Humboldt Universität zu Berlin

Dissertation

Study the possible mechanisms of plant growth promotion by wheat diazotrophic bacteria grown in Uzbekistan soil

Zur Erlangung des akademischen Grades

doctor rerum agriculturarum
(Dr. rer. agr.)

eingereicht an der
Landwirtschaftlich-Gärtnerischen Fakultät
der Humboldt-Universität zu Berlin

von

M.Sc. Genet. Dilafruz   Juraeva

Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies

Dekan der Landwirtschaftlich-Gärtnerischen Fakultät
Prof. Dr. Dr. h.c. Otto Kaufmann

Gutachter:
1. Prof. Dr. Eckhard George
2. Dr. Silke Ruppel

Tag der mündlichen Prüfung: 25.11.2009

Abstract

Plant growth promoting bacteria (PGPB) are ubiquitous in both plant root and shoot, and are important contributors to the nitrogen-input of plants exerting their positive effects on plant growth directly or indirectly through different mechanisms. The present work focuses on a) the isolation of PGPB, which promotes the growth of different plant cultures and controls plant diseases caused by Fusarium species, b) the prospects of PGPB to solve plant nutritional problems, c) developing new molecular methods for the assessment of their diversity and activity.

A total of 780 bacterial strains were isolated from root, rhizosphere and phyllosphere of wheat grown in soil Syrdarya and were tested for their ability to promote the growth of other plants resulting in several universal PGPB strains.

Contributions of PGPB to plant nutrition were investigated with wheat, and several vegetable plants such as cucumber, tomato, paprika, and cauliflower on quartz-sand substrates. Bacterial colonization, plant dry weight, and N concentrations in plants were measured.

Bacterial inoculation effects on plant N nutrition in cucumber and tomato plants were determined by exposing bacterial and non bacterial plants to two, low and high NH⁴⁺NO^3- supply.

In the frame of this thesis, the methods for the description of the diversity of root colonizing PGPB have been developed and improved to provide links between introduced PGPB abundance and activities. The approach used was based on the sensitive real – time PCR detection/quantification of introduced PGBP and the nitrogenase reductase gene (nifH), which served as a marker gene for potential diazotrophs.

The amplified 16S-23S ISR sequences of studied bacteria were subjected to strain – specific primer design and a highly specific bacteria quantification protocol were developed. The bacteria quantification protocol was based on real – time PCR using strain specific primers in order to evaluate the colonization ability of studied bacteria, which were inoculated to plant roots.

The application previously used universal nifH primers to the real – time PCR improved the detection of less abundant diazotrophs in dry land plant root. The protocols were tested and optimized using pure cultures of diazotroph reference strains, and subsequently applied to the analysis of two vegetable plant roots. Real – time analyses of PCR products obtained from plant root DNA extracts revealed that the new nifH PCR protocol differentiated between the diazotroph populations in different plants.

The developed methodology was applied to study nifH abundance of Bacillus licheniformis and Xanthomonas sp. inoculated to cucumber and tomato growing in non – sterile quartz sand. Treatments with nitrogen limiting conditions resulted in more diazotrophic bacteria abundance, as well as, nifH gene pool while nitrogen excess suppressed diazotrophic bacteria abundance in both inoculated and non-inoculated plants. Furthermore, the nifH gene abundance was significantly correlated with measurements of N amount taken by the plant and inoculated bacteria density showing direct contribution of introduced bacteria to plant N nutrition. The results presented in this thesis have shown that monitoring of nifH amount in plant root is a suitable and promising approach to link inoculated diazotrophic bacteria abundance and its potential activity. The study of nifH gene abundance in plant offers the opportunity to identify key players in asymbiotic nitrogen fixation, to study short-term community responses in changing environments, or to analyze the effect of regulation in situ.

