.143 0.143

Burkholderia cepacia (GQ383907) 0.104 0.104 0.104 0.104 0.104 0.104 0.268 0.160 0.160 0.160 0.160 0.158 0.250 0.220 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.112 0.112 0.112 0.112 0.112 0.026

Burkholderia cepacia (FJ652618) 0.104 0.104 0.104 0.104 0.104 0.104 0.268 0.160 0.160 0.160 0.160 0.158 0.250 0.220 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.112 0.112 0.112 0.112 0.112 0.026 0.000

Burkholderia cepacia (FJ887895) 0.104 0.104 0.104 0.104 0.104 0.104 0.268 0.160 0.160 0.160 0.160 0.158 0.250 0.220 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.112 0.112 0.112 0.112 0.112 0.026 0.000 0.000

Burkholderia sp.gx-152 (FJ823011) 0.104 0.104 0.104 0.104 0.104 0.104 0.268 0.160 0.160 0.160 0.160 0.158 0.250 0.220 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.112 0.112 0.112 0.112 0.112 0.026 0.000 0.000 0.000

Burkholderia sp.LDSP-10 (FJ548994) 0.104 0.104 0.104 0.104 0.104 0.104 0.268 0.160 0.160 0.160 0.160 0.158 0.250 0.220 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.112 0.112 0.112 0.112 0.112 0.026 0.000 0.000 0.000 0.000

Clostridium sp. (GU097452) 0.168 0.168 0.168 0.168 0.168 0.168 0.225 0.125 0.125 0.125 0.125 0.124 0.240 0.214 0.142 0.142 0.142 0.143 0.142 0.142 0.142 0.168 0.168 0.168 0.168 0.168 0.210 0.174 0.174 0.174 0.174 0.174

(*Represent the native isolates)


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1 GGAATACATCGGAATCTACCTTTTCGTGGGGGATAA CGTAGGGAAACTTACGCTAATACCGCATACGACC 70

