5 DETECTION AND QUANTIFICATION OF THE nifH GENE IN SHOOT AND ROOT OF CUCUMBER PLANTS

5.1 Abstract

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A real-time polymerase chain reaction method (PCR) method was applied to quantify the nifH gene pool in cucumber shoot and root and to evaluate how N supply and plant age affect the nifH gene pool. In shoots, the relative abundance of the nifH gene was neither affected by different stages of plant growth nor by N supply. In roots, higher numbers of diazotrophic bacteria were found compared to the shoot. The nifH gene pool in roots significantly increased with plant age and, unexpectedly, the pool size was positively correlated with N supply. The relative abundance of nifH gene copy numbers in roots was also positively correlated (r = 0.96) with total N uptake of the plant. The data suggest that real-time PCR-based nifH gene quantification in combination with N-content analysis can be used as an efficient way to perform further studies to evaluate the direct contribution of the N2–fixing plant-colonising plant growth promoting bacteria to plant N nutrition.

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Key words

Real-time PCR – Biological nitrogen fixation – cucumber – N-nutrition, plant growth promoting bacteria

5.2 Introduction

One of the current challenges faced by microbial ecologists is the difficulty to relate information on the abundance of the specific bacterial populations in the environment with their functional activity (Gray and Head 2001). Molecular measurements of the functional gene abundance, as a potential of activity, can link structural and functional data. Most molecular studies on diazotrophic organisms are primarily based on PCR amplification of the nifH gene, a marker gene for biological nitrogen (N2) fixation. In those studies, the abundance and diversity of the nifH gene were investigated using a variety of approaches including PCR amplification followed by denaturing-gradient gel electrophoresis (Nicolaisen and Ramsing 2002, Freitag and Prosser 2003), restriction fragment analysis (Mintie et al. 2003, Stres et al. 2004), DNA hybridisation analysis (Mergel et al. 2001, Neufeld et al. 2001) and cloning-sequencing approaches (Kirshtein et al. 1991, Hamelin et al. 2002). However, despite this technical progress, the relationship between microbial community structure and nitrogen cycling processes in soils or ecosystems is still relatively poorly understood (Wallenstein 2004).

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Several approaches based on quantitative or semi–quantitative PCR, including reverse–transcriptase PCR (Bürgmann et al. 2003), gene hybridisation (Neufeld et al., 2001) and real–time PCR with SYBR® Green I (Wallenstein 2004), have been suggested for the quantification of nifH gene abundance in environmental samples or pure culture DNA. All these approaches have their advantages and disadvantages.

To investigate the relationship between the abundance of nifSKH genes and the corresponding biochemical reaction rates, Neufeld et al. (2001) used a gene hybridisation method based on quantitative gene probing. This method, however, probably underestimated the true number of nif genes present in the samples, because the nif genes may have shown reduced DNA-sequence similarity with the probes used in that study (Neufeld et al. 2001). Moreover, although the reverse–transcriptase PCR approach followed by image analysis of electrophoresis gels used by Bürgmann et al. (2003) was sufficient to detect the nifH gene abundance, the amount of product at the end of the reaction, quantified by end-point analysis (also called semi-quantitative analysis), does not truly represent the initial amount of starting material. In addition to poor precision, end-point PCR analysis produces narrower dynamic ranges than real-time PCR methods (2 to 3 orders of magnitude versus 5 to 8), and has a lower sensitivity and resolution (Schmittgen et al. 2000). For a more reliable quantification of template concentrations, real-time PCR approaches based on direct determination of the amount of product offer an attractive alternative (Boeckman et al. 2000).

Real-time PCR-based methods as developed by Wallenstein (2004) have been 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. To overcome this difficulty, nifH gene copy numbers could be calculated relative to plant housekeeping gene copy numbers. This approach would be more accurate than using adjusted concentrations of DNA extracted from environmental samples.

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In this study, we developed a method to quantify nifH gene copy numbers in plant DNA with a quantitative real-time PCR approach using SYBR® Green I as an intercalating dye. In addition, the preliminary application of this newly developed quantification method in combination with plant N content analyses was used to evaluate the direct contribution of the N2-fixing plant-inhabiting diazotrophic community to plant N nutrition. Finally, we address whether the mineral N availability and/or plant age affect the abundance of the nifH gene pool in cucumber.

