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4. RESULTS AND DISCUSSION

The experimental results derived from the present study are presented and discussed here in light of the existing literature under the following sub-headings: 4.1 4.2 4.3 4.4 4.5 Isolation and screening of nitrogen fixers and phosphate solubilizing bacteria Qualitative assay of phosphate solubilizing activity Quantitative assay of phosphate solubilizing activity Quantitative assay of Nitrogenase activity Detection of Indole acetic acid (IAA) production in nitrogen fixers and phosphate solubilizing bacteria 4.6 Detection of siderophore production in nitrogen fixing and phosphate solubilizing bacteria 4.7 4.8 Detection of ammonia production Characterization, identification and maintenance of isolated microbial strains 4.8.1 4.8.2 4.9 Morphological and Biochemical characterization Molecular characterization of efficient strains

Development of liquid formulations 4.9.1 4.9.2 Liquid carriers for formulations 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 root exudation. 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 C2H4 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).

Table 4.1 Plant

Isolation of nitrogen fixers and phosphate solubilizing microorganisms from the rhizosphere of different crop and medicinal plants No. of soil samples Azotobacter Azospirillum P-solubilizers

No. of isolates obtained

No. of efficient isolates (ARA >150 nmole C2H4 h-1 mg-1 protein) 4

No. of isolates obtained

No. of efficient isolates (ARA >150 nmole C2H4 h-1 mg-1 protein) 5

No. of isolates obtained

No. of efficient isolates (>5 mm zone of solubilization)

Wheat (Triticum aestivum) Maize (Zea mays) Potato (Solanum tuberosum) Aloevera (Aloe barbadensis) Brahmi (Bacopa monnieri) Total

12

18

16

5 5

8 10

3 5

13 15

6 5

11 19

4 7

25

43

18

52

20

61

24

11 9

Table 4.2

Segregation of efficient nitrogen fixers and phosphate solubilizing bacterial isolates obtained from the rhizosphere of different plants

Plant

Codes assigned to Azotobacter WT-A1*, WT-A2, WT-A3, WT-A4 MZ-A1, MZ-A2, MZ-A3

Codes assigned to Azospirillum WT-AS1*, WT-AS2, WT-AS3, WT-AS4, WT-AS5 MZ-AS1, MZ-AS2, MZ-AS3, MZAS4, MZ-AS5, MZ-AS6 PT-AS1, PT-AS2, PT-AS3, PT-AS4, PT-AS5 AV-AS1, AV-AS2

Codes assigned to P-solubilizing bacteria WT-P1*, WT-P2, WT-P3, WT-P4, WT-P5 MZ-P1, MZ-P2, MZ-P3, MZ-P4

Wheat (Triticum aestivum) Maize (Zea mays)

Potato (Solanum tuberosum) Aloevera (Aloe barbadensis) Brahmi (Bacopa monnieri)

PT-A1, PT-A2, PT-A3, PT-A4, PTA5 AV-A1, AV-A2, AV-A3

PT-P1, PT-P2, PT-P3, PT-P4, PTP5, PT-P6, PT-P7 AV-P1, AV-P2, AV-P3, AV-P4

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. 1 isolated from soil sample of Triticum aestivum * WT-P1 represents P-solubilizing bacteria no.1 isolated from soil sample of Triticum aestivum.

12 0

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

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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-solubilization efficiency 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 5th day of incubation and their maximum values were varied from 205.42 to 635.60 g P/ml. Eleven isolates showed maximum solubilization on 7th 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 S. No. Solubilization efficiency of different P-solubilizing bacterial isolates on Pikovskayas agar medium on 10th day of incubation Isolate Zone (mm) Colony diameter (mm) 8.0 %S.E

WT-P1

17.5

118.7

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2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

WT-P2 WT-P3 WT-P4 WT-P5 MZ-P1 MZ-P2 MZ-P3 MZ-P4 PT-P1 PT -P2 PT -P3 PT-P4 PT-P5 PT-P6 PT-P7 AV-P1 AV-P2 AV-P3 AV-P4 BM-P1 BM-P2 BM-P3 BM-P4 P. striata

16.5 20.0 22.0 15.0 15.5 12.0 24.0 19.0 11.0 26.0 17.0 21.0 15.0 21.0 19.0 17.0 17.0 14.0 19.0 16.0 24.0 10.0 21.0 21.0

9.5 10.0 15.0 9.5 10.5 7.0 10.0 11.0 6.0 9.0 12.0 11.5 10.0 12.0 12.5 12.5 11.0 9.0 13.0 11.0 18.0 5.5 13.0 11.0

73.6 100.0 46.6 57.8 47.6 71.4 140.0 72.2 83.3 188.8 41.6 82.6 50.0 75.0 52.0 36.0 54.5 55.5 46.1 45.4 33.3 81.8 61.5 90.9

Table 4.4

Quantitative assay of phosphate solubilization and pH changes exhibited by different bacterial isolates in NBRIP broth
Phosphate solubilization (g/ml) Days of incubation 5 7 360.44 415.46 285.30 380.33 575.57 520.43 490.49 425.56 195.33 240.50 160.52 290.46 395.44 325.65 370.60 585.53 635.60 615.70 180.53 240.38 660.46 685.67 295.67 265.46 320.55 385.62 205.42 180.53 250.83 225.66 310.49 260.37 230.48 270.50 190.48 265.40 280.46 200.38 240.56 325.16 245.37 210.55 495.58 385.74 240.50 180.41 225.40 195.62 475.41 560.61 332.70 345.51 CD (P0.01) 1.027 pH of medium Days of incubation 5 5.43 4.77 4.58 4.81 4.60 5.58 5.04 5.18 5.68 4.92 5.18 4.90 5.31 5.33 4.41 4.83 4.91 5.03 4.83 4.58 5.18 4.40 5.31 6.03 5.54 5.05 CD (P0.01) 0.258

Isolate WT-P1 WT-P2 WT-P3 WT-P4 WT-P5 MZ-P1 MZ-P2 MZ-P3 MZ-P4 PT-P1 PT -P2 PT -P3 PT-P4 PT-P5 PT-P6 PT-P7 AV-P1 AV-P2 AV-P3 AV-P4 BM-P1 BM-P2 BM-P3 BM-P4 P. striata Mean Variant Isolate

3 145.61 105.40 240.45 220.64 90.65 90.46 180.57 130.68 320.57 110.65 375.66 150.64 105.71 120.13 145.56 125.53 120.79 100.44 210.35 145.57 120.34 190.43 95.32 105.48 300.46 161.92 SEm 0.279

11 375.64 290.66 505.39 380.37 185.74 265.34 280.45 410.74 580.49 220.58 620.39 210.51 210.47 140.50 170.64 205.68 240.71 230.25 120.49 255.73 185.44 290.28 240.35 155.60 505.42 291.11

Mean 324.29 265.42 460.46 379.26 178.05 201.69 295.52 374.39 538.09 188.04 585.54 230.57 255.58 161.64 198.17 225.52 215.62 196.64 202.92 241.76 190.42 340.51 189.14 170.52 460.48

3 5.00 5.10 5.53 5.03 4.93 6.03 6.03 5.70 5.33 5.63 5.91 5.31 5.90 6.15 5.72 5.54 5.92 5.71 5.71 5.62 5.62 5.92 5.60 6.21 6.02 5.65 SEm 0.070

7 5.80 4.91 4.81 4.08 5.32 4.33 5.31 4.81 5.05 4.44 4.78 4.60 4.91 5.72 4.70 4.42 5.04 4.82 5.23 4.61 4.54 4.82 5.56 5.73 4.33 4.91

11 5.33 5.28 4.81 4.60 5.57 4.58 5.75 5.20 5.41 5.10 5.32 4.91 5.10 5.91 5.12 4.45 5.65 5.34 5.53 4.82 4.56 5.22 5.83 5.93 4.54 5.19

Mean 5.39 5.01 4.93 4.63 5.11 5.13 5.54 5.22 5.51 5.02 5.15 4.93 5.30 5.78 4.99 4.81 5.38 5.22 5.32 4.91 4.97 5.09 5.58 5.97 5.11

12 4

Day Interaction

0.112 0.559

0.411 2.055

0.028 0.140

0.103 0.517

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thereafter, which continued upto 11th 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 be attributed 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 (585.53 g 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 mL1 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 PTP2 obtained from maize showed a maximum solubilization of 685.67 g P/ml at a minimum pH of 4.78 on 7th 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.

