1 GENERAL INTRODUCTION

1.1 Objectives and outlines of the thesis

1.1.1 Objectives: Towards a better understanding of Plant Growth Promoting mechanisms of diazotrophic bacteria to improve plant N nutrition

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The need for a better understanding of the mechanisms that affect plant N nutrition in natural systems is recurrent theme in the literature discussing the potential application of biological nitrogen fixation (BNF) as performed by free-living diazotrophs. The real-time PCR based quantification has been successfully used to quantify nifH gene pool in soil (Wallenstein 2004) and is applicable to the plant environment as well. However, this method requires implementation of an internal control that prevents the miscalculation of the quantified gene pool due to the presumed different DNA extraction efficiencies from environmental samples. Moreover, this study only used quantification results to describe the effect of environmental factors on nifH gene abundance in soil and did not link the results to N-fixation activity. Methods of molecular ecology are capable of significantly extending our understanding of this process in plant. The establishment of the real-time PCR approach for the quantification of nifH gene abundance in plant will add a possible tool to study the factors that influence BNF in much more detail. The nifH gene quantification parallel with the considered bacteria enumeration, and plant N nutrition makes possible to determine the potential of inoculated bacteria to fix atmospheric nitrogen. Moreover, it will become possible to determine how individual members of the diazotroph community react to environmental factors, and to study some elements of gene regulation in situ in the soil environment. The knowledge gained will allow a focused search for methods of enhancing BNF in the field by inoculation with specifically selected microorganisms. Furthermore, this technology will also allow detailed monitoring and control of the effectiveness of such future applications.

The objective of this thesis is to improve, develop and to apply a number of key methods that are necessary for a successful application of the real - time approach to free-living plant diazotrophs. These methods should provide a sound basis for quantitative studies in the plant environment to obtain new information on the PGP mechanisms and dynamics of inoculated diazotrophs.

1.1.2 Research focus and hypotheses of the thesis

This thesis investigates the growth promoting effects of asymbiotic diazotrophic bacteria, in combination with fertilizers to contribute to growth, nutrient uptake of agricultural plants.

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Four main topics are included in the work:

  1. Isolating local PGPB
    Soil nutritional conditions, microbial diversity may effect a growth promoting efficiency of inoculated bacteria. Therefore, in this study, PGPB were isolated from wheat grown in a nutrient deficient calcisol soil in Syrdarya, Uzbekistan.

  2. Universal plant growth promoting bacteria
    The growth-promoting activity of some bacteria may be highly specific to certain plant species, cultivars and genotypes. Therefore, one of the effective strategies for initial selection and screening of PGPB is the consideration of host plant specificity. In this study, the bacterial strains shown to have a plant growth promoting effect on wheat plants were tested for their stimulatory effect on other plants, such as cauliflower, cucumber, paprika and tomato.

  3. Effect of inorganic N availability on diazotrophic bacteria colonisation in plant
    Understanding the factors involved in controlling the colonisation/distribution of diazotrophic bacteria in the environment may allow identifying the factors controlling N2-fixation in the environment. Therefore it is important to evaluate the response of inoculated bacteria to fertiliser supply to the plant in the presence of N in different levels.

  4. Linking structural and genomic data
    Molecular measurements of the functional gene abundance, as a potential of activity, can link structural and genomic data. To evaluate the direct contribution of the N2-fixing plant-inhabiting diazotrophic bacteria to plant nutrition, the abundance of a marker gene for biological nitrogen fixation, nifH gene can be investigated.

Several approaches based on quantitative or semi-quantitative PCR has been suggested for the quantification of nifH gene abundance in environmental samples or pure culture DNA. All these approaches have their advantages and disadvantages. Real-time PCR-based methods developed by Wallenstein (2004) have shown to be a powerful tool to quantify N2-fixing genes in soil. However, these methods require implementation of an internal control that prevents the miscalculation of the quantified gene pool due to the presumed different DNA extraction efficiencies from environmental samples. Therefore, it was aimed to develop a new method to accurately quantify nifH gene copy numbers in plant DNA.

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From these main topics the flowing hypotheses were generated:

There is a significant correlation between asymbiotic diazotrophic bacteria abundance in plant and N content of plants.

1.1.3 Outline of the thesis

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Chapters, three, four, five and six consist of the content of a manuscript which has been published, has been submitted for publication, or which is nearly ready to be submitted for publication in a refereed academic journal.

Chapter two describes the isolation, phenotypic characterisation and screening the plant growth promoting bacterial strains from wheat plant for producing plant growth promotion in series of plate and pot experiments. Inoculation experiments under greenhouse conditions provides the basis for studies of plant growth promoting effect of used bacteria allowing determination of their effect mechanisms.

Chapter three of this thesis focuses on conventional and molecular based identification of selected bacterial strains. The disagreement between conventional identification and phylogenetic sequence analysis of 16S-23S ISR rDNA amplified from two diazotrophic plant growth promoting bacteria strains was discussed.

