Real-Time PCR Detection of Pseudomonas Cichorii, the Causal Agent of Midrib Rot in Lettuce

Faculty of Sciences Department of Biochemistry, Physiology and Microbiology Laboratory of Microbiology-Ghent

Academic year 2006-2007

Thesis submitted to obtain the degree of Master of Science in ‘Applied Microbial Systematics’

Real-Time PCR detection of Pseudomonas cichorii, the causal agent of midrib rot in lettuce

Ines Verbaendert

Promotor: Dr J. Heyrman Co-promotor: Prof. Dr. P. De Vos

DANKWOORD

Zonder de steun en hulp van een aantal mensen zou ik deze scriptie niet kunnen gerealiseerd hebben. Daarom een kleine attentie voor hen op papier, want hun advies en goede raad was zéér welkom!

Dr. Jeroen Heyrman en Caroline, merci voor alle tijd die jullie hebben gestoken in het aanleren van alle technieken die ik nodig had om deze scriptie tot een goed einde te brengen en voor alle advies die ik van jullie gekregen heb! Jeroen , bedankt ook voor de uurtjes denkwerk en lees- en verbeterwerk die ik u heb aangedaan!

Prof. P. De Vos wil ik heel hartelijk bedanken voor de kans die ik gekregen heb om een tijdje deel uit te maken van het Laboratorium voor Microbiologie.

Kim, Emly, Caroline, An, Jeroen A., Liesbeth, Karen: de sfeer was schitterend! Ik voelde mij na een paar dagen echt op mijn gemak tussen jullie en ik wil jullie bedanken voor alle aanmoedigingen, hulp en het beantwoorden van de meest onmogelijke vragen.

En als laatste wil ik mijn ouders bedanken. Na 6 jaar studeren, zijn ze nog steeds geïnteresseerd in de dingen waar ik mee bezig ben en ik vind het magnifiek dat ze mij nog een jaartje extra wilden ondersteunen om daarna ‘de grote mensenwereld’ in te stappen! Dankuwel!

Ines, mei 2007

Content Dankwoord Index

1. ABSTRACT 8

2. INTRODUCTION 9

2.1. INTRODUCTION TO THE PROJECT 9 2.2. OBJECTIVE OF THE THESIS 10

3. OVERVIEW OF LITERATURE 11

3.1. PSEUDOMONAS CICHORII AS A PLANT PATHOGEN 11 3.1.1. THE HOST RANGE OF P. CICHORII 11 3.1.2. THE INFECTION MODE OF P. CICHORII 11 3.1.3. THE SOURCE OF P. CICHORII 14 3.2. (QUANTITATIVE) REAL‐TIME PCR 15 3.2.1. INTRODUCTION 15 3.2.2. PCR AMPLIFICATION 15 3.2.3. REAL‐TIME MONITORING OF PCR 16 3.2.3.1. Quantification and characterisation 16 3.2.3.2. Detection formats: fluorescence reporters 17 3.2.3.3. Instrumentation of real‐time PCR 19 3.2.4. OPTIMISATION OF REAL‐TIME PCR 20 3.2.5. APPLICATIONS OF REAL‐TIME PCR 20

4. MATERIALS AND METHODS 22

4.1. STRAINS 22 4.1.1. ORIGIN OF THE STRAINS 22 4.1.2. LONG‐TERM PRESERVATION OF THE STRAINS 22 4.2. DNA EXTRACTION 23 4.2.1. QUALITY CONTROL AND STORAGE OF DNA 24 4.2.1.1. Assessment of fragmentation 25 4.2.1.2. Concentration and purity assessment 26 4.3. GRADIENT PCR 28 4.3.1. PCR REACTION 28 4.3.2. GEL ELECTROPHORESIS 29 4.4. PCR 29 4.5. REAL‐TIME PCR ASSAYS 30 4.5.1. INTRODUCTION 30 4.5.2. REAL TIME PCR DEVELOPMENT 32 4

4.5.2.1. Optimisation of the PCR conditions 33 4.5.2.2. Further optimisation 35 4.5.2.3. Exclusivity 36 4.5.2.4. Inclusivity 37 4.5.2.5. Analytical sensitivity (work range) 37 4.5.2.6. Analytical specificity 38 4.5.2.7. TaqMan probe 39

5. RESULTS AND DISCUSSION 40

5.1. CONVENTIONAL PCR 40 5.2. REAL‐TIME PCR 42 5.2.1. SYBR GREEN I 42

5.2.1.1. Optimisation MgCl2 concentration 42 5.2.1.2. Optimisation temperature‐time profile 45 5.2.1.3. Optimisation of the primer concentration 50 5.2.1.4. Further optimisation 51 5.2.1.5. Exclusivity 53 5.2.1.6. Inclusivity 56 5.2.1.7. Analytical sensitivity (work range) 57 5.2.1.8. Analytical specificity 64 5.2.2. ROCHE‐ AND SIGMA‐ KIT FOR REAL‐TIME PCR 66 5.2.3. DEVELOPMENT AND PRELIMINARY TESTING OF A TAQMAN PROBE 67 5.2.3.1. Reverse probe 67 5.2.3.2. Forward probe 68

6. CONCLUDING REMARKS 70

7. REFERENCES 72

List with used abbreviations Addendum

List of figures FIGURE 1: Midrib rot (left) of green house grown lettuce(right) FIGURE 2: Type III secretion structure of P. syringae FIGURE 3: HrcR, hrcS and hrcT of P. syringae pv. syringae FIGURE 4: The PCR temperature cycle FIGURE 5: Quantification of Microcystis aeruginosa PCC 7820 using real‐time PCR FIGURE 6: The chemical structure of SYBR Green I FIGURE 7: The TaqMan probe fluorescent chemistry FIGURE 8: Conventional PCR (segment of photographs)

FIGURE 9: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F1‐R1 and P. cichorii strain LMG 8401

FIGURE 10: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F1‐R1 and P. cichorii strain LMG 2162T

FIGURE 11: Melting curves of the optimisation of the MgCl2 concentration for primer couple F1‐R1 and P. cichorii strain LMG 2162T

FIGURE 12: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F1‐R1 and P. syringae strain LMG 2352T

FIGURE 13: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F2‐R1 and P. cichorii strain LMG 8401

FIGURE 14: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F2‐R1 and P. cichorii strain LMG 2162T

FIGURE 15: Amplification curves of the optimisation of the annealing temperature for primer couple F2‐R1 (0.5µM) and three P. cichorii strains

FIGURE 16: Amplification curves of the optimisation of the annealing temperature for primer couple F2‐R1 (0.5µM) and two non‐P. cichorii strains

FIGURE 17: Melting curves of the optimisation of the annealing temperature for primer couple F2‐R1 (0.5µM) and two non‐P. cichorii strains FIGURE 18: Melting curves of the optimisation of the annealing temperature: 60°C FIGURE 19: Melting curves of the optimisation of the annealing temperature: 62°C FIGURE 20: Melting curves of the optimisation of the annealing temperature: 64°C FIGURE 21: Amplification curves of the optimisation of the annealing time for P. cichorii LMG 2162T and P. cichorii R‐25254

FIGURE 22: Comparison of primer couples F1‐R1 and F2‐R1 with the initial optimised PCR conditions

FIGURE 23: Further optimisation of the primer concentration with primer couple F2‐R1 and P. cichorii strains LMG 2162T, R‐25254 and LMG 8401 FIGURE 24: Further optimisation of the annealing temperature by raising the temperature again up to 64°C for a range of Pseudomonas strains FIGURE 25: The exclusivity assay (amplification curves)

FIGURE 26: The exclusivity assay (melting curves) Pcichrc 2007‐03‐22A FIGURE 27: The exclusivity assay (melting curves) Pcichrc 2007‐03‐22B FIGURE 28: The exclusivity assay (melting curves) Pcichrc 2007‐03‐22C FIGURE 29: The exclusivity assay (melting curves) Pcichrc 2007‐03‐26 FIGURE 30: The inclusivity assay (amplification curves) FIGURE 31: The inclusivity assay (melting curves) FIGURE 32: Amplification curves of a dilution series of P. cichorii LMG 2162T FIGURE 33: Melting curves of a dilution series of P. cichorii LMG 2162T FIGURE 34: Amplification curves of a dilution series of P. cichorii R‐25254 FIGURE 35: Melting curves of a dilution series of P. cichorii R‐25254 FIGURE 36: Amplification curves of a dilution series of P. cichorii R‐31877 FIGURE 37: Melting curves of a dilution series of P. cichorii R‐31877 FIGURE 38: Analytical sensitivity of the SYBR Green I assay determined with serial dilutions of P. cichorii LMG T 2162 genomic DNA using primer couple F2‐R1 FIGURE 39: Analytical sensitivity of the SYBR Green I assay determined with serial dilutions of P. cichorii R‐

25254 genomic DNA using primer couple F2‐R1 FIGURE 40: Analytical sensitivity of the SYBR Green I assay determined with serial dilutions of P. cichorii R‐

31877 genomic DNA using primer couple F2‐R1 FIGURE 41: Analytical specificity for strain P. mediterranea LMG 23075T at a dilution of 108 and 107 (amplification curves) FIGURE 42: Analytical specificity for strain P. mediterranea LMG 23075T at a dilution of 102 (amplification curves) FIGURE 43: Analytical specificity for strain P. mediterranea LMG 23075T at a dilution of 102 and 108

FIGURE 44: Amplification curves of F1‐Rprobe0 for five P. cichorii strains (Pcichrc 2007‐03‐09)

FIGURE 45: Amplification curves for F1‐Rprobe for P. cichorii (Pcichrc 2007‐04‐17)

FIGURE 46: Amplification curves for F1‐Rprobe with additional P. cichorii strains (Pcichrc 2007‐04‐19D)

FIGURE 47: Amplification curves for Fprobe‐R1 for P. cichorii strains (Pcichrc 2007‐04‐17)

FIGURE 48: Amplification curves for Fprobe‐R1 at 65°C for 8 P. cichorii strains

1. ABSTRACT The incidence of bacterial soft rot in salad vegetables caused by Pseudomonas has increased from a rather sporadic to a chronic problem in Flanders and it currently represents a serious threat for the production sector. Early detection of the sources of introduction can guide the implementation of appropriate measures to prevent or counter infections of salad vegetables with P. cichorii. The goal of this study was to participate in the development and mainly in the optimisation of a specific real- time PCR-detection system targeting P. cichorii hrp/hrc genes in order to allow for fast detection of infection at an early stage of the crop. Construction of primers for rapid detection of P. cichorii was already performed. The primers although still had to be extensively tested for: (1) exclusivity (2) inclusivity (3) analytical specificity (4) analytical sensitivity and (5) reproducibility. Also, optimisation of the PCR conditions, e.g. temperature-time profile, concentration of MgCl2, primers and dNTPs, had to be performed. Conclusions: The developed LightCycler PCR assay proved to be quite sensitive and specific for the detection of the hrcRST genes of P. cichorii. An upper limit of detection of 152 genome equivalents per PCR reaction was determined for the detection of P. cichorii organisms in lettuce. A total assay time of about an hour was achieved.