Figure 4.1 Trend of pH changes in context with phosphate solubilizing kinetics as exhibited by efficient isolates (----- represent pH changes and represent P-solubilization) 12 6

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In the present study no relationship could be ascertained with the quantity of Psolubilized 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 (N2) 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 N2 to generate NH3 (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 N2 fixation and is based on the assumption that 34 mol acetylene are reduced to ethylene for every mole of N2 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 C2H4 h-1 mg-1 protein). These six isolates were WT-A1 (441.58 nmole C2H4 h-1 mg-1 protein), WT-A2 (451.45 nmole C2H4 h-1 mg-1 protein), MZ-A2 (440.91 nmole C2H4 h-1 mg-1 protein), PT-A1 (444.02 nmole C2H4 h-1 mg-1 protein), PT-A3 (383.64 nmole C2H4 h-1 mg-1 protein), and BM-A3 (374.44 nmole C2H4 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 S.No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Nitrogenase activity of native isolates of nitrogen fixers isolated from different crop plants Isolate (Azotobacter) WT-A1 WT-A2 WT-A3 WT-A4 MZ-A1 MZ-A2 MZ-A3 PT-A1 PT -A2 PT -A3 PT-A4 PT-A5 AV-A1 AV-A2 AV-A3 BM-A1 BM-A2 BM-A3 A. chroococcum Nitrogenase activity* 441.58a 451.45b 225.48c 256.29d 287.52e 440.91a 194.37f 444.02g 183.23h 383.64i 155.40j 237.63k 241.28l 168.49m 291.60n 151.51o 207.41p 374.44q 372.85r Isolate (Azospirillum) WT-AS1 WT-AS2 WT-AS3 WT-AS4 WT-AS5 MZ-AS1 MZ-AS2 MZ-AS3 MZ-AS4 MZ-AS5 MZ-AS6 PT-AS1 PT -AS2 PT -AS3 PT-AS4 PT-AS5 AV-AS1 AV-AS2 BM-AS1 BM-AS2 Azospirillum brasilense Nitrogenase activity* 202.50a 304.58b 458.33c 219.68d 414.83e 157.53f 462.33g 229.61h 358.47i 175.57j 327.73k 398.46l 153.23m 405.55n 346.32o 428.46p 177.79q 421.38r 312.29s 264.72t 394.48u

Each value represents mean of three replicates. In the same column, significant differences according to LSD at P 0.01 levels are indicated by different letters. Same letters represent that their values are at par. *nmol C2H4 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 C2H4 h-1 mg-1 protein). These isolates were WT-AS3 (458.33 nmole C2H4 h-1 mg-1 protein), WT-AS5 (414.83 nmole C2H4 h-1 mg-1 protein), MZ-AS2 (462.33 nmole C2H4 h1

mg-1 protein), PT-AS1 (398.46 nmole C2H4 h-1 mg-1 protein), PT-AS3 (405.55 nmole

C2H4 h-1 mg-1 protein), PT-AS5 (428.46 nmole C2H4 h-1 mg-1 protein), and AV-AS2 (421.38 nmole C2H4 h-1 mg-1 protein). The most efficient Azospirillum isolate was MZAS2 (462.33 nmole C2H4 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 and phosphate 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 to nitrogenase activity, showed 17.45 g/ml of IAA production which was significantly higher than the standard strain.

Table 4.6 Indole Acetic Acid (IAA) Production by Phosphate solubilizing and nitrogen fixing native bacteria Isolate IAA Conc. Isolate IAA Conc. Isolate IAA Conc. (PSB) (g/ml) (Azotobacter) (g/ml) (Azospirillum) (g/ml) a a 1 WT-P1 14.52 WT-A1 8.65 WT-AS1 2 WT-P2 WT-A2 17.45b WT-AS2 9.32a 3 WT-P3 17.30b WT-A3 10.66c WT-AS3 13.42b 4 WT-P4 WT-A4 WT-AS4 d 5 WT-P5 MZ-A1 18.14 WT-AS5 12.73c 6 MZ-P1 MZ-A2 10.96e MZ-AS1 7 MZ-P2 MZ-A3 MZ-AS2 18.27d 8 MZ-P3 19.15c PT-A1 MZ-AS3 d f 9 MZ-P4 15.91 PT -A2 12.51 MZ-AS4 10 PT-P1 PT -A3 15.14g MZ-AS5 14.16e 11 PT -P2 16.68d PT-A4 16.06h MZ-AS6 7.39f 12 PT -P3 13.28e PT-A5 12.05f PT-AS1 15.87g i 13 PT-P4 AV-A1 20.35 PT -AS2 f j 14 PT-P5 15.29 AV-A2 12.82 PT -AS3 12.26c 15 PT-P6 AV-A3 16.37h PT-AS4 14.78e 16 PT-P7 BM-A1 PT-AS5 17 AV-P1 BM-A2 11.89f AV-AS1 10.56h 18 AV-P2 BM-A3 AV-AS2 8.95a 19 AV-P3 12.51e A. chroococcum 15.51g BM-AS1 20 AV-P4 BM-AS2 20.06i 21 BM-P1 Azospirillum brasilense 14.36e g 22 BM-P2 11.89 23 BM-P3 24 BM-P4 25 P. striata 14.40a Each value represents mean of three replicates. In the same column, significant differences according to LSD at P 0.01 leve ls are indicated by different letters. Same letters represent that their values are at par. S.No.

13 0

131

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

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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 (19.15 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 (Glick 1995). Supplementation of culture medium with tryptophan helps the microorganisms to produce IAA from it. It has been reported by various workers that the precursor, Ltryptophan 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 phosphate solubilizing 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

133

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, PTA3, 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 phosphate solubilizing 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, PTA4, 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, MZAS2, MZ-AS3, MZ-AS4, PT-AS1, PT-AS4, PT-AS5, AV-AS1, AV-AS2, and BM-AS2

Table 4.7 Siderophore Production by Phosphate solubilizing and nitrogen fixing native bacteria S.No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Isolate (PSB) WT-P1 WT-P2 WT-P3 WT-P4 WT-P5 MZ-P1 MZ-P2 MZ-P3 MZ-P4 PT-P1 PT -P2 PT -P3 PT-P4 PT-P5 PT-P6 PT-P7 AV-P1 AV-P2 AV-P3 AV-P4 BM-P1 Siderophore production ++ +++ + + + + ++ +++ + ++ + Isolate (Azotobacter) WT-A1 WT-A2 WT-A3 WT-A4 MZ-A1 MZ-A2 MZ-A3 PT-A1 PT -A2 PT -A3 PT-A4 PT-A5 AV-A1 AV-A2 AV-A3 BM-A1 BM-A2 BM-A3 A.chroococcum Siderophore production ++ + + +++ + + + ++ ++ + Isolate (Azospirillum) WT-AS1 WT-AS2 WT-AS3 WT-AS4 WT-AS5 MZ-AS1 MZ-AS2 MZ-AS3 MZ-AS4 MZ-AS5 MZ-AS6 PT-AS1 PT -AS2 PT -AS3 PT-AS4 PT-AS5 AV-AS1 AV-AS2 BM-AS1 BM-AS2 Azospirillum brasilense Siderophore production + ++ + ++ +++ + + ++ + ++ +++ + +

BM-P2 ++ BM-P3 BM-P4 P.striata ++ - : No Siderophore Production +, ++, +++: represents 8-10, 11-20 and >20 mm orange zone respectively