71 TTCGGGTGAAAGCAGGGGACCTTCGGGCCTTGCGCG GATAGATGAGCCGATGTCGGATTAGCTAGTTGGC 140

141 GGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAG 210

211 ACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCC 280

281 ATACCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAAAGCAGTCAGCTAA 350

351 TACCCGGTTGTTCTGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATAC 420

421 GAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTTGTTTAAGTCTGTTGTG 490

491 AAAGCCCTGGGCTCAACCTGGGAATTGCAGTGGATACTGGGCGACTAGAGTGTGGTAGAGGGTAGTGGAA 560

561 TTCCCGGTGTAGCAGTGAAATGCGTAGAGATCGGGAGGAACATCCATGGCGAAGGCAGCTACCTGGACCA 630

631 ACACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAA 700

701 CGATGCGAACTGGATGTTGGGTGCAATTTGGCACGCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTG 770

771 GGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGT 840

841 TTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATGTCGAGAACTTTCCAGAGATGGATTGGT 910

911 GCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT 980

981 CCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAG CACGTAATGGTGGGAACTCTAAGGAGACCGCCGG 1050

1051 TGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTAC 1120

1121 TACAATGGTAGGGACAGAGGGCTGCAAACCCGCGAGGGCAAGCCAATCCCAGAAACCCTATCTCAGTCCG 1190

1191 GATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTG 1260

1261 AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGC 1330

1331 TTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCG 1366

A

1 GGACAGATGGGAGCTTGCTCCTGATGTTAGCGGCGG ACGGTGGATTAGGACGTGGGTAACCTGCCTGTAA 70

71 GACTGGGATAACTCCGGGAAACCGGGGCTAATACCG GATGCTTGATTGAACCGCATGGTTCAATTATAAA 140

141 AGGTGGCTTCTGGCTACCACTAACAGATGAACCGGCGGGGCTTTACCTGGTTGTGAGGGTACGGGCTCCC 210

211 CAGGCGACCGACTTGGGCCGGCTTCGCTTTTTTTGGCCTTAATAGGGCTTAAAACCCGTCCAAAATCCTA 280

281 CCAAACAACCTTTGGGAATCTTCCGAAATGTACGAAAGGCTTACCGGGGAAGGCAAAAAGATTTTTGAAG 350

351 GTTTTCAGATTGTTAAATTCGGTTGGTGGGGGGGCCGGTTCCGTTCTAATTGGGGGGCCCCTGACGGGAC 420

421 AAAACCCGAAAGCCCCCGCTTACTTCCTGCCAGCAAGCGCGGTAATTCGGAGGTGGGAAGGCTTTTCCGG 490

491 ATTTTGGGCGCTCAAGCCGGCCCCCGCCGGCCCAAAGGCAAAAGGGAAGGAGGGGTTAGACGGGGGGGGT 560

561 CCTTGGAAATTGGGGAACCTAAGGCAAAAGGGGAAAATCGAATTCCCCGGGAGCGGGGAAAATGGTTGGG 630

631 GTTTGGGGAACACCCAGGGGGCAGGCTATTTTTTAGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGG 700

701 AGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCG 770

771 CCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAA 840

841 GGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC 910

911 AGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCA 980

981 TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTGATCTTAGT 1050


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1051 TGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCA 1120

1121 AATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCG 1190

1191 CGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCT 1260

1261 GGAATCGCTAGTAATCGCGGTTCGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACAGACCCCAACAC 1330

1331 ACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGTT 1366

B1 CCGGAATTCGTCAAGTGTGAGCTGTAACAACAGTAA GAAGCTTCGGCTTTAGTGGCGCACGGGTGAGTAA 70

71 CACGTGAGGTCGCTTTTGGTTCGGGATAACGTCTGG AAACGGACGCTAAAACGGATACGCCCTTCAGAGA 140141 GAATGGGCGGAGAAAGTTTACGCCGAGAGAGGGGCCCGCGTCCGATTAGGTATTTGGTGGGGTAATGGCC 210