2. INTRODUCTION 2.1. INTRODUCTION TO THE PROJECT This thesis is part of the IWT project: ‘Characterisation, ecology and epidemiology of Pseudomonads on salad vegetables’ (1). At the start of the project a certain rot in salad vegetables in the field and in green houses was attributed to Pseudomonas, but the precise cause was unknown. The focus of this study is midrib rot of green house grown lettuce (Fig. 1). Symptom is a brownish rot along the midrib of one or more of the inner leaves. Two types of midrib rot can be distinguished and often are found in combination. The first type, soft rot, affects the outermost leaves of the head and looks like a slimy light brown to reddish brown discolouration of the midrib with slimy decay of the leaf blades. The second type, typical midrib rot, affects the inside of the head and looks like a dark brown to greenish black discolouration on the midrib of one or more inner leaves.(2, 3, 4)

a) Figure 1: Midrib rot (left) of green house grown lettuce(right) The high humidity within the inner leaves facilitates the proliferation of the bacteria, which results in rapid disease development. Since the symptoms of typical midrib rot appear typically inside the lettuce, the disease can easily remain unnoticed until harvest. The damage can be extensive and usually results in complete or partial loss of the crop. (2, 3) As the incidence of bacterial soft rot in salad vegetables caused by Pseudomonas has increased from a rather sporadic to a chronic problem in Flanders, it currently represents a serious threat for the production sector.(2,3) Thus, early detection of the sources of introduction can guide the implementation of appropriate measures to prevent or counter such infections. In a first part of the IWT project, isolation and characterisation of bacteria of diseased lettuce originating from different growers throughout Flanders was performed. Isolation of 64 strains and their characterisation via polyphasic taxonomy, showed a clear correlation between midrib rot and the presence of large numbers of fluorescent pseudomonads, such as

Pseudomonas cichorii, P. viridiflava, P. marginalis and P. fluorescens. Also for typical midrib rot, the presence of P. cichorii as a primary pathogen was shown. Additionally, field- experiments were carried out to establish which concentrations could cause infection and what could be disease-promoting or -reducing factors. 2.2. OBJECTIVE OF THE THESIS The goal of this thesis was to participate in the development and mainly in the optimisation of a specific real time PCR-detection system targeting P. cichorii hrp/hrc genes in order to allow for fast detection of infection at an early stage of the crop. Construction of primers for rapid detection of P. cichorii was already performed in the first part of the IWT project. The primers although had to still be extensively tested for: (1) exclusivity (2) inclusivity (3) analytical specificity (4) analytical sensitivity and (5) reproducibility. Also, optimisation of the PCR conditions, e.g. temperature-time profile, concentration of MgCl2, primers and dNTPs, had to be performed.

3. OVERVIEW OF LITERATURE 3.1. PSEUDOMONAS CICHORII AS A PLANT PATHOGEN P. cichorii is a Gram-negative bacterium that belongs to the Gammaproteobacteria. The hierarchy of the different taxa can be found in Table 1. b) Table 1: Hierarchical system of taxa for P. cichorii (5)

Domain Bacteria Phylum Proteobacteria Class Gammaproteobacteria Order Pseudomonadales Family Pseudomonadaceae Genus Pseudomonas (Pseudomonas syringae group) Species Pseudomonas cichorii

3.1.1. THE HOST RANGE OF P. CICHORII

The type strain of P. cichorii in the LMG collection is LMG 2162T, it was originally isolated from endives (Chicorium endivia) (9). Other members of this species though, have a broad host range. An overview of some hosts can be found in Table 2. c) Table 2: Overview of the host range of P. cichorii

Host Scientific name Typical symptoms Reference/ strain Lettuce Lactuca sativa Midrib rot (2,4) Turcmeric Curcuma longa Leaf blight (6) Eggplant Solanum melongena Necrotic leaf spot (7) Coffee Coffea arabica NMa (8) Endives Cichorium endivia Soft rot (9) Celery Apium graveolens Brown stem or bacterial blight (10) Geranium Pelargonium hortorum Leaf spot (11) Cabbage Brassica oleracea var. capitata Lesions (10, 11) Cauliflower B. oleracea Lesions (10,11) Broccoli B. oleracea var. italica Lesions (10) Common chicory Cichorium intybus L. Lesions (10) Chrysanthemum Chrysanthemum morifolium Leaf spot (10,11) Tobacco Nicotiana tabacum - NCPPBb 1039 a: NM : not mentioned in the article b: NCPPB: National Collection of Plant Pathogenic Bacteria, Central Science Laboratory, York, UK

3.1.2. THE INFECTION MODE OF P. CICHORII

The mode of infection of certain plant pathogenic pseudomonads consists of producing pectinolytic enzymes which leads to maceration of the plant tissue. This strategy implies little specificity, hence their broad host range and wide distribution. They are opportunistic bacteria that can cause disease on weakened plants or when the conditions for infection are optimal. (1) In the IWT project however, lab tests showed that for infection, P. cichorii is not dependent on pectinolytic enzymes and that it does not depend on other pseudomonads that dó have them. Probably P. cichorii has to reach a certain number to use his infection system.

Indications for quorum sensing were already found in inoculum concentration tests in chicory. But, that does not imply that P. cichorii has to be present in high initial concentrations to

(1) cause damage. A second possible mode of infection, is infection through a Type III protein secretion system. This is a complex system in the envelope of many Gram-negative bacteria (12, 13), such as P. viridiflava (14) and P. syringae.(15) It is involved in the pathogenicity of many plant pathogenic bacteria.(12) The bacteria depend on the remarkable ability of the Type III secretion system to inject/deliver virulence-associated effector proteins directly into cytoplasm of the eukaryote host cells.(13) Those effector proteins are essential for causing disease in susceptible hosts and defense responses in resistant hosts.(14) Plant pathogens do not enter living host cells, they interact with the host cytoplasm from outside of a ± 100-200 nm thick plant cell wall. The ability to deliver effector proteins via the Type III secretion system is likely to be unique to

(15) plant pathogens. The Type III secretion system (also called the Hrp system) and its effectors are encoded by

1 (14, 15) the hrp/hrc genes. These genes are clustered in large pathogenicity islands (PAI). (14, 15) PAIs often have G+C contents different from the rest of the chromosome and are frequently flanked by mobile DNA elements, suggesting acquisition by horizontal (or lateral) transfer. This is believed to be important because it allows the pathogen to immediately use already-evolved pathogenicity strategies and, thereby, accelerate the pace of pathogen evolution. The idea that PAIs have been transferred horizontally among phylogenetic unrelated bacteria is also supported by the observation that there does not seem to be any similarity between the Type III secretion system-based phylogenetic tree and the rRNA-based phylogenetic tree. The latter of which indicates the evolutionary relationship among bacterial species. (13, 14) The hrp/hrc gene cluster resides at the centre of the Hrp PAI. (15) Type III secretion involves the delivery of effector proteins from the bacterial cytoplasm to the host cell interior, passing both bacterial and host cell walls. So, ‘secretion’ across the bacterial cell wall and ‘translocation’ across the host membrane/cell wall is needed. (13) The structure of the Type III secretion system of mammalian pathogenic bacteria resembles a flagellar basal body, consisting of two outer rings that interact with the outer membrane, two inner rings that interact with the cytoplasmic membrane and an extracellular needle-like extension. However in plant pathogenic bacteria, e.g. P. syringae, the system assembles

1 Hrp stands for hypersensitive response and pathogenicity. Hrc stands for hypersensitive response and conserved. 12

surface structures morphologically resembling bacterial pili, the Hrp-pili (Fig. 2). Type III secretion occurs where the Hrp pilus is assembled. Moreover, the Hrp pilus is likely the functional equivalent of the needle. (13) The needle-like extension and the Hrp pilus however differ drastically in length, but this might reflect the adaptation of each system to overcome the very different barriers they encounter during infection.

d) Figure 2: Type III secretion structure of P. syringae. It is believed to be composed of a needle‐like complex (in pink), which is anchored in the bacterial cell wall; and a translocation complex (translocon; in light blue) in the host cell wall. P. syringae presumably uses the Hrp pilus (made up of the HrpA protein) and possible harpins to penetrate the host cell wall (13) The Type III translocation complex in mammalian pathogenic bacteria has been well studied. In plant pathogenic bacteria though, the Type III translocation complex has been less studied. Evidence points to a mechanism probably similar to that in mammalian pathogenic bacteria, but plant pathogenic bacteria encounter a unique cell wall barrier during type III secretion. Therefore, these pathogens may also produce unique translocators that probably assist the Hrp pilus in overcoming the cell wall barrier.(13) Plant pathogenic bacteria secrete a unique family of type III effectors that are not found in mammalian pathogens: the harpin family of effectors. (13, 15) All plant pathogens secrete proteins of the harpin family, this suggests that these proteins could be involved in assisting the penetration of the plant pathogenic Hrp pilus through the plant cell wall, (13) but there is no genetic evidence yet. The effector proteins probably cross the host cell wall by connecting the pilus to protein complexes in the cell wall to provide a continuous passage for effector proteins. (13) The effectors of the Type III secretion system can regulate the secretion of other Type III secretion system effectors that even can directly alter host structures and functions, resulting in the protein complexes in the cell wall of the host. (16) The assembly and function of the Type III secretion system involves the production of a large number of gene products, which could be very energy-consuming for bacteria. So, bacteria often do not fully express it until they enter host tissues. Once bacteria are inside host tissues,

expression of these genes is tightly regulated. Very important is that the conditions activating the expression of the Type III secretion system-associated genes reflect the host environment that the bacterium enters, e.g. in plant pathogenic bacteria expression of the genes occurs at lower ambient temperatures (20°C) than in mammalian pathogenic bacteria (37°C) (13). In addition, the disease development is dependent on environmental factors. A high relative humidity seemed to promote disease damage. This is quite logical, as bacteria mostly prefer a humid environment. In addition, it has been reported that a higher amount of nitrogen in the soil seems to promote the disease. (1) In summary, actually little is known on the mode of infection of P. cichorii. The use of pectinolytic enzymes by P. cichorii could be excluded in the project, but quorum sensing and infection through a Type III secretion system might still be two possibilities. There is some evidence suggesting the presence of Type III secretion system-like genes in this fluorescent

(12) phytopathogenic pseudomonad and its involvement in the pathogenesis process. More or less 21 genes are associated with optimal Type III secretion, nine are common and share a high level of sequence similarity. These nine highly conserved type III secretion genes have been named hrc in plant pathogenic bacteria and code for the core part of the Type III secretion system. (12, 13) Three of the hrc genes (R, S, T) are of importance in this study, because for hrcRST primers were developed in literature. Modification of these primers allowed amplification and sequencing of hrcRST (Fig 3).

e) Figure 3: HrcR, hrcS and hrcT of P. syringae pv. syringae A real-time PCR assay directed against the Type III secretion system-encoding hrcRST genes of P. cichorii could make detection of the sources of introduction possible and, hence, also the prevention of infection by P. cichorii.

3.1.3. THE SOURCE OF P. CICHORII

Although one presumes that P. cichorii is found in the sprinkler water of lettuce growth farms, there is no scientific evidence for it.(1) It is also speculated that contaminated seeds may

(4,17,18) be the source of introduction of the pathogen or that P. cichorii is able to survive in the

soil for a long time. Although for the latter, there is little evidence and no conclusive results yet. Real-time PCR could facilitate the search for the source of infection. 3.2. (QUANTITATIVE) REAL‐TIME PCR 3.2.1. INTRODUCTION The real-time polymerase chain reaction is based on the method of PCR, developed by Kary Mullis (19) to amplify specific target DNA more than a billion-fold. In real-time PCR the enormous sensitivity of PCR has been coupled to the precision that is reached by ‘real-time’ monitoring of the generation of the amplicons. (20) Higuchi and co-workers accomplished the first demonstration of real time PCR. (21, 22) For this, conventional PCR was coupled to fluorescent chemistries and instrumentation. Both the terms real-time PCR and kinetic PCR refer to amplification of DNA by PCR that is monitored while the amplification is occurring (in real-time). Quantitative real-time PCR refers to the ability of real-time PCR to quantify the starting amount of a specific DNA sequence. (20) The basic goal of real-time PCR is to distinguish and measure a specific target DNA. The most common target DNA sequences for microbial research with real-time PCR are 16S

(23) rDNA and functional genes. Real-time PCR instrumentation and reagents were first made commercially available by Applied Biosystems, but at this moment several other companies also provide them, e.g. Bio-Rad and Roche Applied Science.

