6 QUANTITATIVE REAL-TIME PCR BASED EVALUATION OF THE DIRECT POTENTIAL OF DIAZOTROPHIC BACTERIA TO THE PLANT NITROGEN NUTRITION

6.1 Abstract

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Understanding the factors involved in controlling the colonisation/distribution of nifH gene containing microorganisms in the environment may allow identifying the factors controlling N2 fixation in the environment. Two diazotrophic bacteria were tested for responses in root colonization, growth stimulation, and nitrogen supply to the plant in the presence of N in different levels. We proposed that in the low level of N availability, the diazotrophic bacteria are more abundant than in high N supplied conditions, and that even in high N availability, the application of diazotrophic plant growth promoting bacteria (PGPB) strains can increase the diazotrophic population allowing increased potential for plant N nutrition. The results of performed plant experiments suggested that B. licheniformis BL43 and Xanthomonas sp. Xs148 had the potential to improve the N nutrition of tomato in low level of N availability. B. licheniformis BL43 showed the significant effect on plant N uptake in the presence of high N as well. Furthermore, it was hypothesized that the improved plant N nutrition is due to N2-fixing ability of inoculated bacteria, and that the correlations between plant N content and applied bacteria cell numbers and quantified nifH gene abundance in plant tissue indicate/evaluate the capacity of the applied diazotrophic bacteria to fix atmospheric nitrogen. Quantitative evaluation of nifH gene abundance in the inoculation experiment using real – time PCR based direct quantification approach indicated a high potential for plant-associated N2-fixation in both BL43 and Xs148 strains. Both bacteria showed significantly high abundance in low N availability and were positively correlated to nifH gene abundance in the plant root. We found significant positive correlation between quantified target bacteria abundance, nifH gene copies and plant N nutrition. Relationship of quantified nifH gene to total N nutrition of plant was less close in inoculated plant compared to non – inoculated plant. Those correlation coefficients were affected strongly by N availability.

Key words

Plant growth promoting bacteria – inoculation - biological nitrogen fixation – nifH gene – 16S-23S ISR – quantification – real time PCR

6.2 Introduction

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Asymbiotic biological nitrogen fixation (ANF) can help to meet plant N requirements even in intensive agriculture (Tchan et al. 1988). At present, ANF can only partly meet the N demand of plants since cereals and other non-legumes usually require high N fertilizer amounts for reaching high yields. This is at least partly due to the factor that soil N is mostly negatively correlated with diazotroph abundance, that means the occurrence of bacteria which are able to bind atmospheric nitrogen (Cejudo and Paneque 1986, Limmer and Drake 1998). High soil N availability strongly down regulates the expression the nifH gene which is responsible for the biological N2 fixation in diazotrophs (Triplett et al. 1989, Merrick 1992, Bürgmann et al. 2003). For a more efficiently exploitation of ANF in agriculture, the factors affecting the N2 – fixing ability of diazotrophic bacteria shall be better understood. It is important to gain more detailed knowledge about the colonization ability and stability of diazotrophic populations in the presence or absence of high mineral N levels. In particular, the effect of soil N availability on the potential contribution of different diazotrophic populations should be studied, to select, bacteria that are specifically tolerant to high soil N levels (Ruppel and Merbach 1995, Ruppel and Merbach 1997).

Several crop-inoculation studies have been performed using acetylene reduction, N balance and 15N isotope dilution methods to quantify the contribution of diazotrophic bacteria to the plant N nutrition. The acetylene reduction assay is widely used because of its simplicity and low cost, but measures only nitrogenase activity and reveals no information on whether the fixed N is incorporated into the plant (Boddey et al. 1995). N balance experiments contain two unknown values N-losses and biological N2-fixation and can therefore underestimate the latter one. The 15N isotope dilution and 15N natural abundance techniques are currently accepted as the most useful methods for examining ANF in in vivo conditions (James 2000). However the 15N isotope dilution method is based on the addition of mineral N to the system, which significantly affects the bacterial gross N-transfer rates and biological N2 fixation (Ruppel et al. 2006 a, b). In recent years, a molecular method was employed to detect and quantify the functional genes which are regulation ANF (Buergmann et al. 2003, Juraeva et al. 2006).