211 CACCAAGCCGACGATCGAGAGCTGGTCTGAGAGAATGATCAGCCACACTGGGACTGAGACACTACCCAGA 280

281 CTCCTACGGGGGAATATTGGTGGGGAATATTGAACAATGGGGGGCAACCCTGATCCAGCAATGCCGCGTG 350

351 AGTAGGGTTGTGCCTTAGGGTTGTAAAGCTCTTTCGCACGCGACGATGATGACAGAAGCGTGAGAAGAAG 420

421 CGTGGGCTAACTTTTTTTTTAGCAGCCGCGGTAATACGAAGGGCGCGAATTACTGTTCGTAATTACTGCG 490

491 CGTAAAGGGCGCGTAGGCAGCCCGATCAGCCAGAGGTTAAAGCCCCGGGGCTGAACCTTGAGACCTGCCT 560

561 TTTTTAGTTTCCGGGGTTGAAGTTCCGAAGTCCCCAGGGGAAATCCCAATTTCGAAGGTAAAATTCGGAA 630

631 GAAATTGGGAAGAAACCCGGTGTCTAACCGGCCAATTTGGCCGAAACCTTGGGGACCACCCAGGATTAGT 700

701 TCCCTGGTAGTCCACGCCGTAACGTGAATTCCTAGCGCTGGGGTGCATGCACTCGGGTTTCGCCGCAACG 770

771 CATAAGCATCCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAGGGAATTGACGGGGGCCCGCCCAAG 840

841 CGGGTGGAGCATGTGGTTTTAATTCGGAAGCAACGCGCAGAACCTTACCAACCCTTGACATGTCCCACTA 910

911 CCGGCTCGAGAGATCGGGCTTTCAGTTCGGCTGGGTGGAAAAAAGGTGCTGCATGGCTGTCGTCAGCTCG 980

981 TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG CAACCCCTACCGCCAGTTGCCATCATTCAGTTGG 1050

1051 GCACTCTGGTGGAACTGCCGGTGACAAGCCGGAGGAAGGCGGGGATGACGTCAAGTCCTCATGGCCCTTA 1120

1121 TGGGTTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGATGCGAAGTCGCAAGATGGAGCCAATCC 1190

1191 CCAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGGGTGCATGAAGTTGGAATCGCTAGTAATCGC 1260

1261 GGATCCCCCCGCGGTGAATACGTTCCCGGCCTGTACACAAACACCCCATGGAGTGCTACCGAAGGGTCGC 1330

1331 TATACAAGAGTTGATCATGGCAGCCCCGGGCATTCG 1366

C1 GAGGGGCCCGCGTCCGATTAGGTAGTTGGTGGGGTA ATGGCCCACCAAGCCGACGATCGGTAGCTGGTCT 70

71 GAGAAAATGATCAGCCACAATGGGACTGAGACACGG CCCAGACTCCTACGGGAGGTAGCAGTGGATAATA 140

141 TTGAACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGAGTGATGAAGGACTTAGGTTTGTAAAGCTC 210

211 TTTCGCACGCGACGATGATGACGGTAGCGTGAGAAGAAGCCCCGGCTAATTTTTTTTTCAGCAGCCGCGG 280

281 TAATACGAAGGGGGGGAAGCGCTGTTCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCCCGATCAAGC 350

351 CAGAAGTTAAAGCCCCGGGACTTGAACTTGGGAACTGCATTTTTTTACTTTCCGGGCTTGAGTTCCGGGA 420

421 GAGGATGGTGGAAATTCCCAATTTTGGAGGTGAAATTCGGAAAATATTGGGGAATTTGACTATTGGGGCA 490

491 ACCTGATCAGCATTGCGCGTGAGGATGGACGCCTAGGATGTAAGCTCTTCGCACGCGACGATGATGACGT 560

561 AGCGTGAAGAAGAAGCCCGCTAACCTCGTGCCAGCAGCGCGGTAATACGAAAGGGGGGGCGAGCGTTGTT 630

631 CGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCCGATCAGTCAGATGTGAAAGCCCGGGCTCAACCTGG 700

701 GAACTGCATTTGATACTGTCGGGCTTGAGTTCCCGGAGAGGATGGTGGAATTCCCAGTGTAGAGGTGAAA 770

771 TTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCCATCTGGACGGACACTGACGCTGAGGCGCGA 840


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841 AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGACGCTGG 910

911 GGTGCATGCACTTCGGTGTCGCCGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGTTAA 980

981 AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT GGAGCATGTGGTTTAATTCGAAGCAACGCGCAGA 1050

1051 ACCTTACCAACCCTTGACATGTCCACTATCGGCTCGAGAGATCGGGCTTTCAGTTCGGCTGGGTGGAACA 1120

1121 CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC 1190

1191 CTACCGCCAGTTGCCATCATTCAGTTGGGCACTCTGGTGGAACTGCCGGTGACAAGCCGGAGGAAGGCGG 1260

1261 GGATGACGTCAAGTCCTCATGGCCCTTATGGGTTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGG 1330

1331 ATGCGAAGTCGCAAGATGGAGCCAATCCCCAAAAGC 1366

D

1 GGATGAAGGGAGCTTGCTCTGGATTCAGCGGCGGAC GGGCGGGAAGGCCTAGGAATCTGCCTGGTAGTGG 70

71 GGGATAACGTCCGGAAACGGGCGCTAATACCGCATA CGTCCTGAGGGAGAAAGTGGGGGATCTTCGGACC 140

141 TCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCC 210

211 GTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGC 280

281 AGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGA 350

351 TTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGA 420

421 ATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACT 490

491 GGGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCA 560

561 TCCAAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGAT 630

631 ATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGG 700

701 GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGA 770

771 GATCTTAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAA 840

841 TGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC 910

911 TGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCA 980

981 TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGT 1050

1051 TACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTC 1120

1121 AAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCC 1190

1191 GCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGT 1260

1261 CGGAATCGCTAGTAATCGTGATTCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACTCCCTC 1330

1331 ACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGT 1366

E

1 AAGTAAGTCCATGTGGAACATGTAGACTCCTACGGG AGGCAGCAGTGGGGAATTTTGGACAATGGGCGAA 70

71 AGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGC CTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAA 140

141 TCCTTGGCTCTAATACAGTCGGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCA 210

211 GCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGC 280

281 TAAGACCGTGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGACTGGCAGGCTAGATTATGGCAG 350

351 AGGGGGGTAGAATTCCACGTGTAGCAATGAAATGCGTAGAGATGTGGAGGAAACCGATGGCGAAGGCAGC 420

421 CCCCTGGGCCATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACACTGGTAGTCC 490


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491 ACGCCCTAAACGATGTCAACTAGTTTTGGGGATTCATTTCCTTAGTAACATAGCTAACGCGTGAAGTTGA 560

561 CCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATG 630

631 ATGTGGATTAATTCGATGCACCGCGAAAAACCTTACCTACCCTTGACATGGTCGGAATCCTGCTGAGAGG 700

701 TGGGAGTGCTCGAAAGAGAACCGCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG 770

771 GGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAAGGAGACTGCCG 840

841 GTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCGGACTTCTGAATGCGGCATTACC 910

911 CAGTAGATTC 920

F

Figure 4.2 Partial nucleotide sequence of 16S rRNA gene of efficient isolates: (A) WT-A2, (B) PT-A1,(C) MZ-AS2, (D) WT-AS3, (E) MZ-P4, and (F) PT-P2