5.3 Materials and methods

5.3.1 Greenhouse experiments

Cucumber (Cucumis sativus L. ‘Corona F1’) seeds were germinated for 10 days on trays containing vermiculite under greenhouse conditions (latitude 52° 21`N, longitude 13° 18`E at an altitude of 50 m), close to Berlin, Germany. At the 2-leaf stage, plants were transplanted into pots (one seedling per pot) filled with 800 g quartz sand. 12 replicates were grown for 6 wk (in May-June 2004) at temperatures of 26°C to 28°C during the day and 16°C to 18°C at night without artificial illumination. The substrate water content after planting was approximately 60% water holding capacity and was kept nearly constant throughout the experiment. Percolating water was recycled for each pot. Plants were supplied with half-strength Hoaglands’ nutrient solution lacking nitrogen (Hoagland and Arnon 1950). N was supplied separately at two different concentrations with six replications for each treatment: (1) a high N supply, calculated to fully meet the plant’s nitrogen demand for a 6 week growth period; and (2) a low (N-deficient) treatment, equivalent to approximately 40% of the high level. Two days after planting, 100 mg N were supplied to each plant by injecting NH4NO3 solution into the root zone. Half the plants, selected at random, also received an additional 150 mg N on two further occasions (75 mg N each at 21 and 28 d after planting) and are referred to here as high N supplied plants. As total mineral fertiliser during growth, plants received potassium/nitrogen/phosphorus in a ratio of 10:0.8:1 and 10:2:1 for low and high N supplied plants, respectively.

5.3.2 Harvesting of plant samples and DNA extraction from plant samples

For nifH gene abundance measurements, plants (three replicates for each nitrogen treatment) were harvested on Days 7 and 42 after planting. Roots and shoots were separated and quartz sand particles were carefully removed under a gentle stream of tap water. About 0.1 g (fresh mass) samples from the middle part of roots and youngest leaves were cut, transferred to separate Eppendorf tubes containing 0.1 g of sterile glass beads (0.5 mm diameter), and then frozen at –20°C and lyophilised.

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DNA was extracted from the lyophilised plant samples using the DNeasy Plant Mini Kit (Qiagen, Hilden GmbH, Germany) according to the manufacturer’s instructions. First, the lysis buffer was added to the lyophilised samples, which were then shaken vigorously using the wrist action shaker MM 200 (Retsch, Haan, Germany) at a frequency of 30 shakes per second for 5 min. DNA concentrations were photometrically measured at λ=260 nm using an Eppendorf spectrophotometer.

The additional observations undertaken at harvest d 42 were shoot and root dry mass and total plant N analyses. Six plant samples (for each N supply) were prepared to determine the effect of N supply on plant growth and total N analysis. Plants were separated from substrate as described above for molecular measurements and dried at 80°C for 48 hours. After measurements of shoot and root dry mass, total N was analysed using a CHN-O Rapid elemental analyser (Elementar Analysensysteme GmbH, Hanau, Germany).

5.3.3 Real-time PCR assays

To measure PCR products in real-time PCR, the non-specific intercalating dye, SYBR Green I, was used. In this method, the signal is quenched when the dye is not bound. Thus, as PCR products accumulate, the fluorescent signal increases proportionately, thereby allowing quantification of PCR amplification.

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Quantitative real-time PCR was conducted using an iCycler detection system (Bio-Rad, Laboratories, München, Germany). The fluorescence of the reporter molecule was measured at 520 nm after excitation at 490 nm. Each assay was conducted in a 96-well plate with two replicates for each standard and negative control, and sample in triplicate. Amplification was performed with a reaction mixture containing 12.5 µl of QuantiTect SYBR® Green 2 x Master Mix (Qiagen, Hilden GmbH, Germany), 2.5 µl each 300 nmol·L-1 primer and 2.5 µl DNA template, which was then brought to a final volume of 25 µl by the addition of 5 µl of DNA-free H2O. In order to avoid of the failure in quantification of nifH gene due to too high or low plant DNA concentration used as a template, PCR was performed using undiluted, 1:10 diluted and 1:100 diluted plant DNA. PCR was performed with an initial step at 95°C for 15 min (required to activate the polymerase enzyme) followed by 50 cycles each of 0.5 min at 94°C, 1 min at 50°C and 1.15 min at 72°C.