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Chapter four details the development of strain-specific primers that provide the basis for an accurately evaluation of inoculated bacteria colonization ability in plant root by quantitative detection of bacteria after inoculation.

Chapter five describes the newly developed method for nifH gene quantification that provide the basis for more sensitive and more quantitative the detection of nifH templates in plant root by real-time PCR with universal nifH primers. The laboratory experiment in this study provided data on the nifH gene abundance in plant root growing in non-sterile quartz sand under different conditions, which was significantly positively correlated to inoculated bacteria abundance (chapter 4). The methods described and discussed in chapter 5 are based on peer-reviewed article published in Canadian Journal of Microbiology under the title of ‘Detection and quantification of the nifH gene in shoot and root of cucumber plants’ (Juraeva et al. 2005).

Chapter six presents some preliminary results on the application of the developed methodology that employed to quantify the abundance of considered bacteria after inoculation (as described in chapter 4) parallel with nifH gene quantification (as described in chapter 5) using real – time quantitative PCR, to the study of the capacity of the inoculated diazotrophic bacteria to fix atmospheric N in plant roots and furthermore contains a comprehensive discussion of the achieved results, open questions, and future research opportunities.

1.1.4 Current challenges

1.1.4.1 Improving detection methods for nifH.

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nifH primers in different level of specificity were used to amplify nifH gene fragments from both pure culture DNA and environmental samples (Ueda et al. 1995). The application of PCR to mixed assemblages of diazotrophic organisms requires an unbiased amplification of all nifH gene fragments. Because of the huge phylogenetic differences of nifH genes (Zehr et al. 2003), universal nifH primers (Ueda et al. 1995) have a high degree of sequence degeneracy. Using universal nifH primers developed by Ueda et al. (1995) was successful to amplify nifH genes in rice root. However, this method was not sensitive enough to amplify nifH genes in the dryland plant roots (maize, soybean) suggesting that many less diazotrophic bacteria exist in dryland plant roots than in rice (Ueda et al. 1995). Nowadays, developed molecular approaches and high quality reagents which make approach more sensitive allow amplifying nifH gene even in bulk soil (Wallenstein 2004). The application of previously published primers to more sensitive approaches, such as real-time PCR based direct quantification may allow a more sensitive detection of diazotrophs in dryland plant roots as well. Furthermore, since, quantitative data on nifH abundances obtained with such methods using degenerated primers should be interpreted with caution; the development of real-time PCR protocols with real-time control of PCR product would reduce the risk of PCR biases, making the results more reliable and more amenable to quantitative interpretation of the data. 

1.1.4.2 Development of methods to examine N2 fixation by certain bacteria in complex environments.

While the capability of diazotrophs to fix nitrogen in vitro can be demonstrated easily, efforts to quantify nitrogen fixation in natural associations with plants have produced widely varying results. In the past 30 years many crop inoculation studies, coupled to acetylene reduction measurements, N balance and 15N isotope dilution experiments, have been conducted with root associated bacteria to determine whether the bacteria supply significant amounts of nitrogen to cultivated plants (Boddey et al. 1999; James 2000). A major drawbacks of the acetylene reduction assay, is that it only measures nitrogenase activity and reveals no information on whether the fixed N is incorporated into the plant (Boddey et al. 1995). N balance experiments have the disadvantage that the plant N is not necessarily derived from the air but might also result from improved nutrient uptake by the inoculated plant. Nowadays, the most useful methods for examining N2 fixation in the field and large greenhouse experiments are still the 15N isotop dilution and 15N natural abundance techniques (James 2000).

Assaying the genetic potential of inoculated bacteria for nitrogen fixation, i.e. nifH gene quantification in combination with N-content analysis may be a possible tool to evaluate the direct contribution of the N2–fixing plant-colonizing diazotrophic bacteria to plant N nutrition. Due to stringent regulation of gene is regulated at both pre- and posttranslational levels (Dean and Jacobson 1992) under unfavorable conditions; gene abundance does not always mean the activity of nitrogenase. However, it is an indicator of potential of inoculated bacteria to fix atmospheric nitrogen. Furthermore, the study of gene abundance under environmental conditions is in itself an interesting objective, as the conditions in natural environments (e.g. plant roots) are vastly different from the optimum growth conditions used in laboratory cultures.

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Tab. 1: Comparison of methods of estimating nitrogen fixation

Methods

Advantages

Disadvantages

Sensitivity

1. Total N balance

Simplest

Low sensitivity
including other inputs.

Lowest

2. 15N2 incorporation

Most direct

Expensive, only for short period

High-moderate

3. Acetylene reduction

Simple, highly sensitive

Indirect, semi-quantitative

High

4. 15N dilution

Throughout growing season

Only N Fixation in plant
Varies with reference plants

High-low

4a. Natural abundance

Simple, no disturbance to system

Only slight difference in 15N content

Low

4b. Substrate addition

Difference in 15N content is large

Change of 15N in time and space in soil

Moderate

Note: Adapted from Watanabe 2000.

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Chapter 2. Isolation, phenotypic characterization and screening of wheat inhabiting bacteria for their plant growth promoting effect


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