3.2.2. PCR AMPLIFICATION

To perform a PCR, several reaction components are needed, like double stranded template DNA, a thermostable polymerase, dNTPs (DeoxyNucleotideTriphosphates), two primers and

(24) MgCl2. The DNA polymerase amplifies specific pieces of the dsDNA by using short (±15- 30 bases long) sequence-specific primers. The most commonly used enzyme is Taq DNA polymerase that originates from the bacterium Thermus aquaticus. (20, 21) Since this polymerase is not easily destroyed by heat, it only needs to be added at the beginning of the reaction. During PCR, the target DNA sequence is amplified over a number of denaturation–annealing- extension cycles, called temperature cycling. (23) (Fig. 4): - Denaturation: 92-96°C The dsDNA must be denaturated into single strands by high temperatures. - Annealing: 45-72°C The reaction is cooled to allow the nucleotide primers to anneal to the now single stranded template DNA. 15

- Extension: 72°C This temperature is optimum for the DNA polymerase enzyme to create a new complete strand of DNA by extending the primers with dNTPs. This new dsDNA must be then denatured again before the next cycle of copying can occur.

f) Figure 4: The PCR temperature cycle. (1) The temperature is raised to about 95°C to melt the dsDNA, (2) the temperature is lowered to let the primers anneal and (3) the temperature is set to 72°C to let the polymerase extend the primers (24) . After completion of the PCR reaction, the amplification products in conventional PCR are analysed with the use of gel electrophoresis. In conventional PCR, reactions will reach a plateau. This can be due to (1) depletion of primer, dye or dNTPs, (2) inhibition caused by increased product concentration, (3) the loss of polymerase activity as the number of cycles increases and (4) self- annealing of the accumulating product. The PCR reaction can efficiently amplify DNA only up to a certain quantity before the plateau effect. So, there is no way to reliably calculate the amount of starting DNA by quantifying the amount of amplicon. End-point PCR can only distinguish a positive from a negative sample. It is this characteristic that makes real-time PCR so appealing. (20, 23)

3.2.3. REAL‐TIME MONITORING OF PCR

Quantification and characterisation Real-time PCR measures product formation during the exponential phase of amplification, because amplification only occurs efficiently early in the reaction process. This measurement

(22, 23, 24, 25) correlates to the amount of specific starting DNA, thereby allowing quantification . The threshold cycle Ct (or crossing point Cp) is defined as the cycle at which amplification is first detected above the threshold (Fig. 5a). Ct is inversely correlated to the log value of the initial concentration of the DNA in the original sample (22, 23). So, the sooner the fluorescent signal reaches a threshold level, the higher the amount of original target sequence.

To estimate the concentration/ quantity of an unknown DNA in the sample a standard curve can be constructed. In the real-time PCR standard curve, the Ct values are plotted against the log values of e.g. the dilution factor of a serial dilution of a standard. In this way, the concentration of an unknown DNA sample can be estimated (Fig.5b). As the level of amplification can be assessed in this way, the need for electrophoresis is

(22) eliminated.

g) Figure 5: Quantification of Microcystis aeruginosa PCC 7820 using real‐time PCR. (a) Top: Relative fluorescence intensity of five standard solutions of PCC 7820 throughout amplification cycles where Ct represents the treshold cycle number (b) Bottom: The standard curve for real‐time PCR measurement of PCC 7820 (23)

Detection formats: fluorescence reporters In real-time PCR the concentration of the amplicon is monitored throughout the amplification cycles using fluorescent reagents. These reagents bind with the amplicon and the fluorescence intensity emitted, reflects the amplicon concentration in real time. (20)

(21) Several types of reagents exist, like EtBr or SYBR Green I, hydrolysis probes (5’-nuclease probes/ TaqMan probe), hybridisation probes and molecular beacons. Each type of reagent has its own unique characteristics, advantages and disadvantages, but the strategy for each is simple: they must link a change in fluorescence to amplification of DNA. For this project, SYBR Green I was the first fluorescent chemistry used (Fig. 6). It is an asymmetrical cyanine dye (24) used as a nucleic acid stain. It binds to the minor groove of dsDNA. SYBR Green I could replace the very strong mutagen EtBr.

h) Figure 6: The chemical structure of SYBR Green I This dye has virtually no fluorescence when it is free in solution, but when it binds to the minor groove of dsDNA, not on ssDNA, it becomes brightly fluorescent. During PCR, SYBR Green I binds to DNA products as they are synthesized. Thus, the increase of fluorescence, when measured at the end of each elongation cycle, indicates the amount of PCR product formed during that cycle. Therefore, the higher the amount of dsDNA present in the reaction tube, the higher the amount of DNA binding and fluorescent signal from SYBR Green I. (20, 23, 24) The three most important advantages of SYBR Green I are (1) it is simple to use without the complicated design of a probe and it can be used in combination with any PCR primer set, (2) it is cheaper compared to probes and (3) there is the possibility to do MCA or melting curve analysis: the DNA products can be characterised by subjecting them to increasing temperatures to determine when the dsDNA amplicon denatures again.(24) This melting temperature is a unique property dependent on the product length, product nucleotide composition and %GC.(20) The most important limitations of SYBR Green I are (1) the dye binds with all dsDNA indiscriminately (2) formation of primer-dimers is possible, which influences detection sensitivity (3) aspecific amplification. Thus, accurate primer design and the optimisation of the PCR conditions are crucial when DNA-binding dyes are used.(23) A second fluorescent chemistry used, is the TaqMan system (TaqMan probe). It is a sequence specific oligonucleotide with 2 fluorophores: a 5’ end reporter and a 3’ end quencher. The fluorescence emitted by the reporter is absorbed by the nearby quencher via Fluorescence Resonance Energy Transfer (FRET) (Fig 7, top part).

i) Figure 7: The TaqMan probe fluorescent chemistry (24) The TaqMan probe hybridises with the target DNA. During the extension phase, Taq DNA polymerase hydrolyses the TaqMan probe bound on the target DNA strand, separating the reporter and the quencher (Fig. 7, bottom part). This hydrolysis of the oligonucleotide results in a increase of the reporter’s fluorescence signal. (20, 23) It should be noted that a TaqMan assay, compared to a SYBR Green I assay, is more costly, more difficult in probe design and limited to the detection of shorter PCR product (23), but more specific. Instrumentation of real‐time PCR Because fluorescent chemistries require both a specific supply of energy for excitation ánd detection of a particular emission wavelength, the instrumentation has to be able to do both simultaneously. At present, there are three basic ways in which real-time instrumentation can supply the excitation energy for the fluorophores: (1) by lamp (2) by light emitting diode (LED) (3) or by laser. The Lightcycler® thermal cycler instrument (Roche Applied Science, Mannheim, Germany) used in this study uses a LED. To collect data, the emission energies must be detected at the appropriate wavelengths. The detectors include, e.g. CCD cameras or photomultiplier tubes. (20, 24) A second part of the instrumentation is the thermocycler to carry out PCR. To maintain a consistent temperature among all sample wells, the Lightcycler® thermal cycler instrument (Roche Applied Science) uses heated air. This changes temperature faster, resulting in shorter thermocycling times. Computer hardware and data-acquisition and analysis software completes the real-time instrumentation.

3.2.4. OPTIMISATION OF REAL‐TIME PCR

Optimisation of assay conditions is challenging for conventional PCR. The assessment of different test parameters is a very time consuming and precise process, even when only a single parameter is changed (e.g. optimal MgCl2-concentration). Because real-time PCR is more automated and takes less time, optimisation experiments are not as time consuming. For the development of a real-time PCR assay, a few key components should be optimised (26,27). These factors include:

- MgCl2-concentration, which allows the polymerase enzyme to function at an optimal level - Primer concentrations, which affect the sensitivity and specificity of the assay - Probe concentrations, which also affect the sensitivity and specificity - The type of polymerase enzyme used can even play a significant role, e.g. polymerases that permit a hot start do not function until a critical maximum temperature is used, which reduces the generation of non-specific amplicons - Time-temperature profile: the annealing temperature and the amount of seconds in which annealing or extension occurs, can be varied

Also important is that the primers or probe should be able to: - detect the target in a wide range of strains (inclusivity) - not detect a range of non-target organisms (exclusivity) - detect the target in the presence of non-target organisms (specificity)

3.2.5. APPLICATIONS OF REAL‐TIME PCR

The advantages of real-time PCR are numerous: (20, 28) 1. Quantification of nucleic acids is possible 2. Assay capabilities are greater: with the same instrument qualitative assays as well as quantitative, mutation and multiplex assays can be performed 3. It is highly automated and has a much shorter analytical turnaround time than conventional PCR 4. The closed reaction vessel makes post-PCR manipulations unnecessary, in that way amplicon (cross)contamination is avoided On the other hand, the largest limitation of real-time PCR probably resides in human error: improper assay development, incorrect data analysis or unfounded conclusions. For

microbiology, false positives or negatives must be considered when designing an assay to detect pathogens. (20) However, real-time PCR makes numerous applications possible in many fields of research, e.g. (bio)medical research and molecular diagnostics. Some examples of applications are: - Gene expression analysis (20, 24) - Counting bacterial, viral or fungal loads (20) - Identification of mutations or single nucleotide polymorphisms by melting curve analysis (20, 24)

(24) - Pathogen detection , like detection of o Bacillus anthracis (29) o Bordetella pertussis and Bordetella parapertussis (30) o P. aeruginosa and P. fluorescens (31, 32) - Environmental sample analysis (denitrifying bacteria, cyanobacteria,...) (23)

4. MATERIALS AND METHODS All the practical work was performed at the Laboratory for Microbiology at the Ledeganck complex (fourth floor, second phase), Ghent. 4.1. STRAINS 4.1.1. ORIGIN OF THE STRAINS A total of 118 bacterial isolates was used for this study, they are listed in Addendum A. The list includes Pseudomonas strains collected from diseased lettuce during the IWT project, Pseudomonas (type) strains of the BCCM-LMG2 collection and strains from ILVO-PGB3. Some strains of the publication of Araki et al (14) were requested and included in this study. For most strains DNA was available, for seven strains the procedure for long term preservation and DNA extraction was performed (Table 3). j) Table 3: Strains that were not available in the Ledeganck and arrived later on

Original n° Received R-n° Species Origin/Reference 83-1 R-35806 P. cichorii (14) LMG 23197 --- ‘P. luti’ BCCM-LMG LP23.1a R-35807 P. viridiflava (14) ME3.1b R-35808 P. viridiflava (14) PNA3.3a R-35809 P. viridiflava (14) RMX23.1a R-35810 P. viridiflava (14) RMX3.1b R-35811 P. viridiflava (14)

4.1.2. LONG‐TERM PRESERVATION OF THE STRAINS

A purity check had to be performed on the seven strains. Before long term preservation in the R-collection, the micro-organisms were streaked out on new TSA solid media. After 24h of incubation a purity check under the binocular microscope was performed. For the preparation of TSA medium, see Box 1. Box 1: Protocol preparation of TSA medium - Dissolve 40g Tryptone Soya Agar in 1L distilled water and shake well - Autoclave during 20 minutes at 121°C - After cooling down, put the bottle with the solution in a warm water bath of ± 55°C - Fill the designated petri dishes halfway - Let them dry for ± 30 minutes - Pile them up with the agar side upside down - Pull a bag over the plates - Turn the stack of plates around (to avoid condensation) - Close the bag with tape and write the date and name on the bag

2 BCCM‐LMG collection: Belgian Coordinated Collections of Micro‐organisms – Laboratorium voor Microbiologie, Gent (Belgium) 3 ILVO‐PGB: Instituut voor Landbouw en Visserij Onderzoek – Plant en GewasBescherming 22

Pure isolates were stored using the MicrobankTM system (Pro-Lab Diagnostics, Canada). The MicrobankTM cryovials contain 25 coloured, porous beads and a cryopreservative. After inoculation, the cryovials are kept at -80°C for long time storage (Box 2). When a fresh culture is needed, a single bead is removed from the vial and is used directly to inoculate a suitable bacteriological medium (Box 2). Recovery of new micro-organisms was not needed during this thesis. Box 2: Procedure for preparation of MicrobankTM beads for long term preservation and for recovery of micro-organisms stored on MicrobankTM beads Preparation for long term preservation on MicrobankTM beads

- Work in a disinfected flow - Code the vial (and the respective culture) as desired, using a permanent marker, per vial one organism is to be inoculated - Open the screw cap of the cryovial in aseptic conditions (pass the neck of the vial through a Bunsen burner flame) - Inoculate the cryopreservative fluid with young colonial growth (18-24h), picked up with a swab from a pure culture - Close the vial tightly and invert 4-5 times to emulsify the organism, in that way the micro-organisms will adhere to the porous beads - Leave the plate on the bench and check for contamination the day after - The inoculated cryovials are stored at -80°C

Recovery of micro‐organisms stored on MicrobankTM beads

- Open the screw cap of the cryovial under aseptic conditions - Use a sterile needle to remove one of the beads. Close the vial tightly and return as soon as possible to low temperature storage. - The inoculated bead may then be used to directly inoculate an appropriate medium