16S-23S ISR quantification – asymbiotic diazotrophic bacteria – cauliflower – cucumber – nifH gene quantification – nitrogen uptake – paprika – real-time PCR – tomato – wheat

Keywords: Keywords

Inhaltsangabe

Das Pflanzenwachstum fördernde Bakterien (PGPB) kommen ubiquitär sowohl an der Wurzel als auch am Spross der Pflanzen vor und sie können über direkte oder indirekte Mechanismen einen bedeutenden Beitrag zur Stickstoffernährung der Pflanzen leisten. Die vorliegende Arbeit umfasst a) die Isolierung von PGPB, welche das Wachstum verschiedener Pflanzenarten fördern und durch Fusarien verursachte Pflanzenkrankheiten bekämpfen, b) die Analyse der Möglichkeiten Probleme der Pflanzenernährung durch den Einsatz von PGPB zu lösen, c) die Entwicklung neuer molekularbiologischer Methoden zur Messung der Diversität und Aktivität der PGPB.

780 Bakterienstämme wurden aus der Wurzel, der Rhizosphäre und Phyllosphäre von Weizen, der auf Boden der Syrdarya wuchs, isoliert. Daraus wurden universelle PGPB Stämme ausgewählt, die das Wachstum verschiedenster Pflanzen förderten. Der Beitrag dieser PGPB zur Pflanzenernährung wurde an Weizen und verschiedenen Gemüsepflanzen, wie Gurke, Tomate, Paprika und Blumenkohl in Quarzsand Modellversuchen analysiert. Bakterienbesiedlung der Pflanzen, Pflanzen Trockenmasse und N-Konzentration in den Pflanzen wurden gemessen und bewertet.

Der Bakterieneinfluß auf die pflanzliche N-Ernährung von Gurke und Tomate wurde bei geringer und hoher NH₄NO₃ Versorgung an mit Bakterien inokulierten Pflanzen und nicht inokulierten Kontrollpflanzen analysiert.

Im Rahmen dieser Arbeit wurden Methoden zur Beschreibung der Diversität von rhizosphären PGPB entwickelt und verbessert um Verbindungen zwischen applizierten PGPB und deren Aktivitäten zu prüfen. Die sensitive quantitative real-time PCR Methode wurde zur Quantifizierung bzw. zum Nachweis der inokulierten PGPB und zum Nachweis des nitrogenase-reduktase Gens (nifH), des Markergens für potentiell diazotrophe Bakterien, genutzt.

Bakterienart spezifische Primer wurden aus dem Sequenzvergleich der 16S-23S ISR ausgewählter Bakterienstämme selektiert und Protokolle zur Quantifizierung dieser Bakterienarten erarbeitet. Die Protokolle basierten auf der real-time PCR Methode und dem Einsatz der selektierten artspezifischen Primer. Ziel der Untersuchungen war die Besiedlungsfähigkeit inokulierter Bakterien an Pflanzenwurzeln zu analysieren. Die Anwendung der früher selektierten universellen nifH Primer in der quantitativen real-time PCR verbesserte die Nachweisgrenze von diazotrophen Bakterien signifikant. Somit konnten diazotrophe Bakterien, die nur in geringer Zellzahl an Pflanzenwurzeln in trockenen Regionen vorkommen, entdeckt und quantifiziert werden. Die Protokolle wurden unter Einsatz von diazotrophen Referenzstämmen getestet und optimiert und nachfolgend zur Messung an zwei Gemüsearten angewendet. Die Ergebnisse der real-time PCR Messungen, die an DNA Extrakten aus Pflanzenwurzeln von Gurke und Tomate durchgeführt wurden, zeigten, dass das vorliegende nifH PCR Protokoll zur Differenzierung der Bakterienpopulationen diazotropher Bakterien zwischen verschiedenen Pflanzenarten geeignet ist.

Die neu entwickelten Methoden wurden zum Studium des nifH Vorkommens und der Prüfung der Besiedlungsfähigkeit von Bacillus licheniformis und Xanthomonas sp. an Tomaten Wurzeln eingesetzt, die in nicht sterilisiertem Quarz Sand wuchsen. Unter Stickstoff limitierten Bedingungen waren sowohl die Anzahl diazotropher Bakterien als auch der nifH Genpool erhöht, während bei hoher N-Versorgung der Pflanzen die Anzahl diazotropher Bakterien sowohl in inokulierten als auch nicht inokulierten Varianten reduziert waren. Außerdem bestand eine enge signifikante Korrelation zwischen nifH Gen Vorkommen und N-Aufnahme der Pflanzen und die Dichte der inokulierten Bakterien zeigte einen direkten Beitrag der applizierten Bakterien zur N-Ernährung der Pflanzen.