13 4

Table 4.8 Ammonia Production by Nitrogen and Phosphate solubilizing native bacteria S.No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Isolate Ammonia (PSB) production WT-P1 + WT-P2 + WT-P3 + WT-P4 WT-P5 + MZ-P1 MZ-P2 + MZ-P3 MZ-P4 + PT-P1 + PT -P2 + PT -P3 + PT-P4 + PT-P5 + PT-P6 PT-P7 AV-P1 + AV-P2 AV-P3 + AV-P4 + BM-P1 BM-P2 BM-P3 + BM-P4 P. striata + - : Negative for ammonia Production +: Positive for ammonia production Isolate Ammonia (Azotobacter) production WT-A1 + WT-A2 + WT-A3 + WT-A4 MZ-A1 MZ-A2 + MZ-A3 PT-A1 + PT -A2 PT -A3 PT-A4 + PT-A5 + AV-A1 AV-A2 + AV-A3 + BM-A1 + BM-A2 BM-A3 + A. chroococcum + Isolate Ammonia (Azospirillum) production WT-AS1 WT-AS2 + WT-AS3 + WT-AS4 + WT-AS5 MZ-AS1 + MZ-AS2 + MZ-AS3 + MZ-AS4 + MZ-AS5 MZ-AS6 PT-AS1 + PT -AS2 PT -AS3 PT-AS4 + PT-AS5 + AV-AS1 + AV-AS2 + BM-AS1 BM-AS2 + Azospirillum brasilense +

13 5

136

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 N2 fixing bacteria in crop improvement under tropical and sub-tropical conditions especially with strains excreting a high amount

137

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 BMP3. 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 disease control (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 4.8.1 Characterization and identification of phosphate solubilizing and nitrogen fixing bacterial isolates Morphological and Biochemical characterization

138

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 three isolates (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 the rhizospheric 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 probably adapted to the agroclimatic conditions of Himachal Pradesh in a better way and thus need to be exploited for the preparation of bioinoculants.

139

C 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

Table 4.9 Morphological and Biochemical characteristics of phosphate solubilizing bacteria Gram Morphology Motility O C I MR VP Ci U H2S Utilization of Possible staining organism D M S L R A WT-P1 rods + + + + + - + - + - - Pseudomonas WT-P2 rods + + + - - + - - - Pseudomonas + WT-P3 rods + + + - - - + - - Acinetobacter WT-P4 rods + + + + + - + - - + Pseudomonas WT-P5 rods + + + + + - + - - - - Pseudomonas MZ-P1 rods + + + + - - + + - - U.I MZ-P2 cocci + + - + - - - - Micrococcus + MZ-P3 rods + + - + - + - - Pseudomonas + MZ-P4 rods + + + + + + + + - - Pseudomonas PT-P1 rods + + + + - - + - - - U.I PT -P2 rods + + + - + + + + - Pseudomonas + PT -P3 rods + + + + - - - - - - Pseudomonas PT-P4 rods + + + + + + - - - - Pseudomonas PT-P5 rods + + + + + + - - - - - Pseudomonas PT-P6 rods + + + + - - - - - Alcaligenes + PT-P7 rods + + + + + + - - - - - Pseudomonas AV-P1 rods + + + - - - - - - Pseudomonas AV-P2 rods + + + - - + + - - U.I AV-P3 rods + + + + - - - - - - Flavobacterium AV-P4 rods + + + - - - + - - Acinetobacter BM-P1 rods + + + - - - - - - Pseudomonas BM-P2 rods + + + + + - - - - Pseudomonas BM-P3 rods + + + + - - + + - - U.I BM-P4 rods + + + - - - - - - Pseudomonas O- Oxidase, C-Catalase, I-Indole, MR-Methyl red, VP-Voges Proskauer, Ci Citrate utilization, U- Urease, D-Dextrose, MMannitol, S-Sucrose, L-Lactose, R-Rhamnose, A-Adonitol 14 0 Table 4.10 Morphological and Biochemical characteristics of strains isolated on Jensens medium Isolate

Isolate WT-A1 WT-A2 WT-A3 WT-A4 MZ-A1 MZ-A2 MZ-A3 PT-A1 PT -A2 PT -A3 PT-A4 PT-A5 AV-A1 AV-A2 AV-A3 BM-A1 BM-A2 BM-A3

Gram Morphology Motility staining + rods rods rods rods rods rods rods rods rods rods rods rods rods rods rods rods rods rods + + + + + + + + + + + + + + +

O + + + + + + + + + + + + + + +

C + + + + + + + + + + + + + + + + + +

I -

MR -

VP + + + + + -

Ci + + + + + -

U + -

H2 S F + + + + + + + + + + + + + + + + + +

Utilization of T S Du So + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Ma + + -

O- Oxidase, C-Catalase, I-Indole, MR-Methyl red, VP-Voges Proskauer, Ci Citrate utilization, U- Urease, F-Fructose, TTrehalose, S-Sucrose, Du- Dulictol, So- Sorbitol, Ma- Maltose 141

Table 4.11 Morphological and Biochemical characteristics of strains isolated on NFb medium Isolate WT-AS1 WT-AS2 WT-AS3 WT-AS4 WT-AS5 MZ-AS1 MZ-AS2 MZ-AS3 MZ-AS4 MZ-AS5 MZ-AS6 PT-AS1 PT -AS2 PT -AS3 PT-AS4 PT-AS5 AV-AS1 AV-AS2 BM-AS1 BM-AS2 Gram Morphology Motility O staining rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + rods + + C + + + + + + + + + + + + + + + + + + I MR + VP + + + + + Ci + + + + + + + + U + + + + + + + + + + + + + + + + + + + + H2 S F + + + + + + + + + + + + + + + + + + + + Utilization of Me S Du So + + + + + + + + -

M + + 142

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

143

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 partial sequence 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