4.2. DNA EXTRACTION Strains were grown on a suitable growth medium (TSA) and incubated aerobically at 37°C for 24-48h. DNA extractions were performed according to the method described by Pitcher et al (33) (Box 3 and Box 4). It allows the rapid isolation and purification of genomic DNA from Gram-negative bacteria. Provided that the DNA obtained is of high purity, high molecular weight and double stranded, the resulting DNA preparations can be used for PCR applications. Box 3: Reagents needed for DNA extraction The reagents needed for DNA-extraction were already prepared in the Lab for Microbiology-Ghent: - 0.5M EDTA (pH 8.0): 186.1g EDTA, 20g NaOH and 800mL of MQ are mixed - RS (resuspension) buffer: to 1L MQ 8.8g NaCl and 3.7g EDTA is added (pH 8.0) and the entirety is sterilised - TE (Tris- EDTA) buffer (100x): 121g Tris HCl and 200mL EDTA (pH 8.0) are mixed and diluted up to 1L with MQ and sterilised - GES (guanidinium-thiocyanate-EDTA-Sarkosyl) reagent: 60g guanidiniumcyanate, 20mL 0.5M EDTA (pH 8.0) and 20mL MQ are mixed

and heated at 65°C. After cooling down, 1g sarkosyl and 100mL MQ is added - Ammonium acetate 7.5 M (NH4Ac) - Chloroform/isoamylalcohol: 24/1 (v/v) - Isopropanol - 70% ethanol

Box 4: Protocol for DNA extraction Removal of the majority of exopolysaccharides

- Add 500μL RS buffer into numbered Eppendorf tubes - A loop full of cells is harvested from the growth media using a plastic öse - The cells are suspended in the Eppendorf tubes - The cell suspension is spinned down for 2 minutes at 13000 rpm - Removal of the supernatans - Resuspension of cells in 100μL TE buffer

Lysis of the cells

- The resuspended cells are lysed by the addition of 500μL GES reagent (Guanidium-thiocyanate-EDTA- Sarkosyl) - The suspension is carefully mixed (manually) until a solution with a certain viscosity is obtained and is allowed to stand on ice (cooling block) for 15 minutes

Precipitation of proteins

- 250μL ice-cold NH4Ac (7.5 M) is added and carefully mixed with the lysed cell suspension (manually, until very small bubbles appear) - Keep on ice (cooling block) for 15 min - 500μL chloroform/ isoamylalcohol (24/1) is added (isoamylalcohol is an anti-foaming agent) - The mix is thoroughly shaken until a homogeneous one-phase solution is obtained - The mixture is spinned down for 20 minutes at 11700 rpm - A three phase system is normally obtained: the upper aqueous phase (containing nucleic acids) the inter phase (containing a protein ‘pancake’) the lower phase (containing the chloroform fraction) - Approximately 700μL of the UPPER phase is transferred to new Eppendorf tubes Precipitation of the DNA - 0.54 volumes ice-cold isopropanol is added - By carefully mixing, the nucleic acids are precipitated as a white fluffy cloud. - The precipitated nucleic acids are spinned down for 10 minutes at 13000 rpm - The supernatans is removed and the remaining pellet is washed 3 times (with 1 minute centrifugation and supernatans removal in between) with 150μL 70% ethanol to remove traces of isopropanol - The obtained semi-transparent pellet is allowed to air dry for 20-40 minutes or dried under vacuum and dissolved in 100μL TE buffer - The pellet is allowed to dissolve at 4°C (overnight or longer)

RNAse step a. 25μL RNAse (2 mg/ml) is added per 100 μl DNA solution and incubated for 1 hour at 37°C in a thermoblock b. Store the stock solution at -20°C

4.2.1. QUALITY CONTROL AND STORAGE OF DNA

This comprises on the one hand, assessment of fragmentation of the extracted DNA and on the other hand, assessment of the concentration and of the purity of the extracted DNA.

Assessment of fragmentation To use DNA in a PCR application, the obtained DNA should be of high molecular weight, which means only véry little fragmentation is allowed. The quality of the isolated DNA was analysed with agarose gel electrophoresis. It is a method that separates macromolecules on the basis of size and electrical charge. The electrical current from one electrode repels the molecules, while the other electrode simultaneously attracts the molecules. The frictional force of the gel material acts as a ‘molecular sieve’, separating the molecules by size. During electrophoresis, macromolecules are forced to migrate through the pores when the electrical current is applied. Smaller fragments will migrate faster than larger fragments. The DNA is then visualised with Ethidium Bromide (EtBr, Box 5). EtBr intercalates between DNA base pairs. UV-light at 312 nm causes the EtBr/DNA combination to fluoresce. One can only work with EtBr in a separated, designated space and one should have special attention for personal protection, e.g. special gloves should we worn. After staining of the gel, one clear band at the top of the gel indicates that the DNA is of high molecular weight, while a smear indicates fragmented DNA. The DNA of the 7 isolates was not fragmented and suited to use in PCR applications (data not shown). Box 5: Reagents and protocol for quality control of the extracted DNA Reagents - Agarose: a natural colloid extracted from seaweed, it’s very fragile and can easily be destroyed via handling. Agarose gels have a very large pore size and are used primarily to separate very large molecules with a molecular mass greater than 2000kDa - TAE (Tris-Acetic-EDTA) buffer (50x) (Biorad, Munchen) 50xTAE contains 40mM Tris, 20mM acetic acid, 1mM EDTA and has a pH of 8.3 In the laboratory, 1x TAE is used Preparation Take a volumetric flask of 1L and fill it with 20mL of 50xTAE Fill up the volumetric flask halfway with MQ and close it with parafilm Mix this solution by gently turning the volumetric flask Fill it up with MQ up to 1L Take a bottle of 1L; add the 1xTAE solution, put ‘TAE’ and the date on the bottle - Loading dye (6x buffer contains 4g of sucrose, 25mg of Bromophenolblue, 6 mL of TE-buffer diluted to a total volume of 10mL with MQ) - Smartladder (molecular marker with DNA fragments of a known length, allows to determine the molecular weight of unknown DNA-molecules in the gel, Eurogentec) - EtBr - TBE (Tris-Borate-EDTA) buffer: buffer used in EtBr solution (to stain the gel) and TBE solution (washing step)

Protocol

Preparation of the gel - 1% agarose is mixed with 1x TAE buffer and heated in a microwave until it reaches boiling point. A clear solution should be obtained - Take the gel tray and put the combs in the right position - Make sure that the tray is level - Pour the agarose solution and allow it to cool at room temperature to form a rigid gel - Place the gel into the electrophoresis bath that is filled with 1x TAE buffer

- The gel has to be submerged completely - Remove the combs

Preparation of the sample Take 5µL of DNA of each sample and mix it with 1µL loading dye

Loading of the gel - Pipet in each slot 6µL of the prepared DNA sample, at both ends of the gel 5µL of smartladder is pipetted in the gel - When all slots are filled, the lid is put on the electrophoresis bath and the electrical charge is put at ± 80V - After ± 30 minutes the electrical charge is stopped and the gel is taken out of the bath and slid carefully onto tin foil

Visualisation of the DNA: staining of the gel - put the gel in the EtBr solution for ± 30 minutes - put the gel afterwards in the TBE bath (washing step) for ±10 minutes - get the gel out of the TBE bath and place it onto the UV-apparatus

Procedure for taking a picture of a gel: - turn on the screen, the digitalising system and the UV light (screen down!!) - enlarge/ reduce image and focus. Close the diaphragm - integrate until image is clearly visible - ‘capture’ image - turn on printer and move handle down - ‘print’ - print image and immediately move the handle up and turn of the printer (even in between pictures) - throw away the gel in the waste bins - clean the UV-apparatus or

Take picture with PC (remove contaminated gloves) - click on Colorvision - press ‘ok’ - press the icon with the camera - press the fourth icon to enlarge the image - press the last but one icon to close the window - File: ‘save as’ Æ C:// Æ irisdat. Æ TIFF Æ save file with the date as the filename

Concentration and purity assessment The quality of the DNA can be measured by two ratios:

- OD260/OD280

- OD234/OD260 The first ratio is based on the knowledge that for a pure DNA solution the optical density (OD) at 260 nm is twice as high for DNA and RNA as the OD at 280 nm. Proteins on the other hand have a higher absorbance at 280 nm (especially by the amino acids tyrosin and tryptophan). This also is true for phenol. This ratio has ideally a value between 1.8 and 2.2. RNA has a higher absorbance at 260 nm than DNA, so this could compensate for the effect of the proteins. Therefore, a second ratio is calculated.

The second ratio is a second measurement for the protein-contamination because this is the wavelength at which peptide bonds absorb strongly. This ratio has ideally a value close to 0.5.

Apart from the quality of the DNA, OD260 is also a measurement for the amount of DNA in the solution, because an OD260 of 1 is equal to a DNA-solution of 50 ng/µL. A 1/100 dilution was made for each DNA solution with sterile MQ. The absorbance was measured against a blank at 234, 260, 280 nm in a spectrophotometer (Box 6). From the absorbance values, the DNA concentrations and the ratios could be calculated. The DNA solutions were then further diluted to the desired concentration, OD1, with filtered sterilised TE buffer.

Box 6: Protocol for the preparation of OD1 Protocol - Place the DNA Pitcher stocks into a rack - Make new Eppendorf-tubes - Add 10µL DNA to 990µL MQ - Make a Blank of 1000µL MQ - Measurement in the spectrophotometer: 1. First measure the blank (pipette it into a cuvet) 2. Place the cuvet in the right direction and don’t touch the bottom part of the cuvet 3. Put it in to the spectrometer 4. Click ‘assays’Æ DNA cuvÆ 12 cuv 5. Click on the first window Æ reference 7. Press ‘read’ 8. Do the same procedure for each DNA sample 9. Press ‘Print’ - To calculate the right dilution to become an OD of 1 (e.g. for 100 µL):

100µL/ OD260 (take dilution into account!) = x µL DNA Pitcher

Every time, DNA OD1 of a sample to be used in real-time PCR had run out, after OD measurement, new OD1 had to be prepared (Box 7).

Box 7: Protocol for the preparation of a new OD1 after OD measurement - Spin down the samples (Pitcher stock of the DNA) - dilute the DNA (100 fold) 198 µL MQ + 2 µL DNAPitcher - Vortex the dilutions - Spin down the dilutions (short spin) - Take a microtiterplate and put the blank in the upperrow: 200 µL of MQ - Row under the blank (same column) are for your dilutions: also 200 µL - Cross the row you use(d) on the microtiterplate - Make a second blank in a cuvet: 1000µL - Go to the spectrometer to measure the ODs - Put the cuvet with the blank in the spectrometer (arrow to the right) and close the lid - Get the lid of the microtiterplate and put it in the spectrometer - Click SoftmaxPro 4.8 - Click ‘assays’ DNA-96 -> 1x, 2x, 3x Protocol appears - Click ‘set up’, click ‘strips’, click in blue the amount of columns you need, click the template, click ‘clear’ - Click ‘read’ and eventually print the file

4.3. GRADIENT PCR 4.3.1. PCR REACTION As in regular PCR, gradient PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. Because of the sensitivity of any PCR technique, a sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment. For this, several precautions can be taken: - Preparation of the reaction mixture, the PCR process and the subsequent amplicon analysis should be performed in separate areas (preferably in a separate laminar flow cabinet) - Fresh gloves should be worn for each reaction set-up - The use of dedicated vessels, pipettes and tips for the reaction mixture preparation is strongly recommended - A control reaction, omitting template DNA, should always be performed, to confirm the absence of contamination Construction of primers for rapid detection of P. cichorii was already performed in the first part of the IWT project and they were purchased from Sigma-Genosys, Bornem (Belgium).