However, many studies show that ANF is one of the primary mechanisms responsible for improved plant N nutrition after inoculation with PGPB (Boddey and Döbereiner 1995, Garcia de Salamone et al. 1996, Malik et al. 1997, Boddey et al. 1995, James 2000), diazotrophic PGPBs can also enhance plant N uptake by several other mechanisms, as for example, an increased uptake of mineral N by enhanced root N uptake mechanisms (Bashan 1990, Bashan and Levanony 1991), or by increasing the plant root system as the root branching, root number, thickness, and length (Guanarto et al. 1999, Biswas et al. 2000). There may also exist an antagonistic effect of the bacteria against pathogens, protecting the root from diseases (Bashan and de-Bashan 2002). Therefore, when the role of diazotrophic bacteria for plant growth is studied, it is important to estimate the ANF activity of these bacteria. Although, the presence of nifH genes themself can not quantify the value of ANF, the abundance of this gene indicates the potential of diazotrophic N2 fixation (Juraeva et al. 2006).

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In the present study, the relationship between the numbers of introduced diazotrophic bacteria, the numbers of nifH genes and the plant N nutrition is estimated. A tomato greenhouse experiment was established using two different N fertilization treatments and the inoculation with two diazotrophic bacterial strains. Bacterial colonization and nifH gene quantification was monitored using quantitative real-time PCR and N-balances were calculated.

6.3 Materials and methods

6.3.1 Experimental setup

A two-factorial pot experiment was established with tomato (Lycopersicon esculentum [Mill] L. ‘Counter F1’) in greenhouse conditions (latitude of 52° 21`N, longitude of 13° 18`E at an altitude of 50 m), close to Berlin, Germany. The two factors were mineral N-fertilization values (low nitrogen supply (N1) and high nitrogen supply (N2)) and the application of diazotrophic bacterial strains (control without bacterial application, Bacillus licheniformis BL43, Xanthomonas sp. Xs148), all treatments were 6 times replicated and completely randomized. Tomato seeds were germinated on trays containing vermiculite under greenhouse conditions. Ten days after germination, tomato plants were removed from the trays and vermiculite was gently washed from root system, plants were inoculated with the bacterial strains (see later in this chapter) and transferred into pots (one seedling per pot) filled with 800 g quartz sand. Plants were grown for 6 weeks (in May-June 2004) at temperatures of 26°C to 28°C during the day and 16°C to 18°C at night in a greenhouse without artificial illumination. The water content was adjusted to 60% of maximal water holding capacity and kept during the entire experiment. Plants were supplied with half-strength Hoagland’s nutrient solution (Hoagland and Arnon 1950) lacking N. Nitrogen was supplied separately according the N treatments: N1- low N fertilization, equivalent to approximately 35% of plants N demand and N2 - higher N fertilization, calculated to fully meet the plant N demand for a six week growth period (Tab. 17) at three occasions (2, 21 and 28 days after planting) by injecting NH4NO3 solution homogeneously into the root zone. Plants received potassium/nitrogen/phosphorus in a ratio of 1:0.6:1 and 1:1.8:1 for low and high N supplied plants, respectively.

Tab. 17: Amounts of N fertilized per plant for low (N1) and high N (N2) fertilization treatments, in mg N supplied per plant. The total amount of N was applied at three occasions (2, 21 and 28 days after planting).

N application

(days after planting)

N1 (mg)

N2 (mg)

First (Day 2)

25

50

Second (Day 21)

25

60

Third (Day 28)

25

60

Total

75

170

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6.3.2 Bacterial strains

Two diazotrophic bacterial strains were used: Xanthomonas sp. Xs148 (Xs148) (former Burkholderia sp. 148 (Juraeva and Ruppel 2005a)) isolated from the root of wheat grown on Calcisol (loamy sand) in Uzbekistan (Juraeva and Ruppel 2005b, Juraeva and Ruppel, unpublished data) and Bacillus licheniformis BL43 (BL43) isolated from maize rhizosphere (Culture collection, Institute of Microbiology, Uzbekistan Academy of Sciences). These strains are proven to promote plant growth significantly (Juraeva and Ruppel, 2005a) and to express antagonistic activity against some pathogen Fusarium species.  

