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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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Isolate Gramstaining
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
Isolate Gramstaining
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
Table 4.15 Survival of efficient strains (log CFU/ml) in Matka Khaad on 60 th dayof incubation
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
Table 4.16 Survival of efficient strains (log CFU/ml) in Matka Khaad on 90 th dayof incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.773 10.800 10.820 10.798
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
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.363 10.390 10.410 10.388
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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
Table 4.18 Survival of efficient strains (log CFU/ml) in Matka Khaad on 150 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.233 10.260 10.280 10.258
PVP 9.913 9.943 9.960 9.939 e Glycerol 9.933 9.963 9.983 9.960
PEG 9.803 9.840 9.860 9.834 g Control 6.810 6.843 6.860 6.838Mean 9.339 a 9.370 9.389 c
CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.008
3. Organisms and Tr eatments: NS
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
CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.008
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
CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.008
3. Organisms and Tr eatments: NS
Table 4.21 Survival of efficient strains (log CFU/ml) in Matka Khaad on 240 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 9.733 9.763 9.787 9.761
PVP 9.623 9.650 9.670 9.648 e Glycerol 9.657 9.683 9.703 9.681
PEG 9.560 9.593 9.610 9.588 g Control 2.110 2.140 2.170 2.140Mean 8.137 a 8.166 8.188 c
CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.008
3. Organisms and Tr eatments: NS
Table 4.22 Survival of efficient strains (log CFU/ml) in Matka Khaad on 270 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 9.020 9.030 9.043 9.031
PVP 8.910 8.947 8.960 8.939 e Glycerol 8.933 8.963 8.983 8.960
PEG 8.680 8.713 8.743 8.712 g Control 1.013 1.037 1.060 1.037Mean 7.311 a 7.338 7.358 c
CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.007
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
Table 4.24 Survival of efficient strains (log CFU/ml) in Matka Khaad on 330 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 7.910 7.950 7.967 7.942
PVP 7.850 7.883 7.900 7.878 e Glycerol 7.880 7.913 7.930 7.908
PEG 7.830 7.863 7.880 7.858 g Control 1.013 1.013 1.013 1.013Mean 6.497 a 6.525 6.538 c
CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.008
3. Organ isms and Treatments: 0.014
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
CD at 5%1 .Organisms: 0.0062. Tr eatments: 0.007
3. Organ isms and Treatments: 0.013
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
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.283 10.313 10.333 10.310
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
Table 4.75 Survival of efficient strains (log CFU/ml) in Matka khaad at 15C on30 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.290 10.323 10.343 10.319
PVP 10.283 10.303 10.323 10.303 e Glycerol 10.293 10.313 10.340 10.316
PEG 10.263 10.293 10.310 10.289 g Control 9.183 9.210 9.253 9.216Mean 10.063 a 10.089 10.114 c
CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0073. Organ isms and Treatments: 0.012
Table 4.76 Survival of efficient strains (log CFU/ml) in Matka khaad at 15C on45 th day of incubation
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
CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0083. Organisms and Tr eatments: NS
Table 4.77 Survival of efficient strains (log CFU/ml) in Matka khaad at 15C on60 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.307 10.333 10.353 10.331
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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CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0063. Organ isms and Treatments: 0.011
Table 4.78 Survival of efficient strains (log CFU/ml) in Matka khaad at 25C on15 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.823 10.853 10.883 10.853
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
CD on 5%1 .Organisms: 0.0062. Tr eatments: 0.0083. Organ isms and Treatments: 0.013
Table 4.79 Survival of efficient strains (log CFU/ml) in Matka khaad at 25C on30 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.843 10.870 10.900 10.871
PVP 10.807 10.843 10.873 10.841 e Glycerol 10.833 10.863 10.893 10.863
PEG 10.780 10.810 10.863 10.818 g Control 9.770 9.793 9.823 9.796Mean 10.607 a 10.636 10.671 c
CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0073. Organ isms and Treatments: 0.012
Table 4.80 Survival of efficient strains (log CFU/ml) in Matka khaad at 25C on45 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.820 10.863 10.893 10.859
PVP 10.780 10.813 10.840 10.811 e Glycerol 10.810 10.847 10.860 10.839
PEG 10.753 10.783 10.820 10.786 g Control 9.750 9.783 9.813 9.782Mean 10.583 a 10.618 10.645 c
CD on 5%1 .Organisms: 0.0062. Tr eatments: 0.008
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3. Organisms and Tr eatments: NS
Table 4.81 Survival of efficient strains (log CFU/ml) in Matka khaad at 25C on60 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.790 10.833 10.863 10.829
PVP 10.753 10.790 10.803 10.782 e Glycerol 10.783 10.820 10.833 10.812
PEG 10.713 10.753 10.787 10.751 g Control 9.723 9.750 9.780 9.751Mean 10.553 a 10.589 10.613 c
CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0073. Organ isms and Treatments: 0.012
Table 4.82 Survival of efficient strains (log CFU/ml) in Matka khaad at 40C on15 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.133 10.163 10.180 10.159
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
Table 4.83 Survival of efficient strains (log CFU/ml) in Matka khaad at 40C on30 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.103 10.143 10.163 10.137
PVP 10.053 10.093 10.113 10.087 e Glycerol 10.070 10.110 10.143 10.108
PEG 10.013 10.053 10.073 10.047 g Control 9.033 9.053 9.083 9.057Mean 9.855 a 9.891 9.915 c
CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0063. Organisms and Tr eatments: NS
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Table 4.84 Survival of efficient strains (log CFU/ml) in Matka khaad at 40C on45 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.060 10.100 10.113 10.091
PVP 9.983 10.017 10.040 10.013 e Glycerol 10.043 10.080 10.083 10.070
PEG 9.940 9.980 10.003 9.974 g Control 8.963 8.993 9.013 8.990Mean 9.798 a 9.835 9.851 c
CD on 5%1 .Organisms: 0.0052. Tr eatments: 0.0073. Organisms and Tr eatments: NS
Table 4.85 Survival of efficient strains (log CFU/ml) in Matka khaad at 40C on60 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.033 10.073 10.090 10.066
PVP 9.950 9.990 10.013 9.984 e Glycerol 9.990 10.030 10.043 10.021
PEG 9.900 9.943 9.960 9.934 g Control 8.923 8.953 8.970 8.949Mean 9.759 a 9.798 9.815 c
CD on 5%1 .Organisms: 0.0062. Tr eatments: 0.0073. Organisms and Tr eatments: NS
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
PEG 10.430 10.443 10.460 10.444 g Control 9.273 9.300 9.323 9.299Mean 10.234 a 10.250 10.266 c
CD on 5%1 .Organisms: 0.0062. Tr eatments: 0.0073. Organisms and Tr eatments: NS
Table 4.87 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 4.5 on30 th day of incubation
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
Table 4.88 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 4.5 on45 th day of incubation
B. cepacia S. maltophi li a A. brasilense Mean Trehalose 10.493 10.507 10.533 10.511
PVP 10.430 10.460 10.473 10.454 e Glycerol 10.483 10.493 10.500 10.492
PEG 10.417 10.423 10.443 10.428 g Control 9.263 9.293 9.310 9.289Mean 10.217 a 10.235 10.252 c
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