Table 4.12 Pair wise genetic distance of the six efficient isolates with other selected sequences from the NCBI
ISOLATES Stenotrophomonas maltophilia WT-A2 (GU371215)* Stenotrophomonas maltophilia (DQ257429) Stenotrophomonas sp. CK6 (AJ870967) Stenotrophomonas rhizophila (EU977698) Stenotrophomonas like sp.V4BP15 (AJ244720) Stenotrophomonas maltophilia(AJ131117) Bacillus licheniformis PT-A1 (GU371216)* Bacillus licheniformis (GQ375247) Bacillus licheniformis (GQ375245) Bacillus licheniformis (GQ375244) Bacillus licheniformis (AJ293011) Bacillus licheniformis (AB525389) Azospirillum brasilense MZ-AS2 (GU371217)* Azospirillum brasilense WT-AS3 (GU371218)* Azospirillum brasilense (AY324110) Azospirillum brasilense (Z29617) Azospirillum brasilense (AB480699) Azospirillum brasilense (DQ288688) Azospirillum sp. Ptl3 (GQ284588) Azospirillum sp. 7C (AF411852) Azospirillum sp. DA10-2 (AY118225) Pseudomonas aeruginosa MZ-P4 (GU371219)* Pseudomonas aeruginosa (FJ985806) Pseudomonas sp. YKM-M4 (GU272400) Pseudomonas aeruginosa (GU199190) Pseudomonas aeruginosa (GU181320) Burkholderia cepacia_PT-P2 (GU371220)* Burkholderia cepacia (GQ383907) Burkholderia cepacia (FJ652618) Burkholderia cepacia (FJ887895) Burkholderia sp.gx-152 (FJ823011) Burkholderia sp.LDSP-10 (FJ548994) Clostridium sp. (GU097452) 0.000 0.000 0.000 0.000 0.000 0.249 0.149 0.149 0.149 0.149 0.148 0.256 0.217 0.150 0.150 0.150 0.151 0.150 0.150 0.150 0.084 0.084 0.084 0.084 0.084 0.135 0.104 0.104 0.104 0.104 0.104 0.168 0.000 0.000 0.000 0.000 0.249 0.149 0.149 0.149 0.149 0.148 0.256 0.217 0.150 0.150 0.150 0.151 0.150 0.150 0.150 0.084 0.084 0.084 0.084 0.084 0.135 0.104 0.104 0.104 0.104 0.104 0.168 0.000 0.000 0.000 0.249 0.149 0.149 0.149 0.149 0.148 0.256 0.217 0.150 0.150 0.150 0.151 0.150 0.150 0.150 0.084 0.084 0.084 0.084 0.084 0.135 0.104 0.104 0.104 0.104 0.104 0.168 0.000 0.000 0.249 0.149 0.149 0.149 0.149 0.148 0.256 0.217 0.150 0.150 0.150 0.151 0.150 0.150 0.150 0.084 0.084 0.084 0.084 0.084 0.135 0.104 0.104 0.104 0.104 0.104 0.168 0.000 0.249 0.149 0.149 0.149 0.149 0.148 0.256 0.217 0.150 0.150 0.150 0.151 0.150 0.150 0.150 0.084 0.084 0.084 0.084 0.084 0.135 0.104 0.104 0.104 0.104 0.104 0.168 0.249 0.149 0.149 0.149 0.149 0.148 0.256 0.217 0.150 0.150 0.150 0.151 0.150 0.150 0.150 0.084 0.084 0.084 0.084 0.084 0.135 0.104 0.104 0.104 0.104 0.104 0.168 0.103 0.103 0.103 0.103 0.105 0.322 0.274 0.255 0.255 0.255 0.255 0.255 0.255 0.255 0.261 0.261 0.261 0.261 0.261 0.298 0.268 0.268 0.268 0.268 0.268 0.225 0.000 0.000 0.000 0.001 0.250 0.206 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.154 0.154 0.154 0.154 0.154 0.194 0.160 0.160 0.160 0.160 0.160 0.125 0.000 0.000 0.001 0.250 0.206 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.154 0.154 0.154 0.154 0.154 0.194 0.160 0.160 0.160 0.160 0.160 0.125 0.000 0.001 0.250 0.206 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.154 0.154 0.154 0.154 0.154 0.194 0.160 0.160 0.160 0.160 0.160 0.125 0.001 0.250 0.206 0.152 0.152 0.152 0.153 0.152 0.152 0.152 0.154 0.154 0.154 0.154 0.154 0.194 0.160 0.160 0.160 0.160 0.160 0.125 0.249 0.207 0.151 0.151 0.151 0.152 0.151 0.151 0.151 0.153 0.153 0.153 0.153 0.153 0.192 0.158 0.158 0.158 0.158 0.158 0.124 0.163 0.095 0.095 0.095 0.094 0.095 0.095 0.095 0.249 0.249 0.249 0.249 0.249 0.292 0.250 0.250 0.250 0.250 0.250 0.240 0.063 0.063 0.063 0.064 0.063 0.063 0.063 0.204 0.204 0.204 0.204 0.204 0.243 0.220 0.220 0.220 0.220 0.220 0.214 0.000 0.000 0.001 0.000 0.000 0.000 0.142 0.142 0.142 0.142 0.142 0.189 0.152 0.152 0.152 0.152 0.152 0.142 0.000 0.001 0.000 0.000 0.000 0.142 0.142 0.142 0.142 0.142 0.189 0.152 0.152 0.152 0.152 0.152 0.142 0.001 0.000 0.000 0.000 0.142 0.142 0.142 0.142 0.142 0.189 0.152 0.152 0.152 0.152 0.152 0.142 0.001 0.001 0.001 0.144 0.144 0.144 0.144 0.144 0.190 0.153 0.153 0.153 0.153 0.153 0.143 0.000 0.000 0.142 0.142 0.142 0.142 0.142 0.189 0.152 0.152 0.152 0.152 0.152 0.142 0.000 0.142 0.142 0.142 0.142 0.142 0.189 0.152 0.152 0.152 0.152 0.152 0.142 0.142 0.142 0.142 0.142 0.142 0.189 0.152 0.152 0.152 0.152 0.152 0.142 0.000 0.000 0.000 0.000 0.143 0.112 0.112 0.112 0.112 0.112 0.168 0.000 0.000 0.000 0.143 0.112 0.112 0.112 0.112 0.112 0.168 0.000 0.000 0.143 0.112 0.112 0.112 0.112 0.112 0.168 0.000 0.143 0.112 0.112 0.112 0.112 0.112 0.168 0.143 0.112 0.112 0.112 0.112 0.112 0.168 0.026 0.026 0.026 0.026 0.026 0.210 0.000 0.000 0.000 0.000 0.174 0.000 0.000 0.000 0.174 0.000 0.000 0.174 0.000 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

14 4

0.174

0.174

(*Represent the native isolates)

1 71 141 211 281 351 421 491 561 631 701 771 841 911 981 1051 1121 1191 1261 1331

GGAATACATCGGAATCTACCTTTTCGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGACC TTCGGGTGAAAGCAGGGGACCTTCGGGCCTTGCGCGGATAGATGAGCCGATGTCGGATTAGCTAGTTGGC GGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAG ACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCC ATACCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAAAGCAGTCAGCTAA TACCCGGTTGTTCTGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATAC GAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTTGTTTAAGTCTGTTGTG AAAGCCCTGGGCTCAACCTGGGAATTGCAGTGGATACTGGGCGACTAGAGTGTGGTAGAGGGTAGTGGAA TTCCCGGTGTAGCAGTGAAATGCGTAGAGATCGGGAGGAACATCCATGGCGAAGGCAGCTACCTGGACCA ACACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAA CGATGCGAACTGGATGTTGGGTGCAATTTGGCACGCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTG GGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGT TTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATGTCGAGAACTTTCCAGAGATGGATTGGT GCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT CCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGG TGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTAC TACAATGGTAGGGACAGAGGGCTGCAAACCCGCGAGGGCAAGCCAATCCCAGAAACCCTATCTCAGTCCG GATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTG AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGC TTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCG

70 140 210 280 350 420 490 560 630 700 770 840 910 980 1050 1120 1190 1260 1330 1366

A
1 71 141 211 281 351 421 491 561 631 701 771 841 911 981 GGACAGATGGGAGCTTGCTCCTGATGTTAGCGGCGGACGGTGGATTAGGACGTGGGTAACCTGCCTGTAA GACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAA AGGTGGCTTCTGGCTACCACTAACAGATGAACCGGCGGGGCTTTACCTGGTTGTGAGGGTACGGGCTCCC CAGGCGACCGACTTGGGCCGGCTTCGCTTTTTTTGGCCTTAATAGGGCTTAAAACCCGTCCAAAATCCTA CCAAACAACCTTTGGGAATCTTCCGAAATGTACGAAAGGCTTACCGGGGAAGGCAAAAAGATTTTTGAAG GTTTTCAGATTGTTAAATTCGGTTGGTGGGGGGGCCGGTTCCGTTCTAATTGGGGGGCCCCTGACGGGAC AAAACCCGAAAGCCCCCGCTTACTTCCTGCCAGCAAGCGCGGTAATTCGGAGGTGGGAAGGCTTTTCCGG ATTTTGGGCGCTCAAGCCGGCCCCCGCCGGCCCAAAGGCAAAAGGGAAGGAGGGGTTAGACGGGGGGGGT CCTTGGAAATTGGGGAACCTAAGGCAAAAGGGGAAAATCGAATTCCCCGGGAGCGGGGAAAATGGTTGGG GTTTGGGGAACACCCAGGGGGCAGGCTATTTTTTAGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGG AGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCG CCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAA GGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC AGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCA TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGT 70 140 210 280 350 420 490 560 630 700 770 840 910 980 1050

145

1051 1121 1191 1261 1331 1 71 141 211 281 351 421 491 561 631 701 771 841 911 981 1051 1121 1191 1261 1331 1 71 141 211 281 351 421 491 561 631 701 771

TGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCA AATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCG CGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCT GGAATCGCTAGTAATCGCGGTTCGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACAGACCCCAACAC ACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGTT

1120 1190 1260 1330 1366 70 140 210 280 350 420 490 560 630 700 770 840 910 980 1050 1120 1190 1260 1330 1366 70 140 210 280 350 420 490 560 630 700 770 840