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Burkholderia , thus differentiating the bacterial isolates on the genetic basis. Earlier

workers have also reported the isolation of these genera from the rhizosphere of various

crop plants (Chan et al . 1994; Estrada-de Los Santos et al . 2001; Minkwitz and Berg

2001; Vessey 2003; Bashan et al . 2004). The pair wise genetic distance of the six

efficient isolates with other selected sequences from the NCBI is depicted in Table 4.12.

The pair wise genetic distance of the isolates viz., WT-A2, PT-A1, MZ-AS2, WT-AS3,

MZ-P4, and PT-P2 with other selected sequences ranged from 0.000 to 0.322.

Dendrogram based on phylogenetic analysis presented in Plate 4.6 shows that

except PT-A1, all other bacterial isolates viz., WT-A2, MZ-AS2, WT-AS3, MZ-P4 and

PT-P2 clustered with Stenotrophomonas, Azospirillum, Azospirillum, Pseudomonas and

Burkholderia , respectively, which all belong to Proteobacteria. Whereas, isolate PT-A1

was clustered with Bacillus a typical Firmicute. Based on their affinity with known

sequences in databank, the isolates WT-A2 and MZ-P 4 belong to class -Proteobacteria,

MZ-AS2 and WT- A3 to class -Proteobacteria and PT- P2 to class -Proteobacteria. The

partial nucleotide sequences of these efficient isolates were deposited in Gen Bank given

in Table 4.13 . Various other workers also used this technique for identification and

phylogenetic analysis of the isolates (Catara et al . 2002; Khan and Doty 2009; Islam etal . 2010).

Table 4.13 Molecular characterized (16S rRNA gene sequencing) efficient nativePGPR isolates

S.No. Isolate Accession No.

1. Stenotrophomonas maltophilia GU371215

2. Bacillus licheniformis GU371216

3. Azospirillum brasilense GU371217

4. Azospirillum brasilense GU371218

5. Pseudomonas aeruginosa GU371219

6. Burkholderia cepacia GU371220


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0.02

Plate 4.6 Phylogenetic tree constructed by Neighbor-Joining method derived fromanalysis of the 16S rRNA gene sequences of native isolates and related sequencesobtained from NCBI. Scale bar, 0.02 substitutions per nucleotide position ( representsnative isolates).

In the present study the isolates WT-A2 and PT-P2 were identified as Azotobacter

and Pseudomonas on the basis of biochemical characteristics. But molecular


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characterization of these isolates identified these isolates as: Stenotrophomonas

maltophilia (WT-A2) and Burkholderia cepacia (PT-P2). The reasons for this could be

similar type of phenotypicf characteristics exhibited by Stenotrophomonas and

Burkholderia with respect to Azotobacter and Pseudomonas , respectively. Although

Stenotrophomonas and Burkholderia spp. occurs ubiquitously in the environment, soil

and plants are their main environmental reservoirs and several studies subsequently

demonstrated that these two genus are capable of great metabolic versatility (Tabacchioni

et al . 2002; Ryan et al. 2009). Burkholderia spp. were for many years included in the

genus Pseudomonas owing to its broad and vague phenotypic definition. However,

rRNA DNA hybridization analyses during the early 1970s indicated considerable geneticdiversity among members of this genus (Compant et al . 2008). Therefore, to get reliable

and accurate identification of bacterial isolates, molecular characterization (16S rRNA

gene sequencing) is an important tool.

4.9 Development of liquid formulations

4.9.1 Liquid carriers for formulations

Carrier is an important component of biofertilizer technology and is defined as the

vehicle carrying efficient microbial strains from the laboratory to the field with minimum

damage to the viable cell population. To facilitate introduction of high cell numbers and

increased survival of microorganisms in soil, preparation of carrier based microbial

inoculants is pre-requisite (Bashan 1998). Solid carrier based preparations generally

suffer from short shelf-life, poor quality, high contamination and low and unpredictable

field performances (Vendan and Thangaraju 2006). To overcome these problems, the

liquid carrier based formulations have been introduced (Gupta 2005; Albareda et al .