6.3.3 Inoculation

Pure cultures of bacteria, BL43 and XS148, were grown in Standard I (Merck, Darmstadt, Germany) nutrient solution on a rotary shaker at 28°C for 48h. The bacterial suspensions were centrifuged at 7.000 rpm for 10 min. Growth medium was discarded and the bacterial pellet was resuspended in 0.05M NaCl buffer to a cell density of 109-1010 cells ml-1, checked by plate dilution technique on Standard I nutrient agar (Merck, Darmstadt, Germany) and MPN method. Seedlings were divided into three treatments: control without bacterial inoculation (-B); bacterial strain BL43 (+BBL); and bacterial strain Xs148 (+BXs). At transplanting tomato roots were dipped into the bacterial suspension for 2 min. Control plants were dipped into sterile 0.05M NaCl buffer.

6.3.4 Plant sampling

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Roots and shoots of the plants were sampled at two dates: 7 and 42 days after planting. At day 7 after planting, three replications were harvested, roots and shoots were separated and quartz sand particles were carefully removed from the roots under a gentle stream of tap water.

For molecular measurements 0.1 g fresh samples from the middle part of roots were cut, transferred to separate Eppendorf tubes containing 0.1 g of sterile glass beads (0.5 mm diameter), were frozen at –20°C and lyophilised. Plant DNA was extracted using DNeasy Plant Mini Kit (Qiagen, Hilden GmbH, Germany) as described previously (Juraeva et al. 2006). The rest of plant was used to measure plant fresh mass, total root length and plant dry mass. 42 days after planting, additionally total N content of plants was measured (six replications). Total N concentration in plant tissues was analyzed using a CHN-O rapid elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). At sampling time 42 days after planting a random sub sample: 1 g fresh mass from different parts of root were collected and root length was determined using optical picture analysis system and the KS400 4.0 software (Carl Zeiss Vision GmbH, Hallbergmoos, Germany).

6.3.5 Target bacteria, nifH- and TEF gene quantification

Target bacteria 16S-23S ISR of both inoculated bacterial strains (Juraeva and Ruppel 2005a), nifH gene (Juraeva et al. 2006) and TEF gene copy numbers (Juraeva et al. 2006) in plant samples were quantified using real-time PCR and SYBR® Green I approach, as described previously. The primers used are listed in Tab. 18. Quantitative real-time PCR was performed using an iCycler iQ system (Bio-Rad laboratories, München, Germany) associated with the Icycler iQ Optical System Software (version 3.1; Bio-Rad laboratories, München, Germany). All PCRs were performed in triplicate in a volume of 25 µl. To compensate for any differences in initial template DNA amounts due to variations in different plant sample DNA extraction efficiencies, the 16S-23S ISR and nifH gene copy number were calculated as relative values to the housekeeping TEF gene copy number using the following equation: relative nifH gene copy number = (absolute nifH gene copy number *100)/ TEF gene copy number; relative 16S-23S ISR copy number = absolute 16S-23S ISR copy number *100/ TEF gene copy number.

6.3.6 Statistical analyses

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A two-way factorial analysis of variance was used to gauge the significance of differences in mean values between all factor pairs, such as supplied N level versus vegetation stage, supplied N level versus inoculation, vegetation stage versus inoculation. 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 %. Linear regression analysis was performed and correlation coefficients 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)

Tab. 18: Specificity and nucleotide sequences of PCR primers used in this study. * I= inosine, R= A or G, W= A or T, Y= C or T

Target

Taxon

Primer sequences

Target gene

Product length

Reference

Plant

Teff: 5’-ACT GTG CAG TAG TAC TTG GTG -3’

Tefr: 5’-AAG CTA GGA GGT ATT GAC AAG-3’

TEF

155bp

Wulf et al. 2003

Bacillus licheniformis BL43

BL43F1: 5'-ACCCACCAAGTCTACTGAACAC-3'

BL43R1: 5'-CGAACCGTTGACCTCCTGC-3'

16S-23S ISR

108 bp

Juraeva and Ruppel 2005a

Xanthomonas sp. Xs148

Xs148aF1: 5' GCCGATATGAGAGTCCCTTTTG-3'

Xs148aR1: 5'-GCCTGTCGGGATCGAACC-3'

16S-23S ISR

89bp

Juraeva and Ruppel 2005a

Bacteria

19F*: 5'-GCIWTYTAYGGIAARGGIGG-3'

