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PRODUCTION OF GROWTH HORMONES AND NITROGENASE BY DIAZOTROPHIC BACTERIA AND THEIR EFFECT ON PLANT GROWTH

Anwar, Ghazala (1999) PRODUCTION OF GROWTH HORMONES AND NITROGENASE BY DIAZOTROPHIC BACTERIA AND THEIR EFFECT ON PLANT GROWTH. PhD thesis, University of the Punjab, Lahore.

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Abstract

Nitrogen is a limiting factor in plant productivity. The plant growth promoting bacteria live in association with plants and improve plant growth by nitrogen fixation, growth hormone production, etc. In the present studies, high nitrogen fixing ability was found in association with rice (12-74 umole C2H4 plant/24 h) and wheat (0.6-3.1 umole C2H4 plant/24 h). The in situ acetylene reduction assay (ARA) values for rice grown in nitrogen depleted soil were higher (24- 74 umole C2H4 plant/24 h) than the values obtained with the varieties grown ill fertile soil (12-18 umole C2H4 plant/24 h). The ARA values for rice were higher (12-74 umole C2H4 plant/24h) as compared, to wheat (0.6-3.1 umole C2H4 / plant/24 h). In wheat, the ARA value for non-fertilized soil (1.8 umole C2H4 / plant/24 h and soil supplied with farmyard manure (7.3 umole C2H4 / plant 24 h) were higher as compared to the values obtained from the soil where (0.17 umole C2H4 / plant/24 h) chemical fertilizer was applied. In the soil where nitrogenous fertilizer was applied, the diazotrophic bacterial population was present but the bacteria were not fixing nitrogen. Bacteria isolated by using soil extract based medium belonged to six genera and preponderance of Azospirillum, Flavobacterium, and Pseudomonas strain was observed. Soil extract based medium helped in the isolation of Azotobacter and Flavobacterium strains which had previously not been isolated from these plants. The analysis of the physiological, morphological and biochemical data generated by QTS-20 and comparison by using NTSys with the choice of Jaccard coefficient gave 55-75 % homology among bacterial strains belonging to the same genus and the analysis of the data generated by Biolog and comparison by using NTSys (computer software) with the choice of Jaccard coefficient gave 42-45 % homology among bacterial strains belonging to the same genus. Plant growth promoting rhizobacterial (PGPR) strain K-1 was first identified as Azospirillum brasilense and then as Azoarcus by labeled DNA probe but physiological and biochemical tests by using Biolog plates and their data analysis by NTSys produced different results and this strain proved completely different from the standard Azoarcus strains. The Zooloea Ky-1 showed homology with Pseudomonas strains by QTS and Biolog/ NTSys method which was attributed to the relationship of these two bacterial strains with family Pseudomonadacea, Physiological and biochemical methods showed high similarity between Azospirillum N-4 and ER-20, Intrinsic antibiotic resistance proved useful for the identification/ differentiation of these PGPR strains. Azospirillum ER-20 was resistant to spectinomycin and Azospirillum N-4 was sensitive to spectinomycin. Therefore, this property helped to differentiate these closely related Azospirillum strains. The REP, ERIC and BOX like sequences were identified in bacteria isolated from soil and roots. REP, ERIC and BOX-PCR methods proved a valuable tool for characterization of bacteria. These methods proved equally useful for Gram-negative and Gram-positive bacteria. The REP-PCR generated more products as compared to ERIC-PCR. However, ERlC-PCR was more useful as compared to REP-PCR for the differentiation among bacterial strains. Although PCR based methods are more important for the differentiation among bacterial strains belonging to the same species, however, these methods can be used for the identification of bacteria by using standard bacterial strains. In the present studies, the identification of Azospirillum N-4 and ER-20 was confirmed by BOX- PCR/ Gel Compare method. These strains showed about 80% homology with standard Azospirillum strain. It was confirmed that Azospirillum ER-20 and N-4, Zoogloae Psendomonas 96-51 and Azoarcus K-I were different diazotrophic bacterial strains, It was concluded that in BOX-PCR/ Gel Compare 4.1 analysis by using dice coefficient for analysis, Azospirillum strains showing 60 % homology belonged to the same genus. Pure cultures of bacteria exhibited very high rates of nitrogen fixation (acetylene reduction up to >200 nmole C2H4 /h culture vial). Primary screening for indoleacetic acid (IAA) producing bacteria was done by colorimetric method. The Fe-H2SO4 reagent proved better as compared to Fe-HCIO4 reagent but these reagents gave overestimates of IAA production. The growth hormone production was also identified by using