Die Ergebnisse dieser Arbeit zeigten, dass das Monitoring des nifH Gen Vorkommens in Pflanzenwurzeln eine vielversprechende Methode ist, um die potentielle Luftstickstoffbindungsaktivität inokulierter diazotropher Bakterien zu analysieren. Die nifH Gen Quantifizierung an Pflanzen eröffnet die Möglichkeit Schlüsselorganismen in der assoziativen biologischen Luftstickstoffbindung zu identifizieren und kurzfristige Reaktionen der Bakteriengesellschaften auf Umweltveränderungen und Regulationsmechanismen in situ zu analysieren.

16S-23S ISR Quantifizierung – assoziative diazotrophe Bakterien – Blumenkohl – Gurke – nifH Gen Quantifizierung – Stickstoffaufnahme – Paprika – real-time PCR – Tomate – Weizen

Eigene Schlagworte: Stichworte

Inhaltsverzeichnis

• 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
    • 1.1.2 Research focus and hypotheses of the thesis
    • 1.1.3 Outline of the thesis
    • 1.1.4 Current challenges
      • 1.1.4.1 Improving detection methods for nifH.
      • 1.1.4.2 Development of methods to examine N₂ fixation by certain bacteria in complex environments.
• 2 ISOLATION, PHENOTYPIC CHARACTERISATION AND SCREENING OF WHEAT INHABITING BACTERIA FOR THEIR PLANT GROWTH PROMOTING EFFECT
  • 2.1 Abstract
  • 2.2 Introduction
  • 2.3 Materials and methods
    • 2.3.1 Bacteria isolation from root, rhizosphere and phyllosphere of wheat
      • 2.3.1.1 Soil and Plant
      • 2.3.1.2 Collecting samples
      • 2.3.1.3 Bacteria isolation
      • 2.3.1.4 Morphological characterization and identification of bacteria
    • 2.3.2 Screening of bacterial isolates for their effect on wheat growth
      • 2.3.2.1 Bacteria suspension preparation
      • 2.3.2.2 Screening in Petri dishes
      • 2.3.2.3 Screening in pot experiments
    • 2.3.3 Antagonistic activity of bacteria isolated from wheat root, rhizosphere against pathogenic Fusarium isolates
      • 2.3.3.1 Bacterial and fungal isolates.
      • 2.3.3.2 Selection of bacteria for ability to inhibit in vitro growth of Fusarium culmorum.
      • 2.3.3.3 Specificity of bacterial antagonistic activity against Fusarium isolates.
    • 2.3.4 The influence of beneficial bacteria isolated from wheat rhizosphere on growth promotion of some vegetable plants
      • 2.3.4.1 Plants and bacterial strains
      • 2.3.4.2 Pot experiment setup
      • 2.3.4.3 Preparation of inoculation material
    • 2.3.5 Harvest and plant analysis
    • 2.3.6 Statistical analysis
  • 2.4 Results and Discussion
    • 2.4.1 Bacteria isolation from root, rhizosphere and phyllosphere of wheat
      • 2.4.1.1 Isolation and phenotypic characterization of diazotrophic bacteria
    • 2.4.2 Screening of bacterial isolates for their effect on wheat growth
      • 2.4.2.1 First screening of isolates for their effect on the plant growth.
      • 2.4.2.2 Antagonistic activity of bacteria isolated from wheat root, rhizosphere against pathogenic Fusarium isolates.
      • 2.4.2.3 Selection of bacteria for ability to inhibit in vitro growth of Fusarium culmorum.
      • 2.4.2.4 Specificity of bacterial antagonistic activity against isolates of Fusarium. 
    • 2.4.3 Screening of bacterial isolates for their plant growth promoting effect
    • 2.4.4 The influence of beneficial bacteria isolated from wheat rhizosphere on growth promotion of some vegetable plants
  • 2.5 Conclusion
• 3 EVALUATION OF 16S rRNA AND 16S-23S ISR SEQUENCE-BASED ANALYSES AS A PART OF A POLYPHASIC APPROACH TO IDENTIFY PLANT-INHABITING BACTERIA
  • 3.1 Abstract
  • 3.2 Introduction
  • 3.3 Materials and methods
    • 3.3.1 Bacteria isolation 
    • 3.3.2 Phenotypic characterisation of bacterial isolates
    • 3.3.3 Extraction of bacterial DNA
    • 3.3.4 16S rRNA gene amplification and sequencing
    • 3.3.5 16S-23S ISR amplification and sequencing
    • 3.3.6 Sequence data analysis
    • 3.3.7 Criteria for bacterial isolate identification
  • 3.4 Results
    • 3.4.1 Conventional bacterial identification
    • 3.4.2 16S rRNA sequence-based bacterial isolate identification 