B
CCGGAATTCGTCAAGTGTGAGCTGTAACAACAGTAAGAAGCTTCGGCTTTAGTGGCGCACGGGTGAGTAA CACGTGAGGTCGCTTTTGGTTCGGGATAACGTCTGGAAACGGACGCTAAAACGGATACGCCCTTCAGAGA GAATGGGCGGAGAAAGTTTACGCCGAGAGAGGGGCCCGCGTCCGATTAGGTATTTGGTGGGGTAATGGCC CACCAAGCCGACGATCGAGAGCTGGTCTGAGAGAATGATCAGCCACACTGGGACTGAGACACTACCCAGA CTCCTACGGGGGAATATTGGTGGGGAATATTGAACAATGGGGGGCAACCCTGATCCAGCAATGCCGCGTG AGTAGGGTTGTGCCTTAGGGTTGTAAAGCTCTTTCGCACGCGACGATGATGACAGAAGCGTGAGAAGAAG CGTGGGCTAACTTTTTTTTTAGCAGCCGCGGTAATACGAAGGGCGCGAATTACTGTTCGTAATTACTGCG CGTAAAGGGCGCGTAGGCAGCCCGATCAGCCAGAGGTTAAAGCCCCGGGGCTGAACCTTGAGACCTGCCT TTTTTAGTTTCCGGGGTTGAAGTTCCGAAGTCCCCAGGGGAAATCCCAATTTCGAAGGTAAAATTCGGAA GAAATTGGGAAGAAACCCGGTGTCTAACCGGCCAATTTGGCCGAAACCTTGGGGACCACCCAGGATTAGT TCCCTGGTAGTCCACGCCGTAACGTGAATTCCTAGCGCTGGGGTGCATGCACTCGGGTTTCGCCGCAACG CATAAGCATCCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAGGGAATTGACGGGGGCCCGCCCAAG CGGGTGGAGCATGTGGTTTTAATTCGGAAGCAACGCGCAGAACCTTACCAACCCTTGACATGTCCCACTA CCGGCTCGAGAGATCGGGCTTTCAGTTCGGCTGGGTGGAAAAAAGGTGCTGCATGGCTGTCGTCAGCTCG TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTACCGCCAGTTGCCATCATTCAGTTGG GCACTCTGGTGGAACTGCCGGTGACAAGCCGGAGGAAGGCGGGGATGACGTCAAGTCCTCATGGCCCTTA TGGGTTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGATGCGAAGTCGCAAGATGGAGCCAATCC CCAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGGGTGCATGAAGTTGGAATCGCTAGTAATCGC GGATCCCCCCGCGGTGAATACGTTCCCGGCCTGTACACAAACACCCCATGGAGTGCTACCGAAGGGTCGC TATACAAGAGTTGATCATGGCAGCCCCGGGCATTCG

C
GAGGGGCCCGCGTCCGATTAGGTAGTTGGTGGGGTAATGGCCCACCAAGCCGACGATCGGTAGCTGGTCT GAGAAAATGATCAGCCACAATGGGACTGAGACACGGCCCAGACTCCTACGGGAGGTAGCAGTGGATAATA TTGAACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGAGTGATGAAGGACTTAGGTTTGTAAAGCTC TTTCGCACGCGACGATGATGACGGTAGCGTGAGAAGAAGCCCCGGCTAATTTTTTTTTCAGCAGCCGCGG TAATACGAAGGGGGGGAAGCGCTGTTCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCCCGATCAAGC CAGAAGTTAAAGCCCCGGGACTTGAACTTGGGAACTGCATTTTTTTACTTTCCGGGCTTGAGTTCCGGGA GAGGATGGTGGAAATTCCCAATTTTGGAGGTGAAATTCGGAAAATATTGGGGAATTTGACTATTGGGGCA ACCTGATCAGCATTGCGCGTGAGGATGGACGCCTAGGATGTAAGCTCTTCGCACGCGACGATGATGACGT AGCGTGAAGAAGAAGCCCGCTAACCTCGTGCCAGCAGCGCGGTAATACGAAAGGGGGGGCGAGCGTTGTT CGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCCGATCAGTCAGATGTGAAAGCCCGGGCTCAACCTGG GAACTGCATTTGATACTGTCGGGCTTGAGTTCCCGGAGAGGATGGTGGAATTCCCAGTGTAGAGGTGAAA TTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCCATCTGGACGGACACTGACGCTGAGGCGCGA

146

841 911 981 1051 1121 1191 1261 1331

AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGACGCTGG GGTGCATGCACTTCGGTGTCGCCGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGTTAA AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGA ACCTTACCAACCCTTGACATGTCCACTATCGGCTCGAGAGATCGGGCTTTCAGTTCGGCTGGGTGGAACA CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC CTACCGCCAGTTGCCATCATTCAGTTGGGCACTCTGGTGGAACTGCCGGTGACAAGCCGGAGGAAGGCGG GGATGACGTCAAGTCCTCATGGCCCTTATGGGTTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGG ATGCGAAGTCGCAAGATGGAGCCAATCCCCAAAAGC

910 980 1050 1120 1190 1260 1330 1366

D
1 71 141 211 281 351 421 491 561 631 701 771 841 911 981 1051 1121 1191 1261 1331 GGATGAAGGGAGCTTGCTCTGGATTCAGCGGCGGACGGGCGGGAAGGCCTAGGAATCTGCCTGGTAGTGG GGGATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTGAGGGAGAAAGTGGGGGATCTTCGGACC TCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCC GTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGC AGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGA TTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGA ATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACT GGGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCA TCCAAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGAT ATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGG GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGA GATCTTAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAA TGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC TGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCA TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGT TACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTC AAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCC GCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGT CGGAATCGCTAGTAATCGTGATTCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACTCCCTC ACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGT 70 140 210 280 350 420 490 560 630 700 770 840 910 980 1050 1120 1190 1260 1330 1366

E
1 71 141 211 281 351 421 AAGTAAGTCCATGTGGAACATGTAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAA AGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAA TCCTTGGCTCTAATACAGTCGGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCA GCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGC TAAGACCGTGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGACTGGCAGGCTAGATTATGGCAG AGGGGGGTAGAATTCCACGTGTAGCAATGAAATGCGTAGAGATGTGGAGGAAACCGATGGCGAAGGCAGC CCCCTGGGCCATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACACTGGTAGTCC 70 140 210 280 350 420 490

147

491 561 631 701 771 841 911

ACGCCCTAAACGATGTCAACTAGTTTTGGGGATTCATTTCCTTAGTAACATAGCTAACGCGTGAAGTTGA CCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATG ATGTGGATTAATTCGATGCACCGCGAAAAACCTTACCTACCCTTGACATGGTCGGAATCCTGCTGAGAGG TGGGAGTGCTCGAAAGAGAACCGCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG GGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAAGGAGACTGCCG GTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCGGACTTCTGAATGCGGCATTACC CAGTAGATTC

560 630 700 770 840 910 920

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

148

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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-P4 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 et al. 2010). Table 4.13 Molecular characterized (16S rRNA gene sequencing) efficient native PGPR isolates Isolate Stenotrophomonas maltophilia Bacillus licheniformis Azospirillum brasilense Azospirillum brasilense Pseudomonas aeruginosa Burkholderia cepacia Accession No. GU371215 GU371216 GU371217 GU371218 GU371219 GU371220

S.No. 1. 2. 3. 4. 5. 6.

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0.02

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

151

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, rRNADNA hybridization analyses during the early 1970s indicated considerable genetic diversity 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 4.9.1 Development of liquid formulations 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 the desired 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, 8.166 log 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 30th 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 30th 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 30th and 360th 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 360th day of incubation. After PVP, Polyethylene glycol (PEG) was found effective in maintaining higher microbial load of 5.820 log cfu/ml on 360th 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 180th 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 30th 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 day of incubation

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B. cepacia S. maltophilia 10.933 10.950 Trehalose 10.890 10.920 PVP 10.923 10.950 Glycerol 10.863 10.893 PEG 9.943 9.970 Control a b 10.711 10.737 Mean CD at 5% 1. Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.15

A. brasilense 10.973 10.940 10.967 10.910 9.980 10.754c

Mean 10.952d 10.917e 10.947d 10.889f 9.964g

Survival of efficient strains (log CFU/ml) in Matka Khaad on 60 th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.900 10.930 10.957 10.929d Trehalose 10.807 10.840 10.863 10.837e PVP 10.853 10.883 10.903 10.880f Glycerol 10.760 10.797 10.827 10.794g PEG 9.870 9.903 9.920 9.898h Control 10.638a 10.671b 10.694c Mean CD at 5% 1. Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: NS Table 4.16 Survival of efficient strains (log CFU/ml) in Matka Khaad on 90 th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.773 10.800 10.820 10.798d 10.693 10.720 10.743 10.719e 10.730 10.763 10.780 10.758f 10.230 10.267 10.290 10.262g 8.863 8.897 8.923 8.894h 10.258a 10.289b 10.311c

Trehalose PVP Glycerol PEG Control Mean CD at 5% 1. Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.17 Survival of efficient strains (log CFU/ml) in Matka Khaad on 120th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.363 10.390 10.410 10.388d Trehalose