HPLC. Pseudomonas sp. And Azospirillum ER-2 and ER-20 produced higher amounts of IAA (upto 35 ug/ml) in the liquid medium containing tryptophan and NH4Cl. The IAA production increased (upto 22 ug/ml) with increasing the age of bacterial culture. The lAA production was drastically decreased in semisolid medium without NH4Cl. None of the Azotobacter and Entrobacter strains tested, produced IAA or gibberellic acid (GA). Azospirillum K-I produced higher amount of GA (10 ug/ml) which decreased in later growth stages. This decrease in GA might be due to its hydrolysis by bacterial strain. Azoarcus K-I and Azospirillum ER-2 and ER- 20 also produced indole, acetamide as identified by HPLC. It was observed during lab studies that PGPR fix nitrogen at high rates and contributed 11-38.4% nitrogen to the rice. These strains were also useful for increasing rice biomass, N-uptake and fertilizer-N use efficiency. In green house experiments, inoculation of rice with PGPR increased chlorophyll, leaf area, tiller number, plant height, root shoot biomass and grain yield in rice. Soil, root, leaf and stem inoculation methods were equally useful for plant growth improvement. The use of culture broth with cells and cell free broth for leaf and stem inoculated gave similar growth increasing response. During studies under field conditions, it was observed that the inoculated bacterial strains contribute significant amount of nitrogen (45 kg N/ ha) to increase rice plant biomass and improve nitrogen status of soil. The increase in plant biomass and root shoot proliferation was attributed to the production of plant growth hormones by PGPR. Therefore, growth hormones from the bacteria filtrates were extracted. The effect of these growth hormone containing extracts and pure growth hormones IAA and GA on different growth parameters was studied. Pure IAA and GA concentration range 1-2 ug/ml increased root area, and plant biomass of rice and wheat. The action of IAA was found to be plant species specific as the effect of2 ug/ml of IAA was different on rice and wheat. The growth hormones containing extract from Psendomonas 96-51 increased root area, root length and plant biomass of rice and wheat. Application of growth hormones containing extracts from Azospirillum ER-2 arid ER-20, also increased the root proliferation in wheat. It was observed that the inoculated bacterial strains live in the form of micro colonies in soil and on the roots and survive through out the life of the rice crop. To test the capability of PGPR to live in grasses as endophyte, PGPR were inoculated into leaf and stem tissue of rice and their survival was tested for twelve days. Azospirillum strains were detected in leaf and stem tissues in this period. Highly efficient PGPR were studied to optimize growth conditions for growth in fermenters. The doubling time for different bacterial strains was 2-3 h and highest population of bacteria was 5-7 x 10o colony forming units per milliliter (CFU/ml) of culture broth. The growth of different bacterial strains was equally good in half and quarter strength LB liquid medium. Synthetic auxin 2, 4 dichlorophenoxyacetic acid (2, 4 -D) increased the nitrogenase activity of Azoarcus K-I and proliferation of different bacterial strains in the plant rhizosphere 2, 4 -D (1.5 ppm), induced nodule like structures on wheat roots. The bacteria were found in the nodules in the form of micro colonies/bacterial aggregates. 15N dilution data indicated that 12.6-46.5% atmospheric nitrogen was incorporated into nitrogen pool of inoculated plants. Rhizopine synthesis (mos) and catabolism (mos) genes were discovered in Rhizobium meliloti L5-30. These genes were thought to give a competitive advantage to the bacterial strain harboring these genes among other soil bacteria, During these studies, unique nature of rhizopine catabolism genes was tested by using nucleic acid hybridization, PCR and rhizopine catabolism assay. Among a large number of bacterial strains including LTER-I 00 collection of bacteria, locally isolated bacteria and several Rhizobium meliloti strains, only four bacterial strains showed the rhizopine catabolizing ability, confirming tile unique nature of these genes. It was observed that the presence of moc locus is a unique character which is independent of the geographical origin as moc/mos strains belonged to three different continents,

Item Type:Thesis (PhD)
Uncontrolled Keywords:Growth Hormones, Nitrogenase, Diazotrophic Bacteria, Plant Growth, Azospirillum, Flavobacterium, Pseudomonas, Plant Growth Promoting Bacteria (PGPR), Azoarcus, Zoogloea,
Subjects:Physical Sciences (f) > Chemistry(f2) > Biochemistry(f2.1)
ID Code:314
Deposited By:Mr Ghulam Murtaza
Deposited On:17 Jun 2006
Last Modified:04 Oct 2007 21:00

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