407R*: 5'-AAICCRCCRCAIACIACRTC-3'

nifH gene

~390bp

Ueda et al. 1995

6.4 Results 

6.4.1 Plant growth responses

Shoot N concentrations were in range of 0.65-0.75% in low N supplied plants and 1.36-1.46% in high N supplied plants that is below than threshold for N deficiency (<2%; all expressed on a dry matter basis), indicating that even the high N supplied plants were slightly N-starved (World Fertilizer Use Manual, (Online source)). Thus, in all plants, N was a growth-limiting factor. However, inoculated plants appeared more robust and greener than the non-inoculated. Increased N fertilization of the tomato plants induced significant plant growth responses in shoot and root dry weight and N content in both non inoculated and with diazotrophic bacteria inoculated treatments (Tab. 19). High N supplied control plants resulted in significantly increased root length compared with low N supplied control plants (Tab. 19). Bacterial inoculation induced similar high root growth enhancement in low N1 and in high N2 fertilized treatments.

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The known plant growth promoting activity of the selected diazotrophic bacterial strains B. licheniformis BL43 and Xanthomonas sp. Xs148 was established in the tomato experiment. All parameters measured were significantly increased after bacterial inoculation compared to the non inoculated control in the low N1 fertilized treatment. With higher N2 fertilization, the PGPR effect was still detecTab. in total plant growth, N content and root length, however, BL43 did not significantly increase the shoot dry weight and Xs148 failed to significantly increase root dry weight and root N content (Tab. 19).

6.4.2 Plant root colonization and persistence of inoculated bacteria

Inoculated BL43 cells were shown to colonize the young tomato roots (7 days after planting) independently of the N fertilization level confirming the colonization ability of both inoculated bacterial strains on roots (Tab. 20). The inoculation increased the species specific gene copy numbers significantly over the native population at the non-inoculated plants. However, at the end of the experiment, these bacteria were only able to persist at the roots at low N fertilized plants (N1 42 days after inoculation), where significantly higher BL43 target genes were detected compared to the non-inoculated plants. The lower abundance of BL43 in high N supplied +BBL 

Tab. 19: Effect of N fertilization and diazotrophic bacterial inoculation on plants shoot and root dry wight (d.wt), N content and root length of 42 days old tomato plants. Mean values of 6 replicates ± SE. Asterisk (*) indicates significant bacterial inoculation effects (P < 0.05). Bold numbers indicate significant N fertilization effects (P < 0.05; bold) N1, low N fertilization (75 mg N·plant-1); N2, high N fertilization (170 mg N·plant-1). -B – non-inoculated, +BBL and +BXs – plants inoculated with B. licheniformis BL43 and Xanthomonas sp. Xs148, respectively.

N1

N2

-B

+BBL

+BXs

-B

+BBL

+BXs

Total plant d. wt (g plant-1)

2.07 ± 0.16

 3.15 ± 0.16*

2.95 ± 0.18*

4.86 ± 0.16

5.24 ± 0.12*

  5.35 ± 0.14*

Shoot d. wt (g plant-1)

1.82 ± 0.13

2.59 ± 0.17*

2.48 ± 0.14*

4.12 ± 0.15

4.31 ± 0.11

4.49 ± 0.14*

Root d. wt (g plant-1)

0.35 ± 0.02

0.56 ± 0.04*

0.47 ± 0.04*

0.83 ± 0.03

0.93 ± 0.02*

0.86 ± 0.03

Shoot N (mg plant-1)

13.05 ± 0.96

16.77 ± 1.45*

18.63 ± 3.71*

56.13 ± 2.98

63.00 ± 1.95*

61.20 ± 1.87*

Root N (mg plant-1)

5.21 ± 0.39

7.27 ± 0.48*

6.73 ± 0.47*

21.62 ± 0.76

23.56 ± 0.64*

22.07 ± 0.75

Total plant N (mg plant-1)

18.26 ± 0.65

24.03 ± 1.24*

25.35 ± 1.68*

77.75 ± 3.46

86.56 ± 2.28*

83.90 ± 2.19

Root length (m plant-1)

29.38 ± 6.60

69.92 ±14.22*

57.34 ± 17.18*

51.06 ± 2.50

61.54 ± 5.44*

67.97 ± 5.68*

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Tab. 20: Abundance of inoculated bacterial genes and nifH genes in tomato roots of non inoculated (-B) and with B. licheniformis (BL43) and Xanthomonas sp. (Xs148) (+B) inoculated plants in treatments with low N1 and high N2 fertilization levels, all investigations carried out 7 and 42 days after planting. Gene abundance is expressed relative to the housekeeping TEF gene. Mean values of three replicates ± SE. Asterisk (*) indicates significant bacterial inoculation effects (P < 0.05) Bold numbers indicate significant N fertilization effects (P < 0.05; bold) N1, low N fertilization (75 mg N·plant-1); N2, high N fertilization (170 mg N·plant-1) separately at each sampling time. -B – non-inoculated, +BBL and +BXs – plants inoculated with B. licheniformis BL43 and Xanthomonas sp. Xs148, respectively.