The cycle at which the fluorescence of the target amplicon exceeded the background fluorescence (threshold cycle) was calculated using the iCycler iQ Optical System Software (version 3.1, Bio-Rad, Inc). A melting curve analysis was performed following each assay by measuring fluorescence continuously as the temperature was increased by 0.5°C from 50°C to 97.5°C. Different diazotrophic bacterial pure cultures (Tab. 14) were used to evaluate this method (data not shown). The specificity of amplification was confirmed by running samples on a 1.7 % agarose gel and by checking the melting profile of the PCR product. The lengths of the PCR products from plant samples were compared with that of positive controls of diazotrophic bacterial pure cultures and were estimated using 100 bp DNA Plus Ladder (peqGold, peqLab, Erlangen, Germany).

Tab. 14: Melting temperatures of PCR products amplified using the nifH gene primer pairs for DNA extracted from diazotrophic bacterial pure cultures and from cucumber root and shoot samples.

DNA extracted from

Strain number

Melting T° (°C)

Citrobacter spp. 1

CC322

92.5

Azospirillum sp.   1

D19/1

90.5

Isolate from winter wheat 1

K27

89.5

Citrobacter sp. 1

CC321

87.0

Klebsiella pneumoniae 1

CC2/17

90.5

Serratia rubidea 1

CC12/12

90.0

Pseudomonas aeruginosa 1

CC312

90.0

Azospirillum sp. 1

K22

88.5

Isolate from winter wheat 1

CC307

92.0

Enterobacter radicincitans 1

D5/23

89.0

Arthrobacter simplex 2

43

90.5

Burkholderia spp. 3

148

91.5

Shoot samples of cucumber 4

-

89.5 ±0.5

Root samples of cucumber 4

-

91.5 ±0.9

Shoot samples of cucumber 5

-

89.0 ±0.5

Root samples of cucumber 5

-

90.0 ±1.0

5.3.4 Preparation of the nifH gene standard

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To quantify nifH gene copy numbers in unknown samples, real-time PCR was performed with defined standard samples of the diazotrophic bacterial strains Enterobacter radicincitans sp. Nov. (D5/23) (Kämpfer et al. 2005) and Klebsiella pneumoniae CC 2/17. Cultures were grown in Standard I (Merck, Darmstadt, Germany) broths for 48 h at 28°C and bacterial DNA was extracted using MO BIO Ultra Clean™ Microbial DNA isolation kit (MO BIO laboratories, Inc. Hamburg, Germany). nifH gene amplification from these bacteria was performed with the universal nifH gene primers 19F (5’-GCIWTYTAYGGIAARGGIGG-3’) and 388R (5’–AAICCRCCRCAIACIACRTC-3’) (Ueda et al. 1995). The PCR protocol was the same as described for the real-time PCR assays with the exception of using the QuantiTectTM probe PCR Kit (Qiagen, Hilden GmbH, Germany). The amplified PCR products were analysed on 1.7 % agarose gels and their single products were purified using the QIAquick PCR purification kit (Qiagen, Hilden GmbH, Germany). Purified PCR products of the two bacteria were pooled and mixed, and the standard DNA concentration (µg ml-1) was measured at 260 nmusing an Eppendorf® spectrophotometer. The nifH gene copy numbers per µl were then calculated using the known DNA concentration and the template length of 390 bp. The standard range was set within 2.54E+09 to 2.54E+00 copies per µl as a 10-fold dilution series. The number of nifH gene copies µl-1 of plant DNA was calculated by comparing unknown samples with the nifH gene standard samples of defined gene copy numbers.

5.3.5 Spiking of plant DNA samples

In order to test for competitive or inhibitory effects of plant DNA on nifH gene PCR amplification, an experiment using nifH gene standard samples mixed with plant DNA was performed. Plant DNA extracted from cucumber shoot and root samples (three replicates for each plant part) were used. The recovery of nifH genes from plant DNA samples was measured using the real-time PCR approach specific for nifH genes. Mixture templates contained nifH gene copies ranging from 1.25E+00 to 1.25E+09. Real-time PCR was conducted and the nifH gene amplification of mixed DNA and pure nifH genes were quantified and correlated.