2008). Liquid bioinoculants are special liquid formulations containing not only thedesired microorganisms and their nutrients, but also, special cell protectants or substances

that encourage the longer shelf life and tolerance to adverse conditions (Vora et al . 2008).

Also, a liquid inoculant formulation made from local low cost material may be useful to

the small producers especially in overcoming some of problems associated with

processing of the carrier (Singleton et al . 2002). Before recommending a bioinoculant for

crop production, its shelf life in different carrier materials needs to be addressed. Thus in


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the present investigation, the establishment of bacteria in different liquid carriers for their

survival was studied.

The various liquid carriers used in this study were Biogas Slurry, Vermiwash,

Compost Tea (compost wash), Matka Khaad and a synthetic medium (minimal growth

medium). These liquid carriers were used to study the shelf- life of inoculated efficient

biofertilizer isolates i.e. Stenotrophomonas maltophilia (WT-A2), Azospirillum

brasilense (MZ-AS2) and Burkholderia cepacia (PT-P2).

The results clearly showed that Matka Khaad (Table 4.14 to 4.25) was superior

then the other liquid carrriers [Appendix II (Table 4.26 to 4.73)] in maintaining higher

microbial load. Table 4.21 showed that Matka Khaad maintained 8.137 log cfu/ml, 8.166log cfu/ml and 8.188 log cfu/ml of Burkholderia cepacia (PT-P2), Stenotrophomonas

maltophilia (WT-A2) and Azospirillum brasilense (MZ-AS2), respectively, up to 240

days of incubation which was significantly higher than the other liquid carriers tested. In

Matka Khaad on 30 th day of incubation (Table 4.14), the treatments viz., trehalose and

glycerol were statistically at par with each other, whereas at 60-360 days of incubations,

all the treatments were significantly different. Matka Khaad with tehalose maintained

microbial population of 10.952 log cfu/ml on 30 th day of incubation and 6.798 log cfu/ml

on 360th

day of incubation. Whereas, glycerol as an additive maintained microbial population of 10.947 log cfu/ml and 6.738 log cfu/ml on 30 th and 360 th day of incubation,

respectively. Polyvinylpyrrolidone (PVP) was also effective additive but less efficient

then glycerol. This treatment maintained 5.898 log cfu/ml of inoculated strains on 360 th

day of incubation. After PVP, Polyethylene glycol (PEG) was found effective in

maintaining higher microbial load of 5.820 log cfu/ml on 360 th day of incubation. The

control treatment was found to be least effective in maintaining higher microbial load of

inoculated efficient strains. It maintained microbial population up to 5.308 log cfu/ml on

180 th day of incubation and thereafter the population decreased very rapidly. In the pooled data isolate MZ-AS2 was found to be most efficient and trehalose treatment was

found to be effective in maintaing statistically higher microbial load as compared to other

treatments, except on 30 th day of incubation at which trehalose treatment was at par with

glycerol treatment in Matka Khaad.

Table 4.14 Survival of efficient strains (log CFU/ml) in Matka Khaad on 30 th dayof incubation


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B. cepacia S. maltophi li a A. brasilense M eanTrehalose 10.933 10.950 10.973 10.952

PVP 10.890 10.920 10.940 10.917e

Glycerol 10.923 10.950 10.967 10.947PEG 10.863 10.893 10.910 10.889

Control 9.943 9.970 9.980 9.964 g Mean 10.711 a 10.737 10.754 c

CD at 5%1. Organi sms: 0.0062. Tr eatments: 0.0083. Organi sms and Tr eatments: NS


B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.900 10.930 10.957 10.929

PVP 10.807 10.840 10.863 10.837 e Glycerol 10.853 10.883 10.903 10.880

PEG 10.760 10.797 10.827 10.794 g Control 9.870 9.903 9.920 9.898Mean 10.638 a 10.671 10.694 c

CD at 5%1. Organi sms: 0.0062. Tr eatments: 0.0073. Organi sms and Tr eatments: NS



PVP 10.693 10.720 10.743 10.719 e Glycerol 10.730 10.763 10.780 10.758

PEG 10.230 10.267 10.290 10.262 g

Control 8.863 8.897 8.923 8.894Mean 10.258 a 10.289 10.311 c CD at 5%1. Organi sms: 0.0062. Tr eatments: 0.0083. Organi sms and Tr eatments: NS

Table 4.17 Survival of efficient strains (log CFU/ml) in Matka Khaad on 120 th day of incubation