Two forward primers, one reverse primer and two probes were developed. R-probe0 was adapted into R-probe during the course of this study. The sequences, the lengths and the melting temperature of the primers and the probes, are listed in Table 4. k) Table 4: Primers and probes for PCR and real‐time PCR for the detection of P. cichorii a

Primers Sequence Length Tm Use and probe (5’-3’) (bp) (°C)

F1 GCC GAG GCT TTA TGG AAA CCC TG 23 60.4°C Forward primer 1

F2 GCG GTG ATT GTC GGT GTC ATC AC 23 60.5°C Forward primer 2

R1 ATT CGC TGA CTT CCT TGA ACG GGA G 25 60.8°C Reverse primer 1 R-probe CTC GTY TGA CCG ATC ATG TTG AAA 30 60.8°C - 61.7°C Reverse probe GAC AGG F-probe TTT CAA GCA GGC CAT GTT GCT GGT 27 64,9°C Forward probe R-probe0 CTC GTC TGA CCG ATC ATG TTG AAA 30 61.7°C Reverse probe GAC AGG a: the target gene encodes the P. cichorii Type 3 secretion system (hrc) The species, the primer pairs and the temperature range used to be tested in gradient PCR, were specified (Addendum B). The protocol for (gradient) PCR can be found in Box 8. Box 8: Protocol for (gradient) PCR Set up - Calculate PCR Mastermix 5 µl of 10x PCR-buffer (already contains 1.5mM MgCl2) 3 µl of MgCl2 (25mM) 5 µl of dNTPs 1µl forward primer (50µM) 1µl reverse primer or probe (50µM)

33.75µl MQ (sterilised deionised water, Aq. Dest.) 1.25µl Taq polymerase (1.25U) - Disinfect bench and pipettes with sodiumhypochloride - Take the product-solutions out of the freezer: 10x PCR, MgCl2, dNTPs, forward primer, reverse primer or probe, MQ, Taq polymerase, OD1s of the samples that are to be tested - Put them into a cooling block

Preparation of the reaction mixture - Briefly spin down all solutions after thawing - To perform several parallel reactions, prepare a master mix containing MQ, 10x PCR (buffer), dNTPs, primers and MgCl2 and Taq DNA polymerase in a tube (or several tubes when handling large volumes) that is kept on ice - Take a cooling block for PCR cups out of the freezer - Divide the Mastermix up (49µl) into each PCR cup on ice - Then add the respective template DNA of an OD260 of 1 (1µl) into his respective cup - Place the caps onto the cups - Spin down the samples with the Multifuge - Place the samples into a thermocycler and start PCR

The gradient PCR was performed to determine the annealing-temperature to start with in the PCR and real-time PCR experiments. A temperature range of 56°C to 67°C seemed possible for amplification (Addendum B). Eventually an annealing-temperature of 60°C was used in subsequent PCRs. The thermocycle conditions (PCR program) can be found in Box 9: Box 9: Thermocycle conditions for gradient PCR - an initial denaturation step for 5 minutes at 95°C - followed by 30 cycli, consisting of 30 seconds at 95°C 45 seconds at X°C (gradient: 60.5°C) 45 seconds at 72°C - A last step for 5 minutes at 72°C - Additional cooling was performed of 60 minutes at 4°C

4.3.2. GEL ELECTROPHORESIS

The presence of the specific amplicon was detected by gel electrophoresis (see Box 5). 4.4. PCR For a regular PCR, the same principles and procedures as in gradient PCR are applied. Conventional PCRs were performed to assess whether the hrc gene of P. cichorii was detected or not with the primers and if other Pseudomonas species were detected too or not. The thermocycle conditions differed from the ones used in the gradient PCR (Box 10). The Mastermix for the PCRs were the same as the one used in the gradient PCR (Box7) and the annealing-temperature was set at 60°C. A negative control without DNA and a positive control were included in each PCR run. The strains tested in the fruns are listed in Table 5. The presence of a visible amplicon of the expected size (230bp for F2-R1, 280bp for F1-Rpobe0 and 300bp for F1-R1) was interpreted as a positive result, absence as a negative result. 29

Box 10: Thermocycle conditions for the regular PCRs PCR Program - an initial denaturation step for 5 minutes at 95°C - followed by 30 cycli of 30 sec 95°C 45 sec 60°C 45 sec 72°C - a last step for 5 minutes at 72°C - Additional cooling was performed for 60 minutes at 4°C l) m) Table 5: Strains tested with conventional PCRa

LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG 20220 T 22563 T 21623 T 13190 T 2163 21316 T 23075 T 2229 T 2257 T 1247 T LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG 1242 T 21749 T 23068 T 2172 T 2158 T 21624 T 1223 T 7040 T 2274 T 2336 T LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG 2112 T 21630 T 5096 T 22119 T 2195 T 5054 21632 T 1874 T 21640 T 2342 T LMG LMG LMG LMG LMG LMG R- LMG R- LMG 1224 T 2164 23064 T 19695 T 5034 5868 26431 981 T 25254 22121 T LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG 23134 T 18376 T 2162 T 18387 T 21661 T 21625 T 21609 T 21750 T 22120 T 8401 LMG LMG LMG LMG LMG LMG R- LMG LMG LMG 13184 T 978 T 2162 T 1794 T 980 T 21974 T 31877 21465 T 2209 T 21317 T LMG LMG LMG LMG LMG LMG R- LMG LMG LMG 21629 T 1248 18378 T 2191 T 23066 T 7041 T 25295 1225 T 21615 T 6771 LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG 22709 T 979 T 21466 T 19851 T 5052 21607 T 21614 T 21977 T 11199 T 2352T LMG R- LMG LMG LMG LMG LMG LMG LMG LMG 21611 T 20821 2152 T 11722 T 21604 T 21605 T 21318 T 21606 T 21608 T 21539 T LMG R- LMG LMG LMG R- LMG R- R- R- 2223 T 20805 T 17764 T 21464 T 20222 T 35806 23197 T 35807 35808 35809 R- R- LMG 35810 35811 2162 T a. Top part (first 8 rows): Pcic-hrc F1-R1, Pcic-hrc F2-R1, Pcic-hrc F1-Rprobe0 Bottom part (last 3 rows): Pcic-hrc F1-R1, Pcic-hrc F2-R1/Pcic-hrc F1-Rprobe0

4.5. REAL‐TIME PCR ASSAYS 4.5.1. INTRODUCTION To amplify the P. cichorii hrc gene, a Lightcycler® thermal cycler instrument (Roche Applied Science, Mannheim, Germany) equipped with the MCA (Melting Curve Analysis) program was used with the primers and probes described in Table 4. In the Lightcycler® instrument, the reactions take place in glass capillaries. The design of the glass capillaries facilitates high speed thermal cycling. Their high surface-to-volume ratio permits extremely rapid thermal transfer (34), which minimizes formation of non-specific products that can lead to an overestimate of copy numbers. A single capillary consists out of three parts: - the glass capillary itself - a plastic reservoir at the top of the capillary - a plastic stopper to seal the capillary.

Samples are pipetted into the capillary reservoir and then forced into the glass tube by centrifugation (34).The general protocol to perform a real-time PCR assay can be found in Box 11. Box 11: Protocol for a real-time PCR assay Protocol Calculate PCR Mastermix scheme (dependent on the brand used): Sigma: 10µl ReadyMix (E+) 2µl of each primer (ideal concentration has to be tested) x µl MgCl2 (ideal concentration has to be tested) x µl MQ if necessary Roche: 2µL Master SYBR Green I 2µL of each primer x µL MgCl2 x µL MQ Activate the flow (Gelman Instrument Co., Michigan,USA) push left button, then push the white button until the flow starts, push the light button of the flow: has to run ±10 minutes to be ‘sterile’ Take your product solutions out of the freezer: Sigma: Forward primer Reverse primer or probe MgCl2 E- or E+ (enzyme/mix: TIN FOIL!!) MQ if necessary (Sigma, Steinheim, Germany) Roche: Forward primer Reverse primer or probe MgCl2 Master SYBR Green I (TIN FOIL!!) MQ if necessary (Roche, Mannheim, Germany) Set up computer program for your run on the computer in the real-time PCR room (NO GLOVES) Activate computer, printer and lightcycler Click ‘run’ Let machine run ‘selftest’ (closed lid: if opened only give a slight push to let the lid close itself) ‘selftest passed’ Æ click ‘ok’ Set up computer program you need: ‘add’ Æ activation/ quantification/ melting curves Put on gloves (Kimberly-Clark) and disinfect flow with sodiumhypochloride on kitchen roll Disinfect pipettes (Rainin Instruments LLC, Oakland, USA) with sodiumhypochloride Put the materials you need in the flow: Glass jar for ordinary waste Plastic bag for SYBR Green I waste Cooling block (freezer) Every time you get in or out the flow, clean gloves with ETHANOL Get the cooling block for capillaries (has to be cooled for at least 1h) and the Reference Dye (Sigma) out of the fridge Make if necessary E+(Sigma), or Master SYBR Green I (Roche): Sigma: E- (500µl) + Reference Dye (5µl) Change E- into E+ Reference dye can NOT EVER be put in the freezer!! Put the pipettip in the SYBR Green I waste Cover as much as possible with tin foil en if necessary, put out the light of the flow Roche: 1a (enzyme, 54µL) + 1b (SYBR Green, 10µL) Put a green sticker and a new label on the tube Put the pipettip in the SYBR Green I waste Cover as much as possible with tin foil en if necessary, put out the light of the flow Check the numbers of the capillaries and put them in the cooling block adaptors Dilute the primers and the MgCl2 to the wanted concentration 31

Mix al your products (slightly flick against the Eppendorfs) and spin down (short spin) on the centrifuge (Eppendorf, Hamburg, Germany) Make your Mastermix: add E+/ Master SYBR Green I last to avoid contamination with SYBR Green I Mix your Mastermix (flick and turn gently) and spin down (short spin) Fill the capillaries: 18 µl mix Place the pipet in an oblique way on the edge (pipettip in SYBR Green I waste) 2 µl OD1 DNA Replace gloves in between adding the mix and adding the DNA Pipet a few times up and down (pipettip in SYBR Green I waste) Place caps on the capillaries with the provided pen Spin down the capillaries with the caps: 5 seconds on 700 rcf WITH iron containers WITHOUT lid of centrifuge Carefully put the capillaries in the rotor and push them down Check the volumes in the capillaries (same height) and place the rotor in the LightCycler (click) Give the lid a slight push and remove gloves Press ‘Open experiment file’ Press ‘Edit samples’ Max position = x (maximum 32) Æ done ‘Type’ is unknown (always!) Press ‘ Run’ Æ Press ‘Save’ Æ Press ‘yes’Æ give the file a name Press ‘Enter samples later’ Enter names of the samples and wait until all samples have been recognised Concentration of the primer or MgCl2 behind name Mention primer-couple in ‘notes’ ‘Done’ Disinfect flow and pipettes and put all the material back where it belongs If analysis is done, take floppy disk to real-time PCR room and insert in computer: Quantification: Area between green lines is analysed ReportÆ print summary report QuantificationÆ exportÆ baseline adjustment Window: go to previous window Melting curve: Shift green lines (until line beneath is straight) Melting curve Æ exportÆ melting peak data Get the rotor out of the lightcycler and release capillaries with capillary releaser Remarks Sigma kit: SYBR® Green JumpstartTM Taq ReadyMixTM Roche kit: LightCycler® Faststart DNA Master SYBR Green I (with Hot Start) For SYBR Green the parameter ‘Fluoresce’ in the Lightcycler Software is put on ‘F1’, for TaqMan on ‘F1/F3’

4.5.2. REAL TIME PCR DEVELOPMENT

Optimisation of the PCR conditions, e.g. concentration of MgCl2, temperature-time profile, concentration of primers and probe, had to be performed. In addition, the primers had to be extensively tested for: (1) exclusivity (2) inclusivity (3) analytical specificity and (4) analytical sensitivity. Fluorescence signals were measured once in each cycle at the end of the extension step, except where stated otherwise. Fluorescence data were analysed using the LightCycler

Software (version 3.5; Roche diagnostics, Basel, Switzerland). PCRs were carried out in a final volume of 20 µL, containing 2µL of extracted DNA, except where stated otherwise.