5.3.6 TEF gene quantification

It is likely that DNA extraction efficiency differs among different plant samples. Therefore, it is not feasible to use absolute quantified copy numbers of the nifH gene. The TEF gene (Transcriptional Enhancer Factor), a housekeeping gene for plant DNA analysis (Wulf et al. 2003), was therefore used as an internal control as its expression is not affected by different plant growth stages or treatments. Plant TEF gene was quantified using real-time PCR in conjunction with the SYBR Green I approach with primers TEFf (5’-ACTGTGCAGTAGTACTTGGTG-3’) and TEFr (5’-AAGCTAGGAGGTATTGACAAG-3’) (Wulf et al. 2003). For TEF gene quantification, standard samples for cucumber were prepared from DNA isolated from shoot and root parts using the same procedure as described above for the nifH gene.

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The nifH gene copies quantified from all sampling dates and treatments were calculated as relative values to the housekeeping TEF gene copy numbers (relative nifH gene copy number = (absolute nifH gene copy number *100)/ TEF gene copy number) to compensate for any differences in initial template DNA amounts due to variations in different plant sample DNA extraction efficiencies.

5.3.7 Statistical analyses

Comparison of mean values of three or six replicates for molecular or plant growth measurements, respectively, was performed using Student’s t-test at a P-level of ≤ 5 %. Pearson-type correlations were calculated at a P-level of ≤ 5 %. Where necessary, log transformations were applied to data sets in order to establish homogeneity of variances. All statistical analyses were performed using STATISTICA 6.0 (StatSoft 2001).

5.4 Results 

5.4.1 Effect of mineral N supply on plant growth

Our previous experiments revealed that cucumber could not survive during the 6 weeks of growth without supplying approximately 40% of total N application (Juraeva and Ruppel, Institute for Vegetable and Ornamental Crops, Germany, unpublished data). In this study, although, effects of N supply on plant shoot growth were not significant, strong N deficiency was observed at the end of the experiment for low N supplied cucumber plants. The higher nitrogen supply significantly increased total plant N content and plant root dry mass compared to the low N supply (Tab.15).

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Tab. 15: Effect of nitrogen fertilizer application on dry mass and N content of 42 days old cucumber plants fertilised with low and high nitrogen supply.

N supply

 

Total N uptake

(g plant)-1

Shoot dry mass

(g plant)-1

Root dry mass

(g plant)-1

N1

N2

0.08 ± 0.01

0.12 ± 0.01*

5.09 ± 0.30

5.23 ± 0.98

0.64 ± 0.12

0.86 ± 0.09*

Note:Values are mean ± SE. Values obtained for plants cultivated in the different N availability were compared by a Student’s t-test. N1, low N treatment (100 mgN·plant-1); N2, high N treatment (250 mgN·plant-1). Asterisk (*) indicates significant differences (P < 0.05) in the effect of N fertilization level on plant growth and N nutrition.

5.4.2 New method developed to quantify the nifH gene in plant tissue using quantitative real-time PCR

The universality of primers 19F and 388R (Ueda et al. 1995) was confirmed by amplification of the nifH gene from a range of nitrogenase-positive bacterial strains. Primer concentrations were optimised for the present experimental conditions. In all cases, one specific product was amplified and verified with the melting profile (Fig. 10A) and gel electrophoresis analysis (data not shown).

Using the nifH gene standard, target genes within a range of 2.54E+00 – 2.54E+09 copies µl-1 were detected. The standard curve documents the detection limit of 2.54 nifH gene copies µl-1, measured at the threshold after 50 cycles. It is noteworthy that the quality of the standard curve with a correlation coefficient of 0.997, a curve slope of –3.544 and PCR efficiency of 91.5 % over a range of 9 orders of magnitude is ideal to quantify nifH gene copy numbers within unknown samples (Boeckman et al. 2000).

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Fig. 10: Examples of melting profile analyses of the nifH gene amplified from (A) pure cultures of diazotrophic bacteria belonging to the different genera (see Tab. 1) and (B) cucumber plant DNA to confirm PCR product specificity. (B) The PCR product was amplified from the nifH gene standard sample (melting temperature (Tm) = 90.0°C; black line), plant shoot sample (Tm = 89.5°C; ■) and root sample (Tm = 91.0°C; ▲). The Tm was emperically determined by plotting the change in fluorescence with temperature (dRFU/dT) versus temperature (T). RFU, relative fluorescent units.