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10.130 10.163 PVP 10.273 10.303 Glycerol 9.933 9.963 PEG 7.840 7.870 Control a 9.708 9.738b Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: NS

10.183 10.320 9.983 7.897 9.759c

10.159e 10.299f 9.960g 7.869h

Table 4.18

Trehalose PVP Glycerol PEG Control Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS

Survival of efficient strains (log CFU/ml) in Matka Khaad on 150 th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.233 10.260 10.280 10.258d 9.913 9.943 9.960 9.939e 9.933 9.963 9.983 9.960f 9.803 9.840 9.860 9.834g 6.810 6.843 6.860 6.838h 9.339a 9.370b 9.389c

Table 4.19

Trehalose PVP Glycerol PEG Control Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.20 Survival of efficient strains (log CFU/ml) in Matka Khaad on 210 th day of incubation B. cepacia S. maltophilia A. brasilense Mean

Survival of efficient strains (log CFU/ml) in Matka Khaad on 180 th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.013 10.030 10.050 10.031d 9.903 9.933 9.950 9.929e 9.920 9.943 9.963 9.942f 9.750 9.783 9.800 9.778g 5.280 5.313 5.330 5.308h 8.973a 9.001b 9.019c

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9.880 9.900 Trehalose 9.790 9.823 PVP 9.830 9.863 Glycerol 9.713 9.740 PEG 3.783 3.813 Control 8.599a 8.628b Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.21

9.920 9.850 9.880 9.760 3.830 8.648c

9.900d 9.821e 9.858f 9.738g 3.809h

Trehalose PVP Glycerol PEG Control Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.22

Survival of efficient strains (log CFU/ml) in Matka Khaad day of incubation B. cepacia S. maltophilia A. brasilense 9.733 9.763 9.787 9.623 9.650 9.670 9.657 9.683 9.703 9.560 9.593 9.610 2.110 2.140 2.170 a b 8.137 8.166 8.188c

on 240th Mean 9.761d 9.648e 9.681f 9.588g 2.140h

Trehalose PVP Glycerol PEG Control Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: 0.013 Table 4.23 Survival of efficient strains (log CFU/ml) in Matka Khaad on 300 th day of incubation B. cepacia S. maltophilia A. brasilense Mean 8.880 8.910 8.930 8.907d Trehalose 8.820 8.850 8.873 8.848e PVP

Survival of efficient strains (log CFU/ml) in Matka Khaad day of incubation B. cepacia S. maltophilia A. brasilense 9.020 9.030 9.043 8.910 8.947 8.960 8.933 8.963 8.983 8.680 8.713 8.743 1.013 1.037 1.060 7.311a 7.338b 7.358c

on 270 th Mean 9.031d 8.939e 8.960f 8.712g 1.037h

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8.843 Glycerol 8.517 PEG 1.013 Control 7.215a Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: 0.014 Table 4.24

8.873 8.553 1.017 7.241b

8.890 8.570 1.020 7.257c

8.869f 8.547g 1.017h

Trehalose PVP Glycerol PEG Control Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: 0.014 Table 4.25

Survival of efficient strains (log CFU/ml) in Matka Khaad on 330th day of incubation B. cepacia S. maltophilia A. brasilense Mean 7.910 7.950 7.967 7.942d 7.850 7.883 7.900 7.878e 7.880 7.913 7.930 7.908f 7.830 7.863 7.880 7.858g 1.013 1.013 1.013 1.013h 6.497a 6.525b 6.538c

Trehalose PVP Glycerol PEG Control Mean CD at 5% 1 .Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: 0.013

Survival of efficient strains (log CFU/ml) in Matka Khaad on 360 th day of incubation B. cepacia S. maltophilia A. brasilense Mean 6.770 6.800 6.823 6.798d 5.873 5.900 5.920 5.898e 6.710 6.743 6.760 6.738f 5.793 5.823 5.843 5.820g 1.013 1.013 1.013 1.013h a b c 5.232 5.256 5.272

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 30th 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 15th 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 on 15th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.283 10.313 10.333 10.310d 10.263 10.280 10.300 10.281e 10.270 10.300 10.323 10.298f 10.243 10.273 10.293 10.270g

Trehalose PVP Glycerol PEG

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9.160 Control 10.044a Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: NS Table 4.75

9.193 10.072b

9.220 10.094c

9.191h

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007 3. Organisms and Treatments: 0.012 Table 4.76

Survival of efficient strains (log CFU/ml) in Matka khaad at 15C on 30th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.290 10.323 10.343 10.319d 10.283 10.303 10.323 10.303e 10.293 10.313 10.340 10.316d 10.263 10.293 10.310 10.289g 9.183 9.210 9.253 9.216h a b c 10.063 10.089 10.114

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.77 Survival of efficient strains (log CFU/ml) in Matka khaad at 15C on 60th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.307 10.333 10.353 10.331d Trehalose 10.283 10.307 10.313 10.301e PVP 10.303 10.333 10.343 10.327d Glycerol 10.240 10.280 10.293 10.271f PEG 9.183 9.203 9.233 9.207g Control 10.063a 10.091b 10.107c Mean

Survival of efficient strains (log CFU/ml) in Matka khaad at 15C on 45th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.333 10.350 10.370 10.351d 10.300 10.323 10.350 10.324e 10.320 10.343 10.363 10.342f 10.293 10.313 10.333 10.313g 9.210 9.233 9.250 9.231h 10.091a 10.113b 10.133c

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CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.006 3. Organisms and Treatments: 0.011 Table 4.78 Survival of efficient strains (log CFU/ml) in Matka khaad at 25C on 15th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.823 10.853 10.883 10.853d 10.783 10.820 10.850 10.818e 10.803 10.840 10.880 10.841f 10.750 10.790 10.840 10.793g 9.743 9.773 9.793 9.770h 10.581a 10.615b 10.649c

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: 0.013 Table 4.79

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007 3. Organisms and Treatments: 0.012 Table 4.80 Survival of efficient strains (log CFU/ml) in Matka khaad at 25C on 45th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.820 10.863 10.893 10.859d Trehalose 10.780 10.813 10.840 10.811e PVP 10.810 10.847 10.860 10.839f Glycerol 10.753 10.783 10.820 10.786g PEG 9.750 9.783 9.813 9.782h Control 10.583a 10.618b 10.645c Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.008

Survival of efficient strains (log CFU/ml) in Matka khaad at 25C on 30th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.843 10.870 10.900 10.871d 10.807 10.843 10.873 10.841e 10.833 10.863 10.893 10.863f 10.780 10.810 10.863 10.818g 9.770 9.793 9.823 9.796h 10.607a 10.636b 10.671c

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3. Organisms and Treatments: NS Table 4.81 Survival of efficient strains (log CFU/ml) in Matka khaad at 25C on 60th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.790 10.833 10.863 10.829d 10.753 10.790 10.803 10.782e 10.783 10.820 10.833 10.812f 10.713 10.753 10.787 10.751g 9.723 9.750 9.780 9.751h 10.553a 10.589b 10.613c

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007 3. Organisms and Treatments: 0.012 Table 4.82

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.83 Survival of efficient strains (log CFU/ml) in Matka khaad at 40C on 30th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.103 10.143 10.163 10.137d Trehalose 10.053 10.093 10.113 10.087e PVP 10.070 10.110 10.143 10.108f Glycerol 10.013 10.053 10.073 10.047g PEG 9.033 9.053 9.083 9.057h Control a b c 9.855 9.891 9.915 Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.006 3. Organisms and Treatments: NS

Survival of efficient strains (log CFU/ml) in Matka khaad at 40C on 15th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.133 10.163 10.180 10.159d 10.080 10.113 10.143 10.112e 10.107 10.143 10.160 10.137f 10.060 10.090 10.120 10.090g 9.080 9.110 9.143 9.111h a b c 9.892 9.924 9.949

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Table 4.84

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007 3. Organisms and Treatments: NS Table 4.85

Survival of efficient strains (log CFU/ml) in Matka khaad at 40C on 45th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.060 10.100 10.113 10.091d 9.983 10.017 10.040 10.013e 10.043 10.080 10.083 10.070f 9.940 9.980 10.003 9.974g 8.963 8.993 9.013 8.990h 9.798a 9.835b 9.851c