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PVP 10.130 10.163 10.183 10.159 e Glycerol 10.273 10.303 10.320 10.299

PEG 9.933 9.963 9.983 9.960g

Control 7.840 7.870 7.897 7.869Mean 9.708 a 9.738 9.759 c

CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.007

3. Organisms and Tr eatments: NS



PVP 9.913 9.943 9.960 9.939 e Glycerol 9.933 9.963 9.983 9.960




Table 4.19 Survival of efficient strains (log CFU/ml) in Matka Khaad on 180 th day of incubationB. cepacia S. maltophi li a A. brasilense Mean

Trehalose 10.013 10.030 10.050 10.031PVP 9.903 9.933 9.950 9.929 e

Glycerol 9.920 9.943 9.963 9.942PEG 9.750 9.783 9.800 9.778 g

Control 5.280 5.313 5.330 5.308Mean 8.973 a 9.001 9.019 c


3. Organisms and Treatments: NSTable 4.20 Survival of efficient strains (log CFU/ml) in Matka Khaad on 210 th

day of incubation

B. cepacia S. maltophi li a A. brasilense Mean


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Trehalose 9.880 9.900 9.920 9.900PVP 9.790 9.823 9.850 9.821 e

Glycerol 9.830 9.863 9.880 9.858PEG 9.713 9.740 9.760 9.738 g Control 3.783 3.813 3.830 3.809Mean 8.599 a 8.628 8.648 c





PVP 9.623 9.650 9.670 9.648 e Glycerol 9.657 9.683 9.703 9.681






PVP 8.910 8.947 8.960 8.939 e Glycerol 8.933 8.963 8.983 8.960



3. Organ isms and Treatments: 0.013Table 4.23 Survival of efficient strains (log CFU/ml) in Matka Khaad on 300 th

day of incubationB. cepacia S. maltophi li a A. brasilense Mean

Trehalose 8.880 8.910 8.930 8.907PVP 8.820 8.850 8.873 8.848 e


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Glycerol 8.843 8.873 8.890 8.869PEG 8.517 8.553 8.570 8.547 g

Control 1.013 1.017 1.020 1.017Mean 7.215 a 7.241 7.257 c CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.008

3. Organ isms and Treatments: 0.014



PVP 7.850 7.883 7.900 7.878 e Glycerol 7.880 7.913 7.930 7.908




Table 4.25 Survival of efficient strains (log CFU/ml) in Matka Khaad on 360th

day of incubationB. cepacia S. maltophi li a A. brasilense Mean

Trehalose 6.770 6.800 6.823 6.798PVP 5.873 5.900 5.920 5.898 e

Glycerol 6.710 6.743 6.760 6.738PEG 5.793 5.823 5.843 5.820 g

Control 1.013 1.013 1.013 1.013Mean 5.232 a 5.256 5.272 c



After Matka Khaad, Compost Tea was the next effective liquid carrier for the

formulation development [Appendix II (Table 4.26 to 4.37)]. Biogas slurry was also

found to be effective [Appendix II (Table 4.38 to 4.49)], but less efficient then Compost

Tea. Vermiwash also helped in maintaining higher microbial population [Appendix II

(Table 4.50 to 4.61)], but not as effective as Biogas slurry. Minimal Growth Medium was


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found to be the least effective [Appendix II (Table 4.62 to 4.73)] as compared to the other

liquid carriers tested.

As such no information is available on nutritional status of Matka Khaad in the

literature, but higher survivability in this carrier might be attributed to its nutritional

status. Except for synthetic medium, all liquid carriers tested are rich in nutrients, and

that may be the reason for maintaining higher microbial load for longer duration. For

example, Compost Tea provides soluble nutrients, humic substances, and bioactive

substances that promote plant growth (Diver 2002). Vermiwash is a worm-extract that

has enzymes, secretions of earthworms which have soluble plant nutrients apart from

some organic acids and mucus of earthworms and microbes (Shivsubramanian and

Ganeshkumar 2004). In organic farming, the plant-based extracts are used in the

preparation of liquid manure that may include cow urine, cow dung, molasses, or wood

ashes. This liquid manure is sprayed on plants that provides soluble nutrients, plant

growth-promoting substances, and bioactive compounds that promote growth and help in

controlling insects, pests and diseases of plants (Diver 2002).