5.4.3 Plant DNA spiking

According to plant DNA spiking experiments, the concentration of nifH genes mixed with plant DNA samples was positively correlated with pure nifH genes at a slope of nearly 1 over a range of 7 orders of magnitude (r = 0.992). This indicates that the plant DNA did not inhibit the nifH gene specific real-time PCR within a range of 10E+02 to 10E+08 nifH gene copy numbers µL-1 DNA and that there is a nearly 100 % recovery of added nifH gene copies from plant samples (Fig. 11). The highest concentration of 10E+09 nifH gene copies µL-1 inhibited the PCR reaction. If we transfer these measurements to dimensions per g plant material, then between 10E+04 and 10E+10 nifH gene copies (plant fresh matter)-1 were detectable.

Fig. 11: Result of regression analysis between predefined nifH gene copy numbers (nifH gene added) and the cucumber plant DNA – nifH genomic DNA mixture (nifH-gene-plant). The asterisks (*) indicates significant correlation.

5.4.4 Specificity of the quantitative real-time PCR approach to quantify the nifH gene from plant samples

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In this study, the detection and quantification of nifH gene abundance in cucumber indicated the high sensitivity and reproducibility of the newly developed method based on real–time PCR. In addition, a similar study using tomato plants instead of cucumber further confirmed the ability of this new method to quantify target genes in different plant species (data not shown). PCR amplification with undiluted plant DNA was inhibited in some plant samples. In contrast, for the 1:10 diluted and 1:100 diluted DNA, no inhibition was observed and the 1:10 dilution was quantified precisely. Therefore, 1:10 diluted plant DNA was used for the quantification analysis.

The main disadvantage of the real–time PCR-based quantification approach using SYBR Green I dye is that non-selective detection of any double-stranded DNA molecule could occur and this would lead to a misquantification. However, in this study, this possibility was checked by both melting profiles (Fig. 10B) and gel electrophoresis analysis (Fig. 12). The length of the PCR products amplified from both bacterial pure culture (data not shown) and plant sample DNA was 390 bp (Fig. 12).

Fig. 12: Agarose gel analyses of the nifH gene PCR product (~390 bp) amplified from plant DNA using universal nifH gene primers 19F and 388R. M: size marker peqGOLD 100 bp DNA Ladder Plus. S: standard samples’ amplified nifH gene products from cucumber DNA (1 - shoot; 2 - root).

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Melting profiles of the nifH gene amplicons of various diazotrophic bacterial species differed within a range of 87°C to 92.5°C (Tab. 13, Fig. 10A). In shoot and root samples of cucumber grown under either low or high N availability conditions, the melting point of nifH gene amplicons was detected within the same range as that of bacterial pure cultures of 89.0°C to 91.5°C (Tab. 13, Fig. 10B). The observed different melting temperatures of same-size PCR products could be related to differences in the sequence composition of amplified target DNA.

5.4.5 nifH gene  quantification

The quantitative PCR protocol was used to determine the total copy number of amplifiable nifH sequences in DNA extracted from cucumber shoot and root samples at early and late plant growth stages. nifH gene abundance in plant shoot was found to be relatively stable over time and not affected by mineral N fertilisation ranging between 1.57E+06 – 4.63E+06 copies in 1 g fresh shoot. The results of the statistical analyses for the N supply and plant age effects on relative nifH gene abundance within the plant root as determined by Student’s t-test are summarised in Tab. 16. Relative nifH gene abundance in root increased with increasing plant age (Tab. 16C). Seven days after planting, the nifH gene pool consisted of 1.24E+07 copies (g fresh root)-1, while in 42 d old plant roots, this figure increased to 5.25E+07 and 1.59E+09

Tab. 16: Effects of different factors on nifH gene distribution. Mean values from three replicates were compared by a Student’s t-test. Relative log nifH gene copy numbers per TEF gene copy number (nifH cn) ± standard deviation are shown. Asterisk (*) indicates significant differences (P < 0.05). N1, low N treatment (100 mgN·plant-1); N2, high N treatment (250 mgN·plant-1).  aCopy no. was calculated commonly from harvest 1 (at day 7) and harvest 2 (at day 42).

