ENUMERATION OF TWO DIAZOTROPHIC BACTERIAL STRAINS IN PLANT SAMPLES USING 16S-23S ISR SPECIES SPECIFIC REGIONS AND REAL-TIME PCR 

4.1 Abstract 

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To evaluate the effect of inoculated bacteria, its colonisation ability must be accurately measured. For two diazotrophic bacteria, which have shown PGP effect on different agricultural crops, strain specific primers were developed from the highly variable regions of 16S-23S ISR, amplifying a 109-bp fragment, for Bacillus licheniformis BL43 and yielding a 89-bp product for Xanthomonas sp. Xs148, respectively. The strain specificity of the primer sets was checked by comparison with available ISR sequences in databases and by PCR using DNA from target and reference strains as well as some plant-inhabiting bacterial isolates. The primer system used produced only the single expected fragment for only target bacteria. The detection limits of developed method to quantify B. licheniformis BL43 and Xanthomonas sp. Xs148 were 9.17E+03 and 1.11E+05 gene copies/µl, respectively. In this paper, we reported on the preliminary application of the designed specific primers to the study of the response of inoculated diazotrophic bacteria populations to the N availability.

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Key words

strain-specific primers – 16S-23S ISR – colonisation – Bacillus licheniformisXanthomonas – cucumber

4.2 Introduction 

The diazotrophic bacterial strains Bacillus licheniformis BL43 and Xanthomonas sp. Xs148 were previously shown to promote the growth of some cereals and vegetables (Juraeva et al. 2003, Juraeva and Ruppel 2005a). Plant growth promoting effect of inoculated bacteria take place if the inoculated strains are able to colonize in the rhizosphere, on or inside of host plant root. When redetection of introduced bacteria proves the colonization ability of potential inoculant strain (Tan et al. 2001), accurate quantification analysis of strain colonization after inoculation is of critical importance to verify the association of inoculated bacteria with plant growth promotion quantitatively and to study the response of these bacterial population to some environmental factors, particularly under competitive conditions in non-sterile substrates. Assessments of plant growth promoting bacteria in environmental samples have been mainly based on PCR performed with primers with different degrees of specificity (Bauernfeind et al. 1999; Coenye et al. 1999; Vandamme et al. 1997).  There is a high possibility for the failure of the species-specific primer based quantification, because (i) DNA sequence composition may vary within and between strains of the same species (Bosshard et al. 2002, Clayton et al. 1995), or (ii) on the contrary, the composition of primer targeted genes may be too conserved in a bacterial group (e.g. Bacillus subtilis group) (Nakamura et al. 1999), or (iii) the species-specific primers which have been designed based on the sequences deposited in databases may target the point mutated regions of the studied bacteria DNA. Therefore, for reliable and specific redetection and/or quantification of the bacteria after application to plant, strain-specific primer design is particularly important.

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The high variability of the 16S-23S ISR allowed the designed of genus-, species-, and strain-specific primers (Tyler et al. 1995, Moreira and Amils, 1996, Tilsala-Timisjarvi and Alatossava 1997, Chun et al. 1999, de Olivera et al. 1999, Tan et al. 2001). Our previous studies showed that 16S rRNA sequences of the investigated bacteria in this study showed that this gene in both bacteria was highly conserved (Juraeva and Ruppel, 2005b). Therefore, the development of specific primers designed from a consensus sequence obtained from 16S-23S ISR of used bacteria was aimed. 

In this study, a quantitative real – time PCR technique with newly designed specific primers allowing detection and quantification of Bacillus licheniformis BL43 and Xanthomonas sp. Xs148 in both pure cultures and environmental plant material were developed and the preliminary application of this method to quantify both diazotrophic bacteria after inoculation to plants was performed with cucumber root samples. Moreover, the utility of the developed method in determination of environmental factor effect, such as N application level on inoculated diazotrophic bacteria colonization ability when grown under greenhouse conditions were tested.

4.3 Material and methods

4.3.1 Bacterial strains

Diazotrophic bacteria originally isolated from maize rhizosphere (provided by Microorganism collection, Institute of Microbiology, Uzbekistan Academy of Sciences) and wheat root (Juraeva and Ruppel, 2005b) identified as Bacillus licheniformis and Xanthomonas spp., respectively, using polyphasic approach (see Chapter 2). Inoculation using these diazotrophic bacteria affected the early plant growth of some agricultural crops grown in loamy sand (Egamberdiyeva et al. 2002, Egamberdiyeva et al. 2003, Egamberdiyeva et al. 2004a, 2004b).