Sampling time (days after planting)

N level

Target bacterial abundance

nifH gene abundance

BL43

Xs148

BL43

Xs148

7 (-B)

N1

8.73 ± 0.86

8.58 ± 0.26

9.95 ± 0.76

9.95 ± 0.76

N2

8.27 ± 0.20

10.55 ± 0.08

9.33 ± 0.51

9.33 ± 0.51

7 (+B)

N1

12.30* ± 0.70

10.83*   ± 0.26

11.42* ± 0.17

11.66* ± 0.60

N2

11.07* ± 0.10

10.41 ± 0.07

10.07* ± 0.14

10.65* ± 0.81

42 (-B)

N1

9.43 ± 0.30

9.85 ± 0.00

8.84* ± 0.25

8.84* ± 0.25

N2

9.88 ± 0.22

9.19 ± 0.33

8.15 ± 0.45 

8.15 ± 0.45 

42 (+B)

N1

9.67* ± 0.39

9.65 ± 0.23

8.58 ± 0.30

7.05 ± 0.18

N2

8.30 ± 0.22

10.73* ± 0.23

7.20 ± 0.07

7.90 ± 0.61

plant roots in comparison to low N supplied +BBL plant roots sampled on both sampling times suggested that N availability affected BL43 population in tomato plant root.

The effect of N availability was shown to be stronger on Xs148 abundance. Due to the high native population density of Xanthomonas sp. the persistence of inoculated Xs148 bacterial cells was hardly to show. In early growth stage, with increased N fertilization the native Xanthomonas sp. population was raised (Tab. 20) Therefore, the measurements showed that the inoculated Xs148 cells only increased the cell numbers of the Xanthomonas species at the low N fertilized tomato roots, while this significance was lost in inoculated plants fertilized with high N (Tab. 20). These data indicate a better colonization rate of native Xanthomonas sp. cells at tomato plant roots grown in higher N fertilization level.

6.4.3  nifH gene abundance in inoculated and non-inoculated plant root

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Inoculation with both diazotrophic bacterial strains BL43 and Xs148 increased the nifH gene abundance at inoculated plant roots in low and high N fertilized treatments over the nifH gene abundance at the non-inoculated control roots 7 days after planting (Tab. 20). That increased nifH gene level, which was established 7 days after the inoculation of diazotrophic bacterial cells, did not remain stable during the plant growth. In contrast, at 42-day-old tomato plant roots nifH gene abundance was higher at non-inoculated plant roots than at inoculated ones (Tab. 20). Data from both sampling times of non-inoculated plants revealed that the nifH gene abundance in high N supplied plants was relatively lower when compared with low N supplied plants (Tab. 20). In younger plant roots, gene abundance was relatively higher than older plants. However, those values did not reach the significant level.

6.4.4 Interrelationship between diazotrophic bacterial inoculation, nifH gene abundance and plant N nutrition

The inoculation of tomato plants with the diazotrophic bacterial strains BL43 and Xs148 increased the nifH gene abundance significantly at low N fertilization level at both investigation times 7 and 42 days after planting and inoculation (positive correlation nifH cn vs. ISR cn Tab. 21). Since this positive correlation was observed in most treatments and plant growth stages (Tab. 21), nifH gene concentrations in +B plant roots shown to be controlled by the introduced bacterial population. The only exception was the high N supplied +BXs treatment.

Plants influenced by the inoculum during early stages of development may be affected during early stages of development, even if the inoculum does not persist as a dominant rhizosphere inhabitant throughout plant growth. This effect, for example, been shown in wheat inoculated with Azotobacter, Bacillus and Clostridium (Rovira 1965).