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: NS

Survival of efficient strains (log CFU/ml) in Matka khaad at 40C on 60th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.033 10.073 10.090 10.066d 9.950 9.990 10.013 9.984e 9.990 10.030 10.043 10.021f 9.900 9.943 9.960 9.934g 8.923 8.953 8.970 8.949h a b c 9.759 9.798 9.815

the treatment with trehalose and glycerol was found to be statistically at par upto 30th 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 was low 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 30th 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 30th 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 30th days of incubation (Table 4.88) where trehalose and glycerol treatments were statistically at par. On 30th 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

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: NS Table 4.87

Survival of efficient strains (log CFU/ml) in Matka khaad at pH 4.5 on 15th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.51 0 10.530 10.543 10.528d 10.453 10.463 10.480 10.466e 10.503 10.513 10.523 10.513f 10.430 10.443 10.460 10.444g 9.273 9.300 9.323 9.299h a b c 10.234 10.250 10.266

Survival of efficient strains (log CFU/ml) in Matka khaad at pH 4.5 on 30th day of incubation B. cepacia S. maltophilia A. brasilense Mean

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10.523 10.543 Trehalose 10.463 10.493 PVP 10.520 10.533 Glycerol 10.453 10.470 PEG 9.313 9.343 Control 10.255a 10.277b Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.006 3. Organisms and Treatments: 0.011 Table 4.88

10.560 10.523 10.563 10.523 9.363 10.307c

10.542d 10.493e 10.539f 10.482g 9.340h

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007 3. Organisms and Treatments: 0.012 Table 4.89 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 4.5 on 60th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.463 10.490 10.503 10.486d Trehalose 10.403 10.433 10.443 10.427e PVP 10.440 10.460 10.473 10.458f Glycerol 10.373 10.393 10.400 10.389g PEG 9.230 9.263 9.273 9.256h Control 10.182a 10.208b 10.219c Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007 3. Organisms and Treatments: NS Table 4.90 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 5.5 on 15th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.853 10.880 10.893 10.876d 10.803 10.833 10.843 10.827e

Survival of efficient strains (log CFU/ml) in Matka khaad at pH 4.5 on 45th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.493 10.507 10.533 10.511d 10.430 10.460 10.473 10.454e 10.483 10.493 10.500 10.492f 10.417 10.423 10.443 10.428g 9.263 9.293 9.310 9.289h a b c 10.217 10.235 10.252

Trehalose PVP

165

10.830 Glycerol 10.783 PEG 9.853 Control 10.625a Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.006 3. Organisms and Treatments: NS Table 4.91

10.863 10.813 9.883 10.655b

10.873 10.830 9.903 10.669c

10.856f 10.809g 9.880h

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007 3. Organisms and Treatments: NS Table 4.92 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 5.5 on 45th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.833 10.873 10.890 10.866d Trehalose 10.760 10.800 10.823 10.794e PVP 10.813 10.853 10.870 10.846f Glycerol 10.730 10.770 10.793 10.764g PEG 9.853 9.893 9.923 9.890h Control 10.598a 10.638b 10.660c Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: NS Table 4.93 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 5.5 on 60th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.803 10.850 10.873 10.842d 10.723 10.773 10.793 10.763e 10.773 10.823 10.843 10.813f 10.663 10.713 10.733 10.703g

Survival of efficient strains (log CFU/ml) in Matka khaad at pH 5.5 on 30th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.870 10.903 10.913 10.896d 10.820 10.853 10.860 10.844e 10.853 10.893 10.903 10.883f 10.813 10.833 10.843 10.830g 9.890 9.933 9.950 9.924h 10.649a 10.683b 10.694c

Trehalose PVP Glycerol PEG

166

9.833 Control 10.559a Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.006 3. Organisms and Treatments: NS Table 4.94

9.873 10.607b

9.890 10.627c

9.866h

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007 3. Organisms and Treatments: NS Table 4.95 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 6.5 on 30th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.923 10.943 10.970 10.946d Trehalose 10.883 10.910 10.923 10.906e PVP 10.910 10.947 10.950 10.936f Glycerol 10.850 10.883 10.913 10.882g PEG 9.920 9.953 9.960 9.944h Control 10.697a 10.727b 10.743c Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.96 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 6.5 on 45th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.913 10.933 10.963 10.937d 10.840 10.873 10.890 10.868e 10.893 10.910 10.923 10.909f 10.790 10.830 10.860 10.827g 9.883 9.923 9.943 9.917h 10.664a 10.694b 10.716c

Survival of efficient strains (log CFU/ml) in Matka khaad at pH 6.5 on 15th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.900 10.920 10.943 10.921d 10.863 10.893 10.913 10.890e 10.890 10.907 10.930 10.909f 10.823 10.853 10.880 10.852g 9.893 9.910 9.933 9.912h a b c 10.674 10.697 10.720

Trehalose PVP Glycerol PEG Control Mean

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CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.007 3. Organisms and Treatments: 0.013 Table 4.97 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 6.5 on 60th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.893 10.913 10.943 10.917d 10.793 10.833 10.853 10.827e 10.843 10.873 10.890 10.869f 10.743 10.783 10.803 10.777g 9.853 9.890 9.903 9.882h 10.625a 10.659b 10.679c

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.006 3. Organisms and Treatments: NS Table 4.98 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 7.5 on 15th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.663 10.673 10.693 10.677d Trehalose 10.613 10.630 10.650 10.631e PVP 10.640 10.663 10.673 10.659f Glycerol 10.583 10.603 10.620 10.602g PEG 9.510 9.540 9.560 9.537h Control a b c 10.402 10.422 10.439 Mean CD on 5% 1 .Organisms: 0.006 2. Treatments: 0.008 3. Organisms and Treatments: NS Table 4.99 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 7.5 on 30th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.680 10.703 10.710 10.698d 10.633 10.663 10.673 10.657e 10.663 10.700 10.700 10.688f 10.613 10.640 10.653 10.636g 9.543 9.563 9.583 9.563h 10.427a 10.454b 10.464c

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.007

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3. Organisms and Treatments: NS Table 4.100 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 7.5 on 45th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.653 10.683 10.703 10.680d 10.593 10.623 10.643 10.620e 10.633 10.663 10.683 10.660f 10.570 10.603 10.633 10.602g 9.500 9.533 9.553 9.529h 10.390a 10.421b 10.443c

Trehalose PVP Glycerol PEG Control Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.006 3. Organisms and Treatments: NS Table 4.101 Survival of efficient strains (log CFU/ml) in Matka khaad at pH 7.5 on 60th day of incubation B. cepacia S. maltophilia A. brasilense Mean 10.633 10.660 10.673 10.656d Trehalose 10.553 10.603 10.613 10.590e PVP 10.613 10.653 10.663 10.643f Glycerol 10.523 10.583 10.593 10.567g PEG 9.470 9.493 9.510 9.491h Control 10.359a 10.399b 10.411c Mean CD on 5% 1 .Organisms: 0.005 2. Treatments: 0.006 3. Organisms and Treatments: 0.011

iii.

Effect of Desiccation The tolerance of the efficient isolates to the desiccation at 37 C is shown in

Figures 4.3. It was observed that as the desiccation period prolonged the survivability of the isolates decreased. After 8th day of desiccation, no viable cells were recovered. Among the various additives tested, trehalose was found to be the most effective in maintaining higher microbial load as compared to other additives. It was observed that among the most efficient three isolates, Azospirillum brasilense (MZ-AS2) exhibited more tolerance to desiccation and maintained high microbial load as compared to other two isolates used in the development of formulation. Figure 4.4 shows the survivability

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of the efficient isolates at different desiccation periods in trehalose amended treatment. It was observed that Azospirillum brasilense (MZ-AS2), Stenotrophomonas maltophilia (WT-A2) and Burkholderia cepacia (PT-P2) showed 10.09 log cfu/ml, 9.96 log cfu/ml and 9.95 log cfu/ml, respectively on 0 day, and 6.91 log cfu/ml, 6.80 log cfu/ml and 6.77 log cfu/ml, on 8th day of desiccation, respectively (Figure 4.4) in trehalose amended treatment. It was observed that efficient isolates withstands desiccation up to eight days and Azospirillum brasilense (MZ-AS2) was found out to be the most resistant isolate. This could be due to the fact that azospirilla can persist during unfavorable conditions such as desiccation, temperature and nutrient limitation and convert them into cyst forms (Lamm

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10.2 A.brasilense Stenotrophomonas sp. 10.1
Stenotrophomonas sp.