4.3.2 Design of target bacteria-specific primers 

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To design strain-specific primers, we used the 16S-23S ISR partial sequence data for Bacillus licheniformis BL43 and Xanthomonas sp. Xs148 as determined in our previous study (see Chapter 2). Among the 16S-23S ISR sequences deposited in EMBL-Bank, the entries, which showed the highest similarity to the target bacteria sequences in BLAST search, were selected. These entries with the target bacteria sequences were applied to determine the strain-specific region for primer design. The specific primers were designed using the software Beacon Designer 3.0 (PREMIER Biosoft International).

4.3.3 4.3.3 Greenhouse experiment and plant DNA extraction

The greenhouse experiment was performed as described in our previous study (Juraeva et al. 2006). The bacterial inoculation of plants was performed as following: pure culture of Bacillus licheniformis BL43 and Xanthomonas sp. Xs148 were grown in Standard I (Merck, Darmstadt, Germany) broths for 48h. The bacterial suspensions were centrifuged at 7000 rpm for 10 min. Growth medium was discarded and the bacterial pellet was resuspended in 0.05% NaCl buffer. Seedlings were divided into three treatment groups and were immersed in an appropriate suspension for 2 min prior to placement in pots. The control group seedlings were immersed in sterile 0.05% NaCl. The inoculation treatments were set-up in a randomized design with 12 replicates. Cell densities of bacterial suspensions used for the inoculation were counted by dilution plating and CFU counts and were 1010 in the inoculant material for both bacteria.

Plants harvested (three replicates for each nitrogen treatment) on Days 7 and 42 after planting and plant DNA extraction was performed as described in Juraeva et al. (2006).

4.3.4 Quantitative real-time PCR assay and quantification 

4.3.4.1 Method optimization

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Primer concentrations were optimised for each assay testing different range concentrations, from 50 to 500 nM. The optimised quantitative real-time PCR assay for both bacteria was 300 µM for each forward and reverse primer, 12.5 µl of QuantiTect SYBR® Green 2x mastermix (Qiagen, Hilden GmbH, Germany), 2.5 µl of template, and H2O to a final volume 25 µl. Samples were tested in triplicate. This assay started with 15 min at 95°C followed by 50 cycles of amplification, 30s at 94°C, 30s at 59°C and 1 min 15s at 72°C. A final elongation step consisted of 72°C for 5 min was followed by DNA melting protocol in which 85 cycles of half a degree increase in temperature every 30s beginning at 44°C.

4.3.4.2 Real-time PCR examination of primers

Amplification of target gene from positive and negative control strains was used to confirm primer sensitivity and specificity. The dilutions of a pure culture of appropriate bacteria (B. licheniformis BL43 or Xanthomonas sp. Xs148) were used to determine the sensitivity of the real - time PCR. The assay specificity was tested with approximately 50 ng of genomic DNA extracted from different bacteria species (data not shown) which have been shown to have the closest sequence similarity in CLUSTALW analyses for 16S-23S ISR partly sequence of B. licheniformis BL43 and of Xanthomonas sp. Xs148, respectively. DNA extracted from inoculated cucumber root samples were also tested further to prove the assay with plant samples. PCR products were examined by 2.5 % agarose gel electrophoresis using Marker 5, 100bp: pBR322 DNA/BsuRI (HaeIII)(MBI Fermentas GmbH, St.Leon-Rot, Germany) as size standard.

4.3.4.3 Standard curve generation

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 In order to obtain standard samples to use for the real-time PCR standard curve, DNA from target bacteria (Bacillus licheniformis BL43 or Xanthomonas sp. Xs148) was extracted using UltraClean™ Microbial DNA Isolation Kit (MO BIO Laboratories Inc., Hamburg, Germany) and target gene was amplified with the newly developed specific primer pair (Tab. 13).

PCR cycling parameters and reaction mixtures were the same as described above except that melting protocol was not performed and that Probe master mix (Qiagen, Hilden GmbH, Germany) was used instead of QuantiTect SYBR® Green 2x mastermix (Qiagen, Hilden GmbH, Germany) The PCR product was cleaned using MiniElute™ PCR Purification Kit (Qiagen, Hilden GmbH, Germany) and the DNA concentration photometrically measured at the absorbance of 260 nm. Copy numbers of the standard were calculated using the known DNA concentration and the template length.

The real - time PCR standard curve was generated from the those standards and was adjusted within a range of nine 10-fold dilutions from 9.17E+09 to 9.17E+00 (for B. licheniformis quanti-PCR, Fig. 7a) and from 1.11E+09 to 1.11E+00 (for Xanthomonas sp. quanti – PCR, Fig. 7b) copies per µl DNA and by this way the number of DNA copies could be calculated for each sample.

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Tab. 13: Specificity and nucleotide sequences of PCR primers used in this study.