To establish a melting temperature (Tm) specific for amplicons, the MCA analysis was conducted with positive and negative controls for P. cichorii. A negative control without DNA and a positive control were included in each PCR run. The negative control sample was prepared by replacing the DNA template with sterilised and filtered MQ. A list of all mixed used in this study can be found in Addendum C. At the start of the development of a real-time PCR assay for P. cichorii, the protocol for amplification and melting curve analysis was used as described in Table 6. The parameters indicated in a grey box were varied during the development of the real-time PCR assay for P. cichorii. n) Table 6: Lightcycler amplification and melting curve protocol at the start of the development of the real‐time PCR assay

Program No. of Target temp Hold time Temp Transition Fluorescence cycles (°C) (s) rate (°C/s) acquisition mode Polymerase activation 1 95 600 20 None Three-step PCR 45 Denaturation 95 10 20 None Amplification 60 20 20 None Extension 72 15 20 None Three-step melting curve 1 Denaturation Holding 95 0 20 None Melting 65 15 20 None 95 0 0.1 Continuous Cooling 1 40 30 20 None

Optimisation of the PCR conditions o) MgCl2 concentration

The concentration of MgCl2 affects efficiency as well as specificity of the amplification reaction. The concentration has an influence on (1) Taq DNA polymerase as it prefers Mg2+ as cofactor and (2) the Tm of DNA. High concentrations of MgCl2 stabilize dsDNA and may lead to non-specific primer hybridisation or incomplete denaturation of target DNA (due to an increase in Tm). The optimum MgCl2 concentration for PCR with the LightCycler® varies from 1-5mM. For each primer pair, different concentrations of MgCl2 were tested (Table 7 and Table 8) with the Sigma-kit.

d p) Table 7: Optimisation MgCl2 concentration for F1‐R1

Name/date Primers Mix MgCl2 Temperature-time profile (End concentration) Annealing Annealing timeb a temperature Extension timec

Pcichrc Mix A 5mM 2007-03-05 Pcichrc F1-R1 Mix B Mix B 0.5mM 2007-03-06 Mix C Mix C 1.5mM Mix D Mix D 2.5mM 60°C 20 seconds Mix E Mix E 3.5mM 15 seconds Pcichrc Mix F 2mM 2007-03-06B a. ‘Annealing temperature’ is the target temperature during amplification b. ‘Annealing time’ is the temperature hold time during amplification c. ‘Extension time’ is the temperature hold time during extension d. All three runs were processed in Excel

a q) Table 8: Optimisation MgCl2 concentration for F2‐R1

Name/date Primers Mix MgCl2 Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc Mix F 2mM 2007-03-07A Pcichrc Mix B Mix B 0.5mM 60°C 20 seconds 2007-03-07B F2-R1 Mix C Mix C 1.5mM 15 seconds Mix D Mix D 2.5mM Mix E Mix E 3.5mM a. Both runs were processed in Excel r) Temperature‐time profile The actual annealing temperature of primers during PCR may be much higher or lower than the Tm calculated from the sequence of the primers. To determine it, the target temperature of annealing was varied by raising or lowering it with 2-3°C until the optimal temperature was found. For both primer pairs, 3 temperatures were tested with the Sigma kit: 60°C (Table 7 and Table 8), 62°C and 64°C (Table 9). In addition, to reduce the amount of primer-dimers formed during a run, the annealing time was varied as well (Table 10). s) Table 9: Optimisation annealing temperature (60°CÆ62°CÆ64°C)a

Name/date Primers Mix Primers Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc F1-R1 2007-03-14A 62°C Pcichrc F2-R1 Mix G Mix G 0.25µM 2007-03-14B Mix H Mix H 0.50µM 20 seconds Pcichrc F1-R1 Mix I Mix I 0.75µM 15 seconds 2007-03-15A Mix J Mix J 1.00µM 64°C Pcichrc F2-R1 2007-03-15B a. All runs were processed in Excel t) Table 10: Optimisation annealing and extension timea

Name/date Primers Mix Primers Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc Mix P Mix P 0.10µM 2007-03-16B Mix Q Mix Q 0.15µM 40 seconds F1-R1 Mix R Mix R 0.20µM 60°C 15 seconds Mix S Mix S 0.25µM

Pcichrc 15 seconds 2007-03-26B 15 seconds Pcichrc 10 seconds 2007-03-27 F2-R1 Mix K MgCl2 62°C 15 seconds Pcichrc 2.5mM 5 cycli 20 seconds 2007-03-28B 40 cycli 5 seconds Pcichrc Primers 25 cycli 20 seconds 2007-03-29 0.5µM 20 cycli 5 seconds Pcichrc 15 cycli 20 seconds 2007-03-29B 30 cycli 5 seconds a. Only run Pcichrc 2007-03-26B was processed in Excel u) Primer concentration For the instrumentation used, the optimum concentration of primers is usually 0.1-0.6µM. Higher concentrations may promote mispriming and accumulation of non-specific product. Lower concentrations may be exhausted before the reaction is completed, resulting in lower yields of desired products. Both primer couples were tested for different concentrations with the Sigma-kit: 0.25µM, 0.50µM, 0.75µM and 1.00µM. Primer pair F1-R1 was also tested at lower concentrations: 0.10µM, 0.15µM and 0.20µM (Table 11).

a,b v) Table 11: Optimisation primer concentration for primer pairs F1‐R1 and F2‐R1

Name/date Primers Mix Primers Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc 15 seconds 2007-03-12A Mix G Mix G 0.25µM 12 seconds Pcichrc F1-R1 Mix H Mix H 0.50µM 2007-03-13A Mix I Mix I 0.75µM Pcichrc F2-R1 Mix J Mix J 1.00µM 60°C 2007-03-12B 20 seconds Pcichrc Mix P Mix P 0.10µM 15 seconds 2007-03-16A F1-R1 Mix Q Mix Q 0.15µM Mix R Mix R 0.20µM Mix S Mix S 0.25µM a: Pcichrc 2007-03-12A, Pcichrc 2007-03-12B, Pcichrc 2007-03-12B were processed in Excel b: Pcichrc 2007-03-16A was not processed in Excel, due to deviant amplification curves Further optimisation

Once the MgCl2 concentration and annealing temperature for the best primer pair, F2-R1, were optimised, the four primer concentrations were tested again with the Sigma kit (Table 12 and Table 13). In previous runs, primer-dimer formation was observed. Since primer-dimer products are shorter than the target product, they melt at lower temperature and their presence is easily recognised by MCA. In order to try to avoid the presence and interference of primer- dimers, fluorescence was measured at 83°C in run ‘Pcichrc 2007-03-20’ (Table 12). This was a temperature just below the Tm of the product and above the Tm of the primer-dimers. It was expected that at that temperature, primer-dimers in the reaction mixture would melt before the SYBR Green I fluorescence signal of the target was acquired at each cycle.

After optimisation of the MgCl2 concentration, annealing and extension time and primer concentration, the target temperature of annealing was varied once more by raising it with 2°C to a temperature of 64°C (Table 14). w) Table 12: The 4 primer concentrations with 2.5mM MgCl2 at 62°C a

Name/date Primers Mix Primers and MgCl2 Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc Mix G Mix G 0.25µM 2007-03-20 F2-R1 Mix H Mix H 0.50µM 62°C 20 seconds Mix I Mix I 0.75µM 15 seconds Mix J Mix J 1.00µM MgCl2 All mixes 2.5mM a. Measurement fluorescence at 83°C and processed in Excel a x) Table 13: The 4 primer concentrations with 2.5mM MgCl2 at 62°C

Name/date Primers Mix Primers and MgCl2 Time-temperature profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc Primers 2007-03-21 Mix L Mix L 0.25µM

F2-R1 Mix M Mix M 0.50µM 62°C 20 seconds Mix N Mix N 0.75µM 15 seconds Mix O Mix O 1.00µM

MgCl2 All mixes 2.5mM a. Processed in Excel y) Table 14: Increase of 2°C to an annealing temperature of 64°Ca

Name/date Primers Mix Primers and MgCl2 Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc MgCl2 2007-03-28 F2-R1 Mix K 2.5mM 64°C 20 seconds 15 seconds Primers 0.5µM a. Processed in Excel Exclusivity The designed primers had to be tested for exclusivity. In other words, their ability not to detect non-target organisms that may be present or also the lack of interference from a range of non-target strains (Table 15). The four runs were performed with the Sigma-kit. z) Table 15: Exclusivity assaysa

Name/date Primers Mix Primers and MgCl2 Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc 2007-03-22A MgCl2 Pcichrc 2.5 mM 20 seconds 2007-03-22B F2-R1 Mix K 62°C 15 seconds Pcichrc Primers 2007-03-22C 0.5 µM Pcichrc 2007-03-26 a. All runs were processed in Excel

Inclusivity The designed primers had to be tested for inclusivity or in other words, their ability to detect the correct organisms or to detect the target in a wide range of strains (Table 16). The two runs were performed with the Sigma-kit. aa) Table 16: Inclusivity assaysa

Name/date Primers Mix Primers and MgCl2 Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc MgCl2 2007-03-30 F2-R1 Mix K 2.5mM 62°C 20 seconds Pcichrc Primers 15 seconds 2007-04-16A 0.5µM a. Only run Pcichrc 2007-04-16A was processed in Excel Analytical sensitivity (work range) To determine the assay’s lower limit of detection, real-time PCR experiments were performed on a serial tenfold dilutions of DNA OD1 of the P. cichorii type strain and 3 representatives of each of the three BOX-types previously demonstrated in the IWT (1): LMG 2162T, R-25254, 7 0 R-26430 and R-31877 (10 Æ10 ). The serial dilutions contained 1µL DNA OD1 in 9µL TE- buffer. This run had to be performed in triplicate (Table 17). During the course of the runs to determine the lower limit of detection, a switch from a kit of Sigma-kit to a kit of Roche was necessary. bb) Table 17: Analytical sensitivity of the real‐time PCR assay for P. cichoriia

Name/date Primers Mix Primers and MgCl2 Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc 2007-04-19A Pcichrc 2007-04-19B Pcichrc 2007-04-19C F2-R1 Mix H Pcichrc MgCl2 2007-04-20A 2mM Pcichrc Primers 2007-04-23A 0.5µM Pcichrc Mix H 62°C 20 seconds 2007-04-23B and 15 seconds Mix T Pcichrc Mix H 2007-04-24A Pcichrc 2007-04-27A Pcichrc 2007-05-02A Mix U MgCl2 Pcichrc 2.5mM 2007-05-02B Primers 0.5µM a. Runs Pcichrc 2007-04-19B, Pcichrc 2007-04-19C, Pcichrc 2007-04-23A, Pcichrc 2007-04-24A, Pcichrc 2007-04-26 were not processed in Excel

Analytical specificity

The primers F2-R1 had to be tested for specificity, in other words, to detect the correct organisms in the presence of non-target organisms. This means that there should not be an effect of non-target organisms in a multi-component sample (Table 18). To determine the assay’s specificity, real-time PCR experiments were performed on a serial 7 2 T tenfold dilutions (10 Æ 10 ) of DNA OD1 of LMG 2162 (DNA A) and a second strain

(DNA B). The serial dilutions contained 2µL DNA OD1 in 18µL TE-buffer. The run was carried out in a final volume of 20 µL containing 4µL of extracted DNA (2µL of DNA A and 2µL of DNA B). The work scheme for the run can be found in Table 19. For run Pcichrc 2007-04-27B DNA B was P. mediterranea LMG 23075T, for run Pcichrc 2007-05-03 DNA B was P. poae LMG 21456T and for run Pcichrc 2007-05-7A DNA B was DNA extracted from approximately 20g of a lettuce head. Run Pcichrc 2007-05-8B was performed with both strains LMG 21456T and LMG 23075T in comparison to LMG 2162 T. In 8 this run OD10 (or 10 cells) was included, to assess the concentration effect observed in the preceding runs for analytical specificity. All runs were performed with the Roche-kit. cc) Table 18: Specificity

Name/date Primers Mix Primers and MgCl2 Temperature-time profile (End concentration) Annealing Annealing time temperature Extension time Pcichrc 2007-04-27B Pcichrc F2-R1 Mix U MgCl2 62°C 20 seconds 2007-05-03 2.5mM 15 seconds Pcichrc Primers 2007-05-7A 0.5µM Pcichrc 2007-05-8B a. All runs were processed with Excel dd) Table 19: Work scheme for the real‐time PCR run to determine the specificity of the assaya

107 DNA A 105 DNA A 103 DNA A 102 DNA A MQ 107 DNA B 104 DNA B 105 DNA B 107 DNA A 104 DNA A 103 DNA A 102 DNA A 107 DNA B MQ 105 DNA B 106 DNA B 106 DNA A 104 DNA A 103 DNA A 102 DNA A MQ 104 DNA B 106 DNA B 107 DNA B 106 DNA A 104 DNA A 103 DNA A MQ 106 DNA B 105 DNA B 107 DNA B 107 DNA B 106 DNA A 104 DNA A 102 DNA A MQ 107 DNA B 106 DNA B MQ 106 DNA B 105 DNA A 106 DNA A 102 DNA A MQ MQ 107 DNA B 102 DNA B 105 DNA B 105 DNA A 103 DNA A 102 DNA A MQ 105 DNA B MQ 103 DNA B 104 DNA B 105 DNA A 103 DNA A 102 DNA A 4 µL MQ (negative control) 106 DNA B 103 DNA B 104 DNA B a. Only the first assay was performed with MQ, next assays were performed with TE-buffer instead, because both DNA OD1 of the strains and the dilutions contain TE