Among various additives tested, trehalose (Table 4.14 to 4.73) was found to be

most effective in maintaining higher microbial load for longer period as compared to

other additives used. The reasons for maintaining higher microbial load by these

additives as compared to control are discussed in section 4.9.2. Vendan and Thangaraju

(2006) developed liquid formulation of Azospirillum by using various cell protectants and

found trehalose to be most effective in maintaining higher population. Other workers also

used additives to improve the shelf life of formulation (Larena et al . 2005; Streeter 2006;

Tittabutr et al . 2007).

4.9.2 Effect of stress conditions on liquid formulation

Stress is an inevitable part of the life for all organisms. The bulk soil is generally

a very poor, nutrient-diluted and hostile environment for many microorganisms. In soil,

microorganisms are exposed to a range of variable biotic and abiotic stresses, such as

competition, predation, changes in temperature, osmolarity, availability of water etc.


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(Miller and Wood 1996; van Veen et al . 1997). The performance of inoculants is severely

affected by these stress factors (Zahran 1999; Vriezen et al . 2006) .

The important properties of a good inoculant are having a strain with high plant

growth promoting potential, capability of surviving in stressful conditions such as acidity,

salinity, different temperatures, moisture deficiency, etc. and able to adapt to the

formulation and storage conditions with minimal population reduction (Khavazi et al.

2007).

The efficient native isolates i.e. Stenotrophomonas maltophilia (WT-A2),

Azospirillum brasilense (MZ-AS2) and Burkholderia cepacia (PT-P2) were subjected to

various stress conditions in the best liquid formulation i.e. Matka Khaad.

i. Effect of Temperature

The effect of temperature viz., 15, 25, 40 and 50 C on the survivability of the

efficient strains in the Matka Khaad is shown in Table 4.74 to Table 4.85. It was

observed that at 15 C temperature (Table 4.74 to Table 4.77), the efficient isolates

showed highest survivability upto 45 days of incubation [ Stenotrophomonas maltophilia

(10.113 log cfu/ml), Azospirillum brasilense (10.133 log cfu/ml) and Burkholderia

cepacia (10.091 log cfu/ml)]. Whereas at 25 C (Table 4.78 to Table 4.81), the highest

survivability was observed upto 30 th day of incubation [( Stenotrophomonas maltophilia

(10.636 log cfu/ml), Azospirillum brasilense (10.671 log cfu/ml) and Burkholderia

cepacia (10.607 log cfu/ml)] and the efficient isolates showed highest survivability at 15 th

day of incubation [( Stenotrophomonas maltophilia (9.924 log cfu/ml), Azospirillum

brasilense (9.949 log cfu/ml) and Burkholderia cepacia (9.892 log cfu/ml)] at 40 C

(Table 4.82 to Table 4.85). At 50 C, none of the efficient isolates was able to grow. The

treatment with trehalose was found to be the most effective in maintaining high microbial

load as compared to the other treatments at different incubation intervals. Only at 15 C,

Table 4.74 Survival of efficient strains (log CFU/ml) in Matka khaad at 15C on15 th day of incubation


PVP 10.263 10.280 10.300 10.281 e Glycerol 10.270 10.300 10.323 10.298

PEG 10.243 10.273 10.293 10.270 g


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Control 9.160 9.193 9.220 9.191Mean 10.044 a 10.072 10.094 c

CD on 5%1 .Organisms: 0.0062. Tr eatments: 0.0073. Organisms and Tr eatments: NS



PVP 10.283 10.303 10.323 10.303 e Glycerol 10.293 10.313 10.340 10.316


CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0073. Organ isms and Treatments: 0.012


B. cepacia S. maltophi li a A.

brasilense Mean Trehalose 10.333 10.350 10.370 10.351PVP 10.300 10.323 10.350 10.324 e

Glycerol 10.320 10.343 10.363 10.342PEG 10.293 10.313 10.333 10.313 g

Control 9.210 9.233 9.250 9.231Mean 10.091 a 10.113 10.133 c




PVP 10.283 10.307 10.313 10.301 e Glycerol 10.303 10.333 10.343 10.327

PEG 10.240 10.280 10.293 10.271Control 9.183 9.203 9.233 9.207 g Mean 10.063 a 10.091 10.107 c


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PVP 10.783 10.820 10.850 10.818 e Glycerol 10.803 10.840 10.880 10.841