4.3.4.4 TEF-gene quantification

In order to avoid of the misquantification due to the presumable different DNA extraction efficiencies from plant samples, as a housekeeping gene, TEF gene quantification from plant DNA was performed using the TEFf and TEFr primers (Tab. 13) as described in Juraeva et al. (2006). PCR reaction mixture and PCR program were the same as used in real – time PCR examination of specific primers. All quantified bacteria gene copy numbers were calculated as relative values to the housekeeping TEF gene copy numbers (relative bacteria gene copy number = (absolute 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. Since, the calculation of number of bacteria cells is not practically possible from that calculated 16S-23S ISR gene copies, because of not known rrn operons for B. licheniformis bacteria abundance was defined in gene copy numbers level.

In this study, all PCRs were performed using the iCycler (Bio-Rad, Inc. München, Germany). The quality of the SYBR Green® I quantification method was further verified for each measurement to avoid the possibility of false positive signals induced by primer dimers or other non-specific PCR products. First, a melting profile was recorded after each run, which resulted in one melting peak of the first deviation with the specific melting temperature of the PCR product. Second, PCR products were run on an agarose gel.

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Standard Curve Graph for SYBR-490

Fig. 7a: Standard curve graph (cycles = standard samples) for Bacillus licheniformis BL43 16S-23S ISR copy number calculation of unknown samples from control and inoculated (with B. licheniformis BL43) cucumber plant DNA (squares).

Standard Curve Graph for SYBR-490

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Fig. 7b: Standard curve graph (cycles = standard samples) for Xanthomonas sp. Xs148 16S-23S ISR copy number calculation of unknown samples from control and inoculated (with Xanthomonas sp. Xs148) cucumber plant DNA (squares). The Tm was emperically determined by plotting the change in fluorescence with temperature (dRFU/dT) versus temperature (T). RFU, relative fluorescent units.

4.3.5 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).

4.4 Results and Discussion

In previous studies used bacteria in this study influenced positively on the plant growth promotion of wheat, cotton and maize, tomato. In this work, their growth promoting activities were tested with cucumber and significantly increasing of shoot and root growth was observed in plants inoculated with both bacteria (data not shown). The specific primers for both bacteria B. licheniformis BL43 and Xanthomonas sp. Xs148 were designed and plant samples from this pot experiments were used to test the specific primers to quantify the copy numbers of inoculated bacteria after inoculation to evaluate colonisation ability of the used bacteria.

4.4.1 16S-23S ISR sequences as a means of developing strain-specific primers

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Three primer pair sets for B. licheniformis BL43 and two primer pair sets for Xanthomonas sp. Xs148 were designed based on 16S-23S ISR sequence data (Juraeva and Ruppel 2005b, detailed in Chapter 3). Using pure culture DNA extracted from several bacteria species non-related to the bacteria in query, which produced hits in the BLAST assay were included in the real-time PCR test of primer specificity as the templates showed that the specificities and sensitivities of the primer sets tested varied slightly. Primers BL43aF1 and BL43aR1 appeared to be the most specific while primers BL43aF2 -BL43aR2 and BL43aF3 -BL43aR3 appeared to be the most sensitive (data not shown). Since specificity is more important, the BL43aF1-BL43aR1 primer pair was used for further tests (Tab. 13). Among Xanthomonas sp. Xs148 specific primers, Xs148aF1-Xs148aR1 was selected for its best specificity (Tab. 13) (data not shown).

In addition, the specificity of the selected forward primers were analysed in BLAST search using 16S-23S ISR sequences deposited in the EMBL-Bank database. BLAST searches with B. licheniformis BL43 targeted forward primer BL43aF1 sequence showed only 10 sequence similarities in GenBank database that 2 of obtained 10 to the members of the genus Acinetobacter genomosp. and the rest 8 belonged to non-microbial genera such as Mouse DNA sequences (4), Homo sapiens chromosome (2), and Mus. musculus chromosome (2). The same analysis with Xanthomonas sp. Xs148 targeted forward primer also showed only 10 similar sequences and target organisms were Xanthomonas maltophilia (1), Pseudomonas sp. (1), Stenotrophomonas (2), as-yet-unidentified bacteria (1), Thermococcus kodakarensius (1), Methanosarcina marzei (1), and Homo sapiens (3).

For accurately quantification of the bacteria, the approach based on direct quantification is important. Tan et al. (2001) reported that developed 16S-23S ISR sequence based strain specific primers did not allow detecting target bacteria directly resulting in misamplification in un-nested PCR. In this study, using pure culture DNA and inoculated plant DNA, as a complex DNA mixture, in direct real – time PCR quantification analysis confirmed the excellent specificity and accuracy of approach with melting curve (Fig. 8) analysis followed by agarose gel electrophoresis showing primer pairs gave a single specific product at the expected length (108 bp for B. licheniformis BL43 and 89 bp for Xanthomonas sp. Xs148), but not with DNA samples of non – target bacteria species and produced no PCR product with a blank control (H2O) (data not shown).