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The response of inoculated bacteria to N availability indicating that both bacteria were suppressed by higher mineral N fertilization (Tab. 20), reflected in nifH gene quantification from +B plants showing high degree of correlation between introduced bacteria abundance and nifH gene pool of both bacterial strains in low N fertilized treatments – however, there was no significant correlation in the high N fertilized treatments at Day 42 after planting (Tab. 21). In the low N fertilized treatments, the plant N content was increased with both increased numbers of inoculated bacterial cells and increased nifH gene copy numbers at the end of the experiment, 42 days after inoculation. Such a positive correlation was not detecTab. in the high N fertilized treatments (Tab. 21). In the higher N treatment a positive correlation did only occur between nifH gene copy numbers and the BL43 gene copy numbers immediately (Day 7) after the bacterial inoculation. In the older plants this effect was lost.

Tab. 21: The effect of N fertilisation level on the linear regression relationships between nifH gene copy number (nifH cn) and the BL and Xs bacterial copy numbers (ISR cn), and total plant N content. ND – not determined; BL43, plants inoculated with B. licheniformis BL43; Xs148, plants inoculated with Xanthomonas sp. Xs148. Asterisk (*) indicates significant correlation.

Sampling time (days after planting

N level

 

nifH cn vs ISR cn

 

nifH cn vs plant N content

ISR cn vs plant N content

BL43

 

 

Xs148

    

BL43

 

 

Xs148

BL43

 

 

Xs148

 

7

N1

0.96*

0.82*

ND

ND

ND

 

ND

7

N2

0.89*

-0.23

ND

ND

ND

ND

42

N1

0.86*

0.99*

0.95*

0.99*

0.95*

1.00*

42

N2

0.43

0.80

-0.55

0.55

0.51

-0.06

6.5 Discussion

6.5.1 Plant growth responses

Besides the biological nitrogen fixing activity, a range of other plant growth promoting mechanisms can be induced by bacterial inoculation (Dobbelaere et al. 2003, Bashan et al. 2004) and positive response of the inoculation process in improving plant N nutrition can be related to one of those plant growth mechanisms of the inocula tested. PGPB have a significant impact on nitrogen nutrition by increasing N uptake capacity, indirectly as a consequence of stimulated lateral root development (Okon and Vanderleyden 1997, Bertrand et al. 2000) and possibly directly by stimulating ion uptake systems. In this study, the inoculation process also showed significant effects on plant root resulting in increased root length (Tab. 19). Therefore, the indirect effect of PGPB on nutrient uptake via the increased root surface area due to the stimulation of root development can also be suggested as one possible effect mechanism for the improvement of N uptake (Guanarto et al. 1999, Biswas et al. 2000, Mantelin and Touraine 2004).

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In its turn, the availability of NO-3 is known to affect root branching (Forde et al. 2002). In this study, root measurement results were consistent with the suggestions of previous reports (Dobbelaere et al. 2002, Mantelin and Touraine, 2004) that the total root length is the highest in inoculated plants grown with low N fertilization. In high N fertilized plants, although, the total root length was significantly higher than control plants, the effect of inoculation on root development was much lower than in low N supplied plants.

6.5.2 Interrelation between the abundance of introduced diazotrophic bacteria and plant N nutrition

Effective colonization of plant roots by PGPB plays an important role in growth promotion, irrespective of the mechanism of action (Bolwerk et al. 2003, Raaijmakers et al. 1995).

There are different reports considering the effect of N availability on diazotroph bacterial abundance. For instance, our observations in this study are in agreement with some reports (Cejudo and Paneque 1986, Limmer and Drake 1998, Muthukumarasamy et al. 2002), showing that N availability is negatively correlated with inoculated diazotrophic bacteria abundance tested in this study (Tab. 3). Since the plants select for functional groups in the rhizosphere to be colonised (Grayston et al. 1998, Grayston et al. 2001, Baudoin et al. 2003), it can be suggested that in N limiting conditions, plants select more N2 fixers.

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In this study, we document that N availability affected different diazotrophic bacterial species in different level. The strong effect of N fertilization on diazotrophic bacteria abundance demonstrates the importance to determine optimal N fertilizer levels for efficient inoculation experiments. Measurements of introduced bacteria like bacteria population in +B and -B plants showed that, although, the abundance of both introduced bacteria in +B plants and introduced bacteria like natural bacteria populations in -B plants was significantly decreased in response to high N fertilization, inoculation of plant with diazotrophic bacteria allowed to provide highly abundance of diazotrophic community in inoculated plant root even in high N fertilization providing high potential of diazotrophic community to fix atmospheric N2.