    • 3.4.3 16S-23S ISR-based bacterial isolate identification
  • 3.5 Discussion
    • 3.5.1 16S rRNA sequence-based bacteria identification and conflicting results
    • 3.5.2 Comparison of 16S rRNA sequence-based identification results and conventional bacteria identification
    • 3.5.3 16S-23S ISR-based bacteria identification
  • 3.6 Conclusion
• 4  ENUMERATION OF TWO DIAZOTROPHIC BACTERIAL STRAINS IN PLANT SAMPLES USING 16S-23S ISR SPECIES SPECIFIC REGIONS AND REAL-TIME PCR 
  • 4.1 Abstract 
  • 4.2 Introduction 
  • 4.3 Material and methods
    • 4.3.1 Bacterial strains
    • 4.3.2 Design of target bacteria-specific primers 
    • 4.3.3 4.3.3 Greenhouse experiment and plant DNA extraction
    • 4.3.4 Quantitative real-time PCR assay and quantification 
      • 4.3.4.1 Method optimization
      • 4.3.4.2 Real-time PCR examination of primers
      • 4.3.4.3 Standard curve generation
      • 4.3.4.4 TEF-gene quantification
    • 4.3.5 Statistical analyses
  • 4.4 Results and Discussion
    • 4.4.1 16S-23S ISR sequences as a means of developing strain-specific primers
    • 4.4.2 Optimisation and performance of quantitative real- time PCR protocol
    • 4.4.3 Utility of the developed tool in ecological studies 
  • 4.5 Conclusion
• 5 DETECTION AND QUANTIFICATION OF THE nifH GENE IN SHOOT AND ROOT OF CUCUMBER PLANTS
  • 5.1 Abstract
  • 5.2 Introduction
  • 5.3 Materials and methods
    • 5.3.1 Greenhouse experiments
    • 5.3.2 Harvesting of plant samples and DNA extraction from plant samples
    • 5.3.3 Real-time PCR assays
    • 5.3.4 Preparation of the nifH gene standard
    • 5.3.5 Spiking of plant DNA samples
    • 5.3.6 TEF gene quantification
    • 5.3.7 Statistical analyses
  • 5.4 Results 
    • 5.4.1 Effect of mineral N supply on plant growth
    • 5.4.2 New method developed to quantify the nifH gene in plant tissue using quantitative real-time PCR
    • 5.4.3 Plant DNA spiking
    • 5.4.4 Specificity of the quantitative real-time PCR approach to quantify the nifH gene from plant samples
    • 5.4.5 nifH gene  quantification
  • 5.5 Discussion
    • 5.5.1 Bacterial DNA extraction from plant samples
    • 5.5.2 Productivity and specificity of the quantitative real-time PCR approach to quantify the nifH gene from plant samples
    • 5.5.3  nifH gene quantification
    • 5.5.4 The effect of N amount supplied on nifH-gene abundance in plant  root
    • 5.5.5 Correlation of nifH-gene abundance to plant N nutrition
• 6 QUANTITATIVE REAL-TIME PCR BASED EVALUATION OF THE DIRECT POTENTIAL OF DIAZOTROPHIC BACTERIA TO THE PLANT NITROGEN NUTRITION
  • 6.1 Abstract
  • 6.2 Introduction
  • 6.3 Materials and methods
    • 6.3.1 Experimental setup
    • 6.3.2 Bacterial strains
    • 6.3.3 Inoculation
    • 6.3.4 Plant sampling
    • 6.3.5 Target bacteria, nifH- and TEF gene quantification
    • 6.3.6 Statistical analyses
  • 6.4 Results 
    • 6.4.1 Plant growth responses
    • 6.4.2 Plant root colonization and persistence of inoculated bacteria
    • 6.4.3  nifH gene abundance in inoculated and non-inoculated plant root
    • 6.4.4 Interrelationship between diazotrophic bacterial inoculation, nifH gene abundance and plant N nutrition
  • 6.5 Discussion
    • 6.5.1 Plant growth responses
    • 6.5.2 Interrelation between the abundance of introduced diazotrophic bacteria and plant N nutrition
    • 6.5.3 Native diazotrophic bacteria abundance in tomato plants 
  • 6.6 Conclusion
• 7 GENERAL DISCUSSION
  • 7.1 Importance of a polyphasic approach in identifying bacterial isolates
  • 7.2 Potential of different databases and search programs in molecular identification of bacteria
  • 7.3 Effect of supplied nitrogen level on the abundance and activity of natural and introduced diazotrophic bacteria on plant root
  • 7.4 Relationship between nifH-gene abundance and plant N nutrition 
  • 7.5 PGP effect of inoculated bacteria
  • 7.6 Perspectives for further research
• 8 SUMMARY
• Abbreviations
• ACKNOWLEDGEMENTS
• References