4.4.2 Optimisation and performance of quantitative real- time PCR protocol

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The optimal primer concentrations and annealing temperatures to obtain a sensitive, repetitive and specific test using specific primers was 300 nM and 57°C of each primer set. Under these conditions, the standard curve had a slope in the range -3.75 to -3.00 and a correlation coefficient greater than 0.97 (Fig. 7a and Fig. 7b) and could therefore be accepted for quantification (Boeckman et al. 2001). The assessment of detection limit of the assay using the same dilution series of external standard in quantitative real time PCR with standard curve analyses showed that up to 9.17E+03 and 1.11E+05 gene copies/µl of B. licheniformis BL43 and Xanthomonas sp. Xs148, respectively, could be detected. In order to test for competitive or inhibitory effects of plant DNA on PCR amplification performed experiments using original DNA of some plant samples, the inhibition of PCR amplification with original plant DNA was observed and the amplification with 1:10 diluted and 1:100 diluted plants DNA showed that PCR with 1:10 dilutions was not inhibited. Therefore, 1:10 diluted plants DNA was used for quantification analysis.

Melt Curve Graph for SYBR-490

Fig. 8: Melting profile analyses to confirm only single PCR product amplification from tomato plant DNA during the PCR: bacteria (Bacillus licheniformis BL43 as an example) specific gene PCR product amplified from the standard sample (line with highest curve) and DNA extracted from non-inoculated and inoculated tomato plant. Melting temperatures for all PCR products are the same (81.0°C). The Tm was empirically determined by plotting the change in fluorescence with temperature (dRFU/dT) versus temperature (T). RFU, relative fluorescent units.

4.4.3 Utility of the developed tool in ecological studies 

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Real-time PCR based bacteria specific quantification enabled us to re-detect and accurately quantify introduced bacteria abundance in plant root 7 and 42 days after inoculation. The quantification of significantly higher copy numbers of target bacteria from plant root sampled 7 days after inoculation showed that both B. licheniformis BL43 and Xanthomonas sp. Xs148 were able to colonise in cucumber root (Fig. 9).

Fig. 9: The effect of N availability on colonisation of the introduced bacteria like population in both inoculated and non – inoculated plant root samples 2 days (Day 7) and 37 days (Day 42) after inoculation. Bacteria 16S-23S ISR copy numbers are calculated relative to housekeeping TEF gene copy numbers.

Evaluation of the factor effects, which may alter the inoculated bacteria abundance (or colonisation ability) is critical to achieve successful inoculation experiements and to assess the effect of inoculated bacteria on plant growth parameters. It is well recognized that N availability is mostly negatively correlated with diazotrophic bacteria abundance (Cejudo and Paneque 1986, Limmer and Drake 1998; Tan et al. 2003). Quantification results showed that N availability was negatively correlated to Xanthomonas sp. Xs148 abundance showing relatively less bacteria abundance in high N supplied plants (Fig. 3), while B. licheniformis BL43 abundance in high N supplied plants were significantly higher than low N supplied plants. Unexpectedly, B. licheniformis BL43 abundance in high N supplied plants were significantly higher even in comparison to 2 days after inoculation suggesting that the response of diazotrophs to N availability is different (Juraeva et al. 2006) and N stimulated B. licheniformis BL43 abundance in cucumber plants root. While this technique can provide accurate measurements of gene copy numbers per unit of plant DNA, to extrapolate those values to cell density, the knowledge in operons contained in considered species is required. At the present time, rRNA database of microorganisms (Klappenbach et al. 2000) has very limited information about rrn operons. Of Xanthomonas genus, it has been reported for only Xanthomonas that rRNA consists 6 operons and the number of operons contained in B. licheniformis species are unknown. However, this approach is still powerful tool in quantification analysis.

4.5 Conclusion

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One of the remarkable conclusion of this study is that if re-detection or/and quantification of inoculated bacteria has been aimed, the specific primer designing in either genus or species level based on sequence alighments available in EMBL-Bank database could be unsuccessful due to one or another level of misidentification or highly different sequence composition in the species in one genus.

Our study shows that rRNA ISR of bacteria has significantly high variability to design specific primers in comparison to 16S rRNA gene sequences.

In addition, amplification and sequencing of the considered bacteria can allow finding DNA regions which are unique to the considered bacteria which make the PCR enable for more reliable quantification of the inoculated bacteria in strain level.

Chapter 5. Detection and quantification of the nifH gene in shoot and root of cucumber plants


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