TaqMan probe A TaqMan probe was developed during this study. A reverse probe (R-probe) and a forward probe (F-probe) were developed (Table 4). At the start of the study a first reverse probe (R- probe0) was tested in gradient PCR, regular PCR and real-time PCR. It gave a positive signal for P. cichorii LMG 2162 T in gradient PCR and in regular PCR for all P. cichorii strains. In real-time PCR however, it did not detect all P. cichorii strains (data not shown). That’s why a new, degenerate R-probe was developed. The F-probe and R-probe were tested as primers in combination with primers R1 and F1 before the sequence was ordered as a TaqMan probe (Table 20). All runs were performed with the Sigma-kit. ee) Table 20: Assays performed with F‐probe and R‐probe before purchase of the TaqMan probea

Name/date Primer-probe Mix End concentrations Temperature-time profile Annealing Annealing time temperature Extension time Pcichrc F1-Rprobe0 Mix F Primers/probes 2007-03-09 1µM MgCl2 2mM 60°C 20 seconds Pcichrc Fprobe-R1 Primers/probes 15 seconds 2007-04-17 F1-Rprobe 0.5µM Pcichrc F1-Rprobe Mix K 2007-04-19D MgCl2 Pcichrc Fprobe-R1 2.5mM 65°C 2007-04-20B a. All assays were processed in Excel

5. RESULTS and DISCUSSION 5.1. CONVENTIONAL PCR

Conventional PCR assays were performed for the different primer couples F1-R1, F2-R1 and

F1-Rprobe0. The aim was to assess whether under optimal PCR conditions (e.g. high primer concentrations (1µM)), all P. cichorii would show a 230bp (F2-R1), a 280bp (F1-Rprobe0) or a

300bp (F1-R1) hrc gene product. In addition, it was checked whether or not non-target strains showed the amplicon on gel. Electrophoresis of amplicons obtained by conventional PCR showed that for all primer couples, there was an amplicon of the right size for all P. cichorii strains. In addition, no DNA template amplification was observed in the negative controls for all primer coules. T For assay Pcic-hrc F1-R1, two non P. cichorii strains, P. orizyhabitans LMG 7040 and P. viridiflava LMG 2352T, also exhibited the 230-300bp hrc gene product. Aspecific amplicons were observed for P. jinjuensis LMG 21316T, P. luteola LMG 7041T, P. mandelii LMG 21607T, P. mediterranea LMG 23075T, P. mendocina LMG 1223T, P. nitroreducens LMG 21614T, P. oleovorans LMG 2229T, P. pertucinogena LMG 1874T, P; pictorum LMG 981T, P. putida LMG 2257T, P. resinovorans LMG 2274T, P. rhizosphaera LMG 21640T, P. teatrolens LMG 2336T, P. tremae LMG 22121T and P. umsongensis LMG 21317T. T For assay Pcic-hrc F2-R1, one non P. cichorii strain, P. citronellosis LMG 18378 , also exhibited the hrc gene product. Aspecific amplicons were observed for most strains. T For assay Pcic-hrc F1-Rprobe0, two non P. cichorii strains, P. citronellosis LMG 18378 and P. viridiflava LMG 2352T, also exhibited the hrc gene product. No aspecific amplicons were observed. The strains used in each conventional PCR can be found in Table 6. Examples of the results of the conventional PCRs can be found in Fig.8.

ff) Figure 8: Conventional PCR (segment of photographs). On the left: 1=LMG 21609T, 2=R‐31877, 3=R‐25295, 4=LMG 21614T, 5=molecular marker, 6=LMG 2229T, 7=LMG 7040T, 8=LMG 1874T In the centre: 1=molecular marker, 2= LMG 21623T, 3=LMG 23068T, 4= LMG 5096T, 5=LMG 23064, 6= LMG 21630T, 7=LMG 2162 T, 8=LMG 18378T On the right: 1=molecular marker, 2= LMG 21623T, 3=LMG 23068T, 4= LMG 5096T, 5=LMG 23064, 6= LMG 21630T, 7=LMG 2162 T, 8=LMG 18378T From these results, fourteen strains were chosen to perform the first real-time PCR run (Pcichrc 2007-03-05): a) three P. cichorii strains with strong 230-300bp band for all three primer couples: LMG 1248, LMG 5054, LMG 2164 b) three P. cichorii strains with a weak 230-300bp band for all three primer couples: R- 25254, LMG 8401, LMG 5052 c) three Pseudomonas strains, other than P. cichorii, that gave a band close to the 230-300bp band: T - for primer couple F1-R1: P. pertucinogena LMG 1874 T - for primer couple F1-R1 and F2-R1: P. mediterranea LMG 23075 T - for primer couple F1-R1: P. rhizosphaerae LMG 21640 d) two Pseudomonas strains, other than P. cichorii, that also exhibited a 230-300bp band: P. T T orizyhabitans LMG 7040 for primer couple F1-R1 and P. citronellosis LMG 18378 for

both primer couples F1-R1 and F2-R1 e) P. cichorii type strain: LMG 2162T f) P. syringae type strain: LMG 1247 T g) the P. viridiflava type strain that exhibited a positive band for primer couples F2-R1 and T F1-Rprobe0 LMG 2352

For primer couples F1-R1 and F2-R2 optimisation of real-time PCR conditions was carried out, with SYBR Green I as the chosen fluorescent chemistry (see 4.2.1). To assess the ability of the TaqMan probe to bind on all P. cichorii strains, it was also tested as a primer in SYBR Green I assays (see 4.2.2).

5.2. REAL‐TIME PCR 5.2.1. SYBR GREEN I

Optimisation MgCl2 concentration a) Primer couple F1‐R1 (Pcichrc 2007‐03‐05, Pcichrc 2007‐03‐06 and Pcichrc 2007‐03‐6B) P. cichorii strains LMG 2162T and LMG 8401 are taken as representatives for the performed runs.

For strain LMG 8401 there is no reaction at 0.5mM MgCl2, at 1.5mM MgCl2 the curve is less steep and at 3.5 and 5mM MgCl2, the curves are levelled off (Fig. 9). For P. cichorii LMG T 2162 there is no reaction at 0.5Mm MgCl2 and at 3.5 and 5mM MgCl2 the curves are levelled off. At 1.5mM MgCl2 an amplification curve is observed with a higher Ct value. Melting curve analysis showed that this amplification curve is due to the presence of primer-dimers, which are due to aspecific amplification (Fig. 10 and Fig. 11). For both strains the amplification curves are optimal at 2.0-2.5mM MgCl2. All other strains in the assay, showed a similar profile (Addendum D). The non-target strains included in the runs (P. viridiflava LMG 2352T, P. mediterranea LMG 23075T, P. orizyhabitans LMG 7040T and P. syringae LMG 1247 T) showed a similar trend, but their Ct values were much higher (28, 40-31, 12). In Fig. 12, P. viridiflava LMG 2352T is taken as a representative for the non-target strains.

gg) Figure 9: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F1‐R1 and P. cichorii strain LMG 8401

hh) Figure 10: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F1‐R1 and P. cichorii strain LMG 2162T

ii) Figure 11: Melting curves of the optimisation of the MgCl2 concentration for primer couple F1‐R1 and P. cichorii strain LMG 2162T

jj) Figure 12: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F1‐R1 and P. syringae strain LMG 2352T b) Primer couple F2‐R1 (Pcichrc 2007‐03‐07A, Pcichrc 2007‐03‐7B) P. cichorii strains LMG 2162T and LMG 8401 are taken as representatives for the performed runs.

For both strains, there is no reaction at 0.5mM MgCl2 and 1.5mM MgCl2, at 3.5mM MgCl2 the curve is levelled off (Fig. 13 and Fig. 14). Furthermore, the fluorescence curves are optimal at 2-2.5mM MgCl2. All other strains in the assay showed a similar result. (Addendum E) The non-target strains included in the runs showed a similar trend, but their Ct values were in the range of 29,03 to 31,42 (data not shown, for similar results see Fig. 12).

kk) Figure 13: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F2‐R1 and P. cichorii strain LMG 8401

ll) Figure 14: Amplification curves of the optimisation of the MgCl2 concentration for primer couple F2‐R1 and P. cichorii strain LMG 2162T

In conclusion, a concentration of 2mM up to 2.5mM MgCl2 was the most optimal for both primer couples F1-R1 and F2-R1. These results are similar to the results obtained for the 16S rDNA genes of P. cichorii. Optimisation temperature‐time profile a) Optimisation of the annealing temperature (Pcichrc 2007‐03‐14A, Pcichrc 2007‐03‐14B, Pcichrc 2007‐03‐15A, Pcichrc 2007‐03‐15B) Further optimisation was performed at three different temperatures and at four different primer concentrations. By testing the effect of annealing temperature and primer concentration together, a possible coupled effect could have been revealed. As this was not the case, both are discussed separately.

P. cichorii strains LMG 2162T, LMG 8401 and R-25254 are taken as representatives for the target species (Fig. 15). Strains P. viridiflava LMG 2352T and P. orizyhabitans LMG 7040T are taken as representatives for the non-target organisms (Fig. 16). The assays at primer concentrations 0.25µM, 0.75µM and 1.00µM for all other strains can be found in Addendum F. Amplification of the P. cichorii hrc gene was most optimal at 62°C at primer concentrations of 0.5µM. Primer pair F2-R1 was the best primer pair at 62°C. At 60°C and 64°C, amplification curves were less steep and Ct values were higher in comparison to Ct values at 62°C.

For F1-R1 at 60°C and 62°C, Ct values were not consistent with the different primer concentrations and both runs did not yield reproducible results. An annealing temperature of 64°C led to Ct values between 29,90 and >41 for all strains (data not shown).

Figure 15: Amplification curves of the optimisation of the annealing temperature for primer couple F2-R1 (0.5µM) and three P. cichorii strains

mm) Figure 16: Amplification curves for the optimisation of the annealing temperature for primer couple F2‐R1 (0.5µM) and two non‐P. cichorii strains On the basis amplification curves in Fig. 16 one could conclude that 62°C is also an optimal annealing temperature for amplification of the non-target strains. However, the melting curve analysis (Fig. 17) of P. viridiflava LMG 2352T and P. orizyhabitans LMG 7040T for 62°C shows that the observed amplification is due to primer-dimers (aspecific amplification).

nn) Figure 17: Melting curves of the optimisation of the annealing temperature for primer couple F2‐R1 (0.5µM) and two non‐P. cichorii strains At 60°C, more melting peaks that were different from primer-dimer peaks were observed than at other temperatures as can be seen in Fig. 18-20. At 64°C results were suboptimal for P.cichorii when compared with 62°C. In all three figures (Fig. 18-20) four non P. cichorii strains (P. viridiflava LMG 2352T, P. mediterranea LMG 23075T, P. oryzihabitans LMG 7040T, P. trivialis LMG 21464T) and the negative control are included at the four tested primer concentrations (0.25µM, 0.50µM, 0.75µM, 1.00µM).