Factor

nifH cn

(A) Plant part a

Shoot

5.83 ± 0.42

Root

8.18 ± 1.05*

(B) N supply

N1

6.96 ± 0.46

N2

8.61 ± 0.87*

(C) Plant age

7 days old

6.39 ± 0.20

42 days old

8.62 ± 0.67

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in both low and high N-supplied plants, respectively. Interestingly, at the end of the experiment, nifH gene abundance in high N supplied plants was significantly higher compared to low N supplied plant roots. According to the regression analysis, a strong correlation (r = 0.96) was observed between the relative nifH gene copy numbers measured for the plant and the plant N content (Fig. 13).

Fig. 13: Result of regression analysis between the nifH gene copy numbers relative to housekeeping TEF gene copy numbers (nifH gene, measured as relative cn) and the plant N content (Plant N, measured as mg N plant-1) of cucumber plants.

5.5 Discussion

5.5.1 Bacterial DNA extraction from plant samples

From cucumber plant samples, DNA could be obtained in the range of 55.08-140.50 ng/ mg fresh shoot and 12.58-52.08 ng/ mg fresh root. Efficiency of DNA isolation with DNeasy Plant mini kit was different for plants grown in different level of N availability and of different age. Our previous study performed to quantify inoculated bacteria abundance in tomato plant shoot and roots using a real-time PCR approach revealed that DNeasy Plant mini kit enabled us to recover bacterial DNA within plant samples precisely (Juraeva and Ruppel 2005).

5.5.2 Productivity and specificity of the quantitative real-time PCR approach to quantify the nifH gene from plant samples

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Ueda et al. (1995) reported that nifH gene amplification from dryland plant DNA (maize and soybean) failed; whereas using the same protocol, the nifH genes from rice root DNA could be amplified. The lack of amplification could result from either a target gene concentration below the detection limits of the employed assay or from inhibition of PCR due to PCR inhibitors in template DNA. In this study, the detection and quantification of nifH gene abundance in cucumber indicated the high sensitivity and reproducibility of the newly developed method based on real–time PCR. In addition, a similar study using tomato plants instead of cucumber further confirmed the ability of this new method to quantify target genes in different plant species (data not shown).

In this study, the advantages and disadvantages of employed approach for gene quantification purposes were carefully taken into consideration. The main disadvantage of the real–time PCR-based quantification approach using SYBR® Green I is that any double-stranded DNA molecule is measured and this would lead to misquantification if primer dimers occurred. However, in this study, product specificity was checked by both melting profiles (Fig. 10B) and gel electrophoresis analysis (Fig. 13). The length of the PCR products amplified from bacterial pure culture (data not shown) and plant DNA were within the same range of previously published nifH gene sequences amplified using the same primer pair (Ueda et al. 1995) and thus, confirmed the accuracy of the method to amplify the target gene.

One advantage of the real–time PCR-based approach used in combination with SYBR Green I is that melting profile analyses allows checking of the specificity of PCR products and discovery of unspecific primer dimers by generating one single melting peak with the known specific melting temperature of the product, thereby testing correct gene quantification. Additionally, shifts in specific melting temperatures may be indicative of differences in amplified PCR product sequence composition. The observed different melting temperatures of same-size PCR products amplified from both pure bacterial culture DNA and plant DNA could be related to differences in the sequence composition of amplified target DNA. Moreover, different melting temperatures of nifH gene sequences amplified from the shoot and from root samples probably indicate that different dominating diazotrophic bacterial communities colonise the shoot and root parts of the plant. However, whether or not the nifH-gene-specific real-time PCR approach with SYBR Green I can be used to measure nifH gene diversity in environmental samples has to be proven in further experiments.

5.5.3  nifH gene quantification

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Quantified nifH gene copies were calculated relative to the quantified copy numbers of the housekeeping TEF gene. Although the DNA-based PCR method is not directly related to nifH gene expression, it does allow comparison of the effects of different treatments on the N2–fixing potential of the microbial community in plant samples by quantification of nifH gene abundance in plant. These nifH gene values reflect the abundance of diazotrophic populations associated with the plant.

For the first time, the nifH gene pool in the plant shoot has been quantified and found to be lower compared to root samples. Various studies indicated that the abundance of total diazotrophs or of specific populations in both pure culture and environmental samples can be influenced by the amounts of inorganic N applied (Cejudo and Paneque 1986, Herridge and Brockwell 1988, Limmer and Drake 1998, Fuentes-Ramirez et al. 1999). We found that although, high N supply did significantly increased N amount in both plant shoot and root (data not shown), it did not affect the gene abundance in plant shoot. Therefore, we suggest that plant N status did not influence the abundance of diazotrophic bacteria inhabiting plant tissue. Moreover, the stability of the nifH gene in plant shoot over time suggests that in contrast to plant roots, physiological changes occurring during the plant shoot development did not significantly affect diazotrophic population abundance in plant shoot.