PEG 10.750 10.790 10.840 10.793 g Control 9.743 9.773 9.793 9.770

Mean 10.581a 10.615 10.649

c




PVP 10.807 10.843 10.873 10.841 e Glycerol 10.833 10.863 10.893 10.863





PVP 10.780 10.813 10.840 10.811 e Glycerol 10.810 10.847 10.860 10.839


CD on 5%1 .Organisms: 0.0062. Tr eatments: 0.008


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PVP 10.753 10.790 10.803 10.782 e Glycerol 10.783 10.820 10.833 10.812





PVP 10.080 10.113 10.143 10.112 e Glycerol 10.107 10.143 10.160 10.137

PEG 10.060 10.090 10.120 10.090 g

Control 9.080 9.110 9.143 9.111Mean 9.892 a 9.924 9.949 c CD on 5%1 .Organisms: 0.0062. Tr eatments: 0.0083. Organisms and Tr eatments: NS



PVP 10.053 10.093 10.113 10.087 e Glycerol 10.070 10.110 10.143 10.108




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PVP 9.983 10.017 10.040 10.013 e Glycerol 10.043 10.080 10.083 10.070





PVP 9.950 9.990 10.013 9.984 e Glycerol 9.990 10.030 10.043 10.021



the treatment with trehalose and glycerol was found to be statistically at par upto 30 th day

of incubation. The interaction between treatments and microorganisms was found to be

non-significant at 15 C (Table 4.76), 25 C (Table 4.79) and 40 C (Table 4.82) on the

incubation day at which isolates showed highest survivability.

It was observed that (Table 4.74 to Table 4.85) population of efficient strains waslow at 15 C and 40 C as compared at 25 C. This might be due to the fact that organisms

grow well and multiply at 25 C which was nearer to optimum growth temperature.

ii. Effect of pH

pH is an important aspect of bacterial cell physiology over which the cell exerts

relatively tight regulation (Booth 1985).To examine the effect of different pH on the

survivability of the efficient isolates in the Matka Khaad, the bacterial isolates were


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grown in varying pH viz., 4.5, 5.5, 6.5 and 7.5. Tables 4.86 to 4.101 depict the effect of

different pH on the shelf life of the efficient isolates. It was observed that on 30 th day of

incubation all the isolates showed highest survivability at all the tested pH values (Table

4.87, Table 4.91, Table 4.95 and Table 4.99). The least survival was observed at pH 4.5

(Table 4.88) . The highest survivability at various tested pH was observed at pH 6.5

which was 10.697 log cfu/ml, 10.727 log cfu/ml and 10.743 log cfu/ml (Table 4.95) for

Burkholderia cepacia , Stenotrophomonas maltophilia and Azospirillum brasilense ,

respectively on 30 th day of incubation. Trehalose was found to be best additive that

maintained statistically higher microbial load as compared to the other additives used,

except at pH 4.5 on 30 th days of incubation (Table 4.88 ) where trehalose and glycerol

treatments were statistically at par. On 30 th day of incubation, the interaction between

treatments and microorganisms was found to be significant at pH 4.5 whereas, it was

non-significant at other pH values (Table 4.87, Table 4.91, Table 4.95 and Table 4.99).

Tables 4.86 to 4.101 show that pH 4.5, 5.5 and 7.5 maintained lower microbial

load as compared to pH 6.5. This could be due to the fact that pH 6.5 is closer to neutral

and is optimum for microbial growth as compared to other tested pH values.

Table 4.86 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 4.5 on15 th day of incubation

B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.51 0 10.530 10.543 10.528

PVP 10.453 10.463 10.480 10.466 e Glycerol 10.503 10.513 10.523 10.513




B. cepacia S. maltophi li a A. brasilense Mean


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Trehalose 10.523 10.543 10.560 10.542PVP 10.463 10.493 10.523 10.493 e

Glycerol 10.520 10.533 10.563 10.539PEG 10.453 10.470 10.523 10.482 g Control 9.313 9.343 9.363 9.340Mean 10.255 a 10.277 10.307 c CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0063. Organ isms and Treatments: 0.011



PVP 10.430 10.460 10.473 10.454 e Glycerol 10.483 10.493 10.500 10.492


CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.007

3. Organ isms and Treatments: 0.012Table 4.89 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 4.5 on

60 th day of incubationB. cepacia S. maltophi li a A. brasilense Mean

Trehalose 10.463 10.490 10.503 10.486PVP 10.403 10.433 10.443 10.427

Isolation and Screening of Nitrogen Fixers and Phosphate Solubilizing Bacteria

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