The significantly close relationship between introduced bacterial abundance and nifH gene copy number in plant roots (Tab. 21) indicated the presence of a high potential of introduced bacteria to fix atmospheric N2 in low N conditions. The positive correlation between introduced bacterial cells and the nifH gene copy numbers and the plant N content strongly suggest a positive impact of inoculated diazotrophic bacteria on plant N nutrition in young plant growth stages under N limited conditions.

Regression analysis to determine the relationships of those introduced diazotrophic bacteria and nifH gene abundance, and the significant correlation of their abundance to plant N content (Tab. 21) could be employed to identify the possible contribution of biological N2-fixation by introduced bacteria to plant N nutrition. Introducing the active members of diazotrophs to plant root increased the amount of this contribution, however, relatively less close relationships of nifH gene abundance and plant N nutrition of +B plants in comparison to -B plants was observed (Tab. 21). It may be due to the secondary mechanisms, like root growth promotion by phytohormonal effects (Tab. 19), of introduced bacteria contributed to general N nutrition of plant (Bashan et al. 1989, Hurek et al. 1994).

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Regression analysis allowed to evaluate the effect of supplied N to the supposed activity of nifH gene existed in plant root. The correlation between nifH gene and plant N nutrition for low N supplied plants was stronger in comparison to high N supplied plants (Tab. 21) indicating that an increased N input does not only induce changes in the abundance of nifH gene, but also influences the potential activity of this gene suggesting that the proportion of biological N2-fixation was higher at low N levels.

Plant experiments performed using the non-nitrogen fixing (Nif -) Azoarcus sp. mutant as a negative control, combined with 15N – based balance studies (Hurek et al. 1998, Hurek et al. 2002) can provide the direct evidence whether plant benefits from the N2 fixed by introduced diazotrophic strains. However, as it is observed in this study that increasing of nifH gene pool in plant root was not due to the contribution of introduced bacteria (because, it was not colonized significantly), but it was shown to be the effect of introduced diazotrophic bacteria on the abundance of natural diazotrophic population, such methods which centres on only introduced diazotrophs, would ignore the possible contribution of inoculated bacteria on the native diazotrophic population inhabiting the plant and can led to the wrong conclusion that plant N nutrition was improved by another plant growth promoting effects of introduced bacteria.

6.5.3 Native diazotrophic bacteria abundance in tomato plants 

It is often claimed that the presence of nifH gene does not directly related to its activity, because nifH gene expression is highly regulated (Hoover 2000), at both transcriptional (Chen et al. 1998) and post-translational levels (Kim et al. 1999). However, the assumption that “genes are ultimately not retained by microorganisms, unless they are functional and thus, are selected for in the environment” (Zehr et al. 2003) should be true for nitrogenase, since N2 fixation can involve around 20 genes. Therefore, it can be suggested that significantly close relationships of nifH gene abundance and plant N content (correlation coefficient of 1.00 and 0.71 for low and high N supplied, respectively; data not shown) can indicate evidence for direct contribution of natural diazotrophic population to plant N nutrition.

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The population of native diazotrophic bacteria may vary in different plant species, even in different varieties (Hoefsloot et al. 2005). We earlier reported (Juraeva et al. 2006) that natural diazotrophic community abundance inhabiting cucumber plant root was positively correlated with N availability. It is likely that the effect of N availability on diazotrophs may be plant-species dependent, as we have observed in measurements of both inoculated-like-native-diazotrophic bacteria and nifH gene abundance, the lower availability of inorganic nitrogen was favorable to tomato-root-inhabiting-native-diazotrophic bacteria.

6.6 Conclusion

The observed significant effect of N availability on the colonization ability of inoculated bacterial strains underlines the importance to optimize the mineral N fertilization level for a successful application of diazotrophic biofertilizers. Factorial experiment studies of nifH gene abundance and plant N nutrition can serve to evaluate the contribution of diazotrophic bacteria to the N nutrition of plants. One of the remarkable conclusions of this study is that if three data, (i) considered bacteria, (ii) nifH gene abundance and (iii) total plant N nutrition, are significantly positive correlated to each other, a direct contribution of inoculated bacteria to the improved plant N nutrition due to its N2 - fixing ability can be assumed.


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