5.5.4 The effect of N amount supplied on nifH-gene abundance in plant  root

The significantly higher nifH gene copy numbers in high N- compared to low N-supplied cucumber plant roots (Tab. 16B) run contrary to previous reports (Tan et al. 2003; D. Juraeva and S. Ruppel, Institute for Vegetable and Ornamental Crops, Germany, unpublished data). Our data suggest that (1) higher N supply stimulated the growth of diazotrophic bacteria in cucumber roots and (2) the effect of N availability on diazotrophs may be plant-species dependent, as in rice (Tan et al. 2003) and tomato roots (D. Juraeva and S. Ruppel, Institute for Vegetable and Ornamental Crops, Germany, unpublished data) where the diazotrophic communities were suppressed by higher mineral N fertilisation. In our experiment, high N supplied cucumber plants developed a significantly larger root system (Tab. 15) than the low N supplied plants, which provides favorable conditions for microorganism growth, including those for diazotrophic bacteria. Seldin et al. (1984) reported no repression of nitrogenase genes in some diazotrophs and Piceno and Lovell (2000) even documented increased nitrogenase gene expression with increasing N availability. Therewith, it seems imaginable that an increased abundance of certain diazotrophic groups in higher N supplied plants lead to an increased N nutrition of plants due to biological N2 fixation. Since the number of nifH gene operons contained in various organisms may vary and is usually unknown, it is not possible to extrapolate quantified nifH gene copy numbers to cell density values exactly. However, a rough estimation of the population size of diazotrophic bacteria in cucumber plant shoot and roots at early and late plant growth stages can be calculated. Assuming the DNA extraction efficiency of 100% from plant material and the presence of two nifH gene copies per bacterial cell (Yeager et al. 2004), diazotrophic bacterial numbers exceeded between 1.57E+06 – 4.63E+06 in cucumber shoot parts, and 1.24E+07 – 1.59E+09 in root parts. From day 42, diazotrophic bacterial numbers inhabiting high N supplied cucumber roots were 15-fold higher than those in low N supplied plants. Comparative analysis of previous reports on diazotrophic bacteria abundance in plant rhizoplane and inside of roots (Reinhold et al. 1986, Barraquio et al. 1997), and approximate calculation of diazotrophic bacteria cells in cucumber shoot and root based on the present study’s results, demonstrated that our results in nifH gene copy numbers quantified from the plant DNA using real – time PCR approach was reasonable. Reinhold et al. (1986) reported 2.0E+07 diazotrophs (g dry weight)-1 of rice roots, while Barraquio et al. (1997) enumerated the size of populations of diazotrophic bacteria in different rice genotypes ranged from 10E+03 and 10E+07 per g roots.

5.5.5 Correlation of nifH-gene abundance to plant N nutrition

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In this study, the combination of nifH gene quantification and plant N-uptake measurements was shown to be a possible tool to evaluate the contribution of the N2–fixing plant-inhabiting diazotrophic community to plant N nutrition. The positive correlation between nifH gene abundance and plant N nutrition highlights the potential value of studying functional genes in the context of ecosystem processes. However, these results are only suggestive of this relationship, and future studies should focus on measuring the relationships of gene abundance to the target gene expression and activity simultaneously. Additionally, the relationship between the diversity of special diazotrophic bacterial populations and their sensitivity to environmental changes should be examined.

New technologies and methods to investigate microbial communities are being developed at a rapid pace and provide new opportunities to link community structure to ecosystem processes. The herein described DNA-based real-time PCR quantification of nifH gene abundance in plant tissues can be extended to RNA-based approaches as DNA is more likely to reflect the standing biomass of a particular community and mRNA should be more closely related to activity rates (Bürgmann et al 2003). Therefore, these real-time PCR approaches offer an additional promising avenue for linking microbial communities to environmental processes.

Chapter 6: Quantitative real – time PCR based evaluation of the direct potential of diazotrophic bacteria to the plant N nutrition


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