9.4

A.brasilense

B.cepacia

9.2 B.cepacia

10

9.9

8.8

Log CFU/ml

Log CFU/ml

8.6

9.8

8.4

9.7
8.2

9.6
8

9.5

Trehalose

PVP

Glycerol

PEG

Control

7.8 Trehalose PVP Glycerol TREATMENTS PEG Control

TREATMENTS

A.brasilense Stenotrophomonas sp. B.cepacia

8
8.4 A.brasilense Stenotrophomonas sp. 8.2 B.cepacia

Log CFU/ml

Log CFU/ml

7.8

7.6

7.4

C
7.2 Trehalose PVP Glycerol PEG Control

0
Trehalose PVP Glycerol PEG Control

TREATMENTS

TREATMENTS

A.brasilense Stenotrophomonas sp.

B.cepacia

Log CFU/ml

E
0

Trehalose

PVP

Glycerol
TREATMENTS

PEG

Control

Figure 4.3 Survival of efficient strains in Matka Khaad on: (A) 0 day of desiccation, (B) 2nd day of desiccation, (C) 4th day of desiccation, (D) 6th day of desiccation and (E) 8th day of desiccation

17 0

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12 A.brasilense Stenotrophomonas sp. 10 B.cepacia

Log CFU/ml

4
DAYS

10

Figure 4.4 Survival of efficient strains in Matka Khaad with trehalose treatment on different days of desiccation

171

and Neyra 1981; Sadasivan and Neyra 1985). The ability of microorganisms to survive desiccation depends on their ability to cope up with radiation stresses, reactive oxygen species (ROS), certain salts and solutes, and temperature extremes (Welsh 2000; Ramos et al. 2001; Deaker et al. 2004). However, some of the cells may be nonculturable upon exposure to different stresses (Potts 2001). The similar types of stress tolerance patterns have also been reported by several researchers (Vriezen et al. 2006; Vriezen et al. 2007; Chaiharn and Lumyong 2009; Vyas et al. 2009). Nature has developed numerous strategies for the long-term survival of organisms. Among the most intriguing are the biological mechanisms that preserve living organisms exposed to damaging conditions like extreme cold, dryness, or heat, or the absence of oxygen (Feofilova 2003). The intracellular accumulation of large quantities of a particular group of small, organic osmolytes is a common and flexible strategy of adaptation for eukaryotic and prokaryotic microorganisms when they need to cope up with stress conditions. These compounds, which function as protectants, are termed compatible solutes since they can be amassed by the cell in very high concentrations providing stress tolerance without disturbing essential cellular functions (Galinski and Truper 1994). It is generally assumed that uptake of external osmoprotectants in the medium is energetically preferred over de novo synthesis (Bremer and Kramer 2000; Galinski 1995; da Costa et al. 1998; Roberts 2005). There is increasing evidence indicating that compatible solutes are multifunctional molecules with stabilizing properties, hence protecting cell components from several abiotic stresses such as high salinity, freezing, desiccation, high temperature, pressure, or oxygen radicals, functioning as chemical chaperones (Welch and Brown 1996). These compatible solutes stabilize enzymes, DNA, membranes, and whole cells against different kinds of stresses such as freezing, drying, and heating (Lippert and Galinski 1992; Louis et al. 1994). In the natural environment, compatible solutes can be released upon death of organisms or during efflux processes, rendering these compounds accessible to others that can scavenge them for osmoadaptation or as carbon source, provided they have the appropriate mechanisms for their uptake and catabolism (Poolman and Glaasker 1998). In i

ii

response to stress, microbial cells undergo physiological changes that are likely to optimize their survival under these conditions. The physiological changes include the accumulation of disaccharides, such as trehalose, which serve as protectants of macromolecules within the cell; the accumulation of water-stress proteins; alterations in lipid composition of membranes to maintain membrane fluidity; enhanced production of extracellular polysaccharides; and morphological changes that maximize volume-tosurface ratios of bacterial colonies (Potts 1994, Managbanag and Torzilli 2000). Tables 4.14 to 4.101 show that trehalose was the best additive in maintaining higher microbial load for longer duration. After trehalose, glycerol was an effective additive followed by PVP and PEG which helped in survivable of efficient isolates. The reasons for this are discussed as below. The most widely distributed and studied compatible solute is trehalose. Trehalose is a disaccharide that is ubiquitous in the biosphere. The success of trehalose in nature compared to other sugars can be explained by its peculiar structure. In addition to being nonreducing, it possesses several unique physical properties, which include high hydrophilicity and chemical stability, non-hygroscopic glass formation and the absence of internal hydrogen bond formation. These features account for the principal role of trehalose as a stress metabolite (Arguelles 2000). During stress, the presence of trehalose lowers the transition temperature ( Tm) of the dry membranes by replacing the water between the lipid headgroups, preventing the phase transition and its accompanying leakage upon rehydration (Crowe et al. 1988; Crowe et al. 1989). In addition to lowering the Tm of membranes, trehalose has been shown to preserve both structure and function of proteins during drying (Carpenter et al. 1987a; Carpenter et al. 1987b; Crowe et al. 1987). Trehalose interacts directly with oxidation-sensitive parts of unsaturated fatty acids, protecting it from the auto-oxidation (Oku et al. 2003; Nery et al. 2008). Besides, several Gram-positive and Gram-negative bacteria use exogenous trehalose as sole source of carbon and energy (Arguelles 2000). Glycerol is a well-known metabolite formed by many microorganisms including bacteria, yeasts, molds, and algae (Spencer 1968; Vijaikishore and Karanth 1986; Rehm ii

iii

1996). Glycerol has a high water binding capacity and may protect cells from the effect of desiccation by slowing the rate of drying (Lorda and Balatti 1996). It often serves the function of an osmolyte, balancing external osmotic pressure (Brown 1978; Blomberg and Adler 1992; Sunder et al. 1996). PVP is believed to detoxify the media by complexing with the phenolic-type, self-limiting toxins in the media (Singleton et al. 2002). PVP also has a high water binding capacity, which could maintain water around the cells for their metabolism (Singleton et al. 2002; Deaker et al. 2004; Vendan and Thangaraju 2006). Moreover, some polymeric additives such as PVP, PVA and starch have stabilization properties. PEG has also been reported to decrease the cell permeability during rehydration, avoiding intracellular losses and therefore increasing viability (Beker et al. 1984). The studies showed that the addition of PEG to media enhanced trehalose concentration in the cells (Harman et al. 1991).

Polyhydroxyalkanoates (PHAs), a family of optically active polyesters, are accumulated intracellularly by microorganisms during unbalanced growth condition as a means of storing of carbon as energy reserves (Anderson and Dawes 1990). Addition of PEG into culture medium causes a change in PHA biosynthesis involving the enzyme system and results in the incorporation of ethylene glycol (EG) monomer repeat units into the PHA polymer (Saha and Paul 2006). In conclusion, efficient native Nitrogen Fixers and Phosphate solubilizers are isolated from the rhizospheric soil samples and tested for PGPRs activities. On the basis of 16S rRNA gene sequencing, the efficient isolates i.e. WT-A2, PT-A1, MZ-AS2, WTAS3, MZ-P4 and PT-P2 are found to be Stenotrophomonas maltophilia (GU371215), Bacillus licheniformis (GU371216), Azospirillum brasilense (GU371217), Azospirillum brasilense (GU371218), Pseudomonas aeruginosa (GU371219) and Burkholderia cepacia (GU371220), respectively. The most efficient isolates are WT-A2, MZ-AS2 and PT-P2. Out of five liquid carriers tested, Matka Khaad is found to be most effective in the development of liquid formulation. It maintain the microbial poupulation of 9 log CFU/ml up to 240th day of incubation, and 8 log CFU/ml up to 300th day of incubation. The efficient native isolates survive well under stress (temp., pH and desiccation) conditions in the developed liquid formulation. In the present study, Stenotrophomonas iii

iv

and Burkholderia are found as efficient nitrogen fixer and P-solubilizer, respectively. However, these isolates require further studies and testing under pot culture as well as field conditions before recommending them as a bioinoculant.

iv