Tabellen

• Tab. 1: Comparison of methods of estimating nitrogen fixation
• Tab. 2: Physical conditions of the pot experiment in growth chambers.
• Tab. 3: Natural colonisation of the studied isolates in wheat root, phylossphere and rhizosphere as counted on the master plate with a visual inspection.
• Tab. 4: The effect of bacterial strains on plant growth and development
• Tab. 5: Relative inhibition of growth of Fusarium species by selected strains.
• Tab. 6:  The effect of inoculation of wheat with diazotrophic PGPB on the length and weight of shoots and roots in nonsterile soil 4 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.
• Tab. 7: Cauliflower shoot, root length and dry matter 8 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.
• Tab. 8: Cucumber shoot, root length and dry matter 8 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.
• Tab. 9: Paprika shoot, root length and dry matter 8 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.
• Tab. 10: Tomato shoot, root length and dry matter 8 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.
• Tab. 11: Specificity and nucleotide sequences of PCR primers used in this study.
• Tab. 12a: Results obtained from phenotypic and molecular biological methods used to identify plant-inhabiting bacteria.
• Tab. 12b: Results obtained from phenotypic and molecular biological methods used to identify plant-inhabiting bacteria.
• Tab. 13: Specificity and nucleotide sequences of PCR primers used in this study.
• 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.
• 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.
• 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).
• 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).
• 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
• 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, +B^BL and +B^Xs – plants inoculated with B. licheniformis BL43 and Xanthomonas sp. Xs148, respectively.
• 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, +B^BL and +B^Xs – plants inoculated with B. licheniformis BL43 and Xanthomonas sp. Xs148, respectively.
• 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.

Bilder

• Fig 1: The effect of diazotrophic bacteria Xanthomonas sp. Xs148 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.
• Fig. 2: The effect of diazotrophic bacteria Bacillus licheniformis BL43 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.
• Fig. 3: The effect of diazotrophic bacteria Bacillus sp. WR2 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.
• Fig. 4: The effect of diazotrophic bacteria Bacillus sp. WR9 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.
• Fig. 5: The effect of diazotrophic bacteria Bacillus sp. WR22 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.
• Fig. 6: Gel electrophoreses analysis of amplified 16S rRNA from bacterial isolates BL43 and Xs148. Lane M: Marker SMO 0328; lane 1 – E.coli (positive control), lane 2 – isolate BL43, lane 3 – isolate Xs148.
• 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).
• 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 T_m was emperically determined by plotting the change in fluorescence with temperature (dRFU/dT) versus temperature (T). RFU, relative fluorescent units.
• 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 T_m was empirically determined by plotting the change in fluorescence with temperature (dRFU/dT) versus temperature (T). RFU, relative fluorescent units.
• 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.
• 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 (T_m) = 90.0°C; black line), plant shoot sample (T_m = 89.5°C; ■) and root sample (T_m = 91.0°C; ▲). The T_m was emperically determined by plotting the change in fluorescence with temperature (dRFU/dT) versus temperature (T). RFU, relative fluorescent units.
• 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.
• 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).
• 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.