Figure 18: Melting curves of the optimisation of the annealing temperature: 60°C The figure includes several P. Cichorii strains, four non P. cichorii strains (P. viridiflava LMG 2352T, P. mediterranea LMG 23075T, P. oryzihabitans LMG 7040T, P. trivialis LMG 21464T) and the negative control

Figure 19: Melting curves of the optimisation of the annealing temperature: 62°C.The figure includes several P. cichorii strains, four non-P. cichorii strains (P. viridiflava LMG 2352T, P. mediterranea LMG 23075T, P. oryzihabitans LMG 7040T, P. trivialis LMG 21464T) and the negative control

oo) Figure 20: Melting curves of the optimisation of the annealing temperature: 64°C.The figure includes several P. cichorii strains, four non‐P. cichorii strains (P. viridiflava LMG 2352T, P. mediterranea LMG 23075T, P. oryzihabitans LMG 7040T, P. trivialis LMG 21464T) and the negative control It was concluded that 62°C was the most optimal temperature for annealing of the primers. b) Optimisation of the annealing and extension time For a non-target strain, a Ct value higher than 40 or a flat amplification curve is preferred. The non-target strains gave Ct values higher than 30 and some even showed melting curves in the range of the target. This reduced the analytical sensitivity or work range. In order to try to improve specificity, two assays with a shorter annealing time were performed. In addition, three assays were performed in which the annealing time was reduced in the first cycles and in which all other cycles had a normal annealing time of 20 seconds. The latter were also performed to improve specificity. Only the run Pcichrc 2007-03-26B (indicated with the grey box) could be compared with a similar run (with 20 seconds annealing time and 15 seconds extension time) (Table 21). pp) Table 21: Runs performed for the optimisation of annealing and extension time

Run Annealing time Result and processing Extension time Pcichrc 2007-03-16B 40 seconds Gave rise to flat amplification curves. 15 seconds Pcichrc 2007-03-26B 15 seconds No significant difference with 20 seconds 15 seconds annealing time and 15 seconds extension time Pcichrc 2007-03-27 10 seconds Gave rise to flat amplification curves. 15 seconds Pcichrc 2007-03-28B 5 cycli 20 seconds The insertion of two quantification rounds is 40 cycli 5 seconds possible in the LightCycler Software Program, but Pcichrc 2007-03-29 25 cycli 20 seconds such data can not be processed into one good 20 cycli 5 seconds single Excel graph. Pcichrc 2007-03-29B 15 cycli 20 seconds 30 cycli 5 seconds

As can be seen in Fig. 21, there is no significant difference between an annealing time of 20 seconds or one of 15 seconds. Thus, in subsequent assays an annealing time of 20 seconds was kept. P. cichorii strains LMG 2162T and R-25254 are representatives for the runs.

qq) Figure 21: Amplification curves of the optimisation of the annealing time for P. cichorii LMG 2162T and P. cichorii R‐25254 It was concluded that: (1) lower primer concentrations are not advisable because even at 40 seconds annealing time no amplification occurs, (2) an annealing time of 10 seconds probably is too short and (3) an annealing time of 20 seconds or 15 seconds does not yield much difference in result. In conclusion, an annealing time of 20 seconds was kept. The Roche LightCycler manual does mention that reduction of the annealing time can improve specificity, but gives 20 seconds as a standard setting. Optimisation of the primer concentration (Pcichrc 2007-03-12A, Pcichrc 2007-03-13A, Pcichrc 2007-03-12B, Pcichrc-2007-03-16A) Both primer pairs were tested at four different concentrations: 0.25µM, 0.50µM, 0.75µM and

1.00µM. Primer couple F1-R1 was also tested at lower concentrations: 0.10µM, 0.15µM, 0.20µM and 0.25µM. A 0.50µM concentration seemed to work best, whereas 0.25µM and 1.00µM respectively, were too low and too high. Though, the best suited concentration was not unequivocal for both primer pairs F1-R1 and F2-R1 (data not shown). For that reason, the real-time PCRs Pcichrc 2007-03-20 and Pcichrc 2007-03-21 were performed at different primer concentrations (See 5.2.1.2, Optimisation of annealing temperature).

Further optimisation

The initial optimisation showed that primer pair F2-R1 was the most optimal for detection of the P. cichorii hrc gene in this assay (Fig. 22). Ct values for P. cichorii with primer couple

F1-R1 are overall too high with the same parameters as for F2-R1.

rr) Figure 22: Comparison of primer couples F1‐R1 and F2‐R1 with the initial optimised PCR conditions ( P. cichorii strains R‐25254, LMG 2162T and LMG 8401) In previous runs, primer-dimer formation was observed. Since primer-dimer products are shorter than the target product, they melt at lower temperature and their presence is easily recognised by MCA. In order to try to avoid the presence and interference of primer-dimers in the amplification curves, fluorescence was measured at 83°C in run ‘Pcichrc 2007-03-20’.

This was a temperature just below the Tm of the product and above the Tm of the primer- dimers. It was expected that at that temperature, primer-dimers in the reaction mixture would melt apart before the SYBR Green I fluorescence signal of the target was acquired at each cycle. One would expect that for the strains with with primer-dimers in their melting curves, amplification curves would be more levelled off than before. This approach however did not result in such an effect (data not shown).

Once the MgCl2 concentration and annealing temperature for primer pair F2-R1 were optimised, the four primer concentrations were tested again (Pcichrc 2007-03-21). A concentration of 0.5µM (and also 0.75µM and 1.00µM) gave the best result. The lower primer concentration of 0.5µM was chosen. In theory, lower primer concentrations improve specificity, as annealing of the primers on non-target strains would decrease. P. cichorii strains R-25254 and LMG 8401 are representatives of the target species for these runs (Fig.23). The assays with the other strains included, can be found in Addendum G.

ss) Figure 23: Further optimisation of the primer concentration with primer couple F2‐R1 and P. cichorii strains LMG 2162T, R‐25254 and LMG 8401

After optimisation of the MgCl2, annealing and extension time and primer concentration, the target temperature of annealing was varied once more by raising it with 2°C to a temperature of 64°C (Pcichrc 2007-03-28) (Fig. 24). A target annealing temperature of 64°C though, raises the Ct values of P. cichorii: LMG 2162T from 12.47 to 12.86 and of P. cichorii R- 25254 from 12.90 to 17.31.

tt) Figure 24: Further optimisation of the annealing temperature by raising the temperature again up to 64°C for a range of Pseudomonas strains In conclusion, optimisation of the real-time PCR conditions yielded the results listed in Table 22. In subsequent real-time assays, these conditions were used accordingly. uu) Table 22: Optimised conditions after optimisation with SYBR Green I

Primers MgCl2 and primer Time-temperature profile

(End concentrations) Annealing Annealing time temperature Extension time MgCl2 F2-R1 2-2.5mM 62°C 20 seconds Primers 15 seconds 0.5 µM

Exclusivity (Pcichrc 2007-03-22A, Pcichrc 2007-03-22B, Pcichrc 2007-03-22C, Pcichrc 2007-03-26) The designed primers had to be tested for exclusivity, in other words their ability not to detect non-target organisms present. Four runs were performed, Fig. 25 is a combination of the four graphs that were processed in Excel. Two representatives for P. cichorii were chosen: LMG 2162T for run 2, 3 and 4 and LMG 8401 for run 1. The negative control and the non P. cichorii strains are visible at the right side of Fig. 25, all non P. cichorii strains used in the runs are listed in Addendum H. Most of the amplification curves at the right side of Fig. 25 are caused by primer-dimers.

vv) Figure 25: The exclusivity assay (amplification curves) The melting curves from the four runs can be found in Fig 26-29. Ct values for the non-target strains ranged from 29.18 up to 37.18 and most values were in between 31 and 34.

Figure 26:The exclusivity assay (melting curves) Pcichrc 2007-03-22A

ww) Figure 27: The exclusivity assay (melting curves) Pcichrc 2007‐03‐22B

xx)

yy) Figure 28: The exclusivity assay (melting curves) Pcichrc 2007‐03‐22C

zz) Figure 29: The exclusivity assay (melting curves) Pcichrc 2007‐03‐26

The strains that showed an indistinguishable melting peak from the one of P. cichorii were: Pcichrc 2007-03-22A: P. cichorii R-25254, P. cichorii R-25295, P. cichorii R-26430, P. cichorii R-31877, P. cichorii LMG 8401, P. cichorii LMG 2162T Pcichrc 2007-03-22B: P. cichorii LMG 5868, P. cichorii LMG 5052, P. cichorii LMG 2162T, P. cichorii LMG 5054, P; cichorii LMG 6771 Pcichrc 2007-03-22C: P. cichorii LMG 2162T, P. cichorii LMG 2164, P. cichorii LMG 5034, P; cichorii LMG 2163, P. cichorii LMG 1248 Pcichrc 2007-03-26: This run was performed two times, but the same aspecific products were observed. Only P. cichorii LMG 5052 shows the correct melting peak. For two strains, LMG 23197T (‘P. luti’) and LMG 21465T (P. poae), melting peaks without primer-dimer peaks and close to the melting peak of P. cichorii were observed in Pcichrc 2007-03-22A. Al other peaks observed were two primer-dimer peaks of the same height.

Inclusivity (Pcichrc 2007-04-16A) The designed primers had to be tested for inclusivity as well, in other words their ability to detect all strains from the target organism, P. cichorii. On the left side of Fig. 30, P. cichorii strains can be seen (Addendum I), on the right side the negative control, LMG 2152T and LMG 17764T, can be seen. The amplification curves of the inclusivity run showed a variation in Ct values, ranging from 9.926 to 14.63. This might be a consequence of small variations in OD1 solution of DNA that was used in this project.

aaa) Figure 30: The inclusivity assay (amplification curves)

The melting curves of the inclusivity run showed that there is a range in Tm from 87,64°C- 89,23°C for the P. cichorii strains (Fig. 31). This was expected, as the hrcRST genes are also

variable in sequence (black arrow Fig. 31). The primer-dimers seen in Fig. 31 only occur in the non-target strains, P. cichorii strains do not exhibit primer-dimer peaks.

bbb) Figure 31: The inclusivity assay (melting curves). ccc) The black arrow shows melting curve variation due to sequence variation

Analytical sensitivity (work range) a) Detection limit As with every new PCR protocol in microbiology, the sensitivity of the assay has to be carefully examined. To determine the lowest number of P. cichorii cells detectable by the assay, the real-time PCR experiments were performed on serial dilutions of DNA OD1 of P. cichorii type strain LMG 2162T and three P. cichorii representatives (R-25254, R-26430 and R-31877) of each of the three BOX-types previously demonstrated in the IWT project. This test had to be performed in triplicate, but during the course of testing the analytical sensitivity, some difficulties turned up. The runs not marked in grey (Table 23) showed very T high Ct values or no Ct values at all. Even the positive control (LMG 2162 OD1) did not give the expected amplification curve. That is the reason why first a small-scale comparison of two kits, the Sigma kit and the Roche kit, was performed (Pcichrc 2007-04-23B). The Sigma kit did not yield reliable results anymore. As a consequence, subsequent runs were performed with the Roche kit. ddd) Table 23: Runs performed to determine the assay’s analytical sensitivity

Run Kit Strains Results Pcichrc 2007-04-19A Sigma LMG 2162T, R-25254, R- Normal Ct values 26430, R-31877 Pcichrc 2007-04-19B Sigma LMG 2162T, R-25254, R- Abnormal Ct values (no Ct values at all or very 26430, R-31877 high Ct values)

Pcichrc 2007-04-19C Sigma LMG 2162T, R-25254, R- Abnormal Ct values ( mostly no Ct values at all 26430, R-31877 and a small number of very high Ct values), Pcichrc 2007-04-20A Sigma LMG 2162T, R-25254, R- Only Ct values of LMG 2162T normal 26430, R-31877 Pcichrc 2007-03-04 LMG 2162T, R-25254, R- Abnormal Ct values (no Ct values at all or very 23A 26430, R-31877 high Ct values) Pcichrc 2007-04-23B Sigma LMG 2162T Comparison of 2 kits due to previous problems and with Sigma kit. (also see 5.2.2) Roche Pcichrc 2007-04-24A Sigma LMG 2162T, R-25254 Abnormal Ct values (no Ct values at all or very high Ct values) Pcichrc-2007-04-26 Sigma LMG 2162T, R-25254 Abnormal Ct values (no Ct values at all or very high Ct values) Pcichrc 2007-04-27A Roche LMG 2162T, R-25254 Good run, processed in Excel Pcichrc 2007-04-30 Roche LMG 2162T, R-25254, R- Good run, processed in Excel 26430, R-31877 Pcichrc 2007-05-02A Roche LMG 2162T, R-25254, R- Good run, processed in Excel 31877 Pcichrc 2007-05-02B Roche LMG 2162T, R-25254, R- Good run, processed in Excel 31877

A close inspection of the amplification curves (Fig. 32, Fig. 34, Fig. 36) revealed a lower limit of detection of 102 cells for P. cichorii LMG 2162T, R-25254 and R-31877 for primer 1 0 couple F2-R1. The dilutions of 10 and 10 gave amplification curves, sometimes even with expected Ct values. However, the amplified product is the consequence of primer-dimers, as observed in the melting curves (Fig. 33, Fig. 35, Fig. 37). In this study, DNA templates of 7 OD1 were used. The assumption was made that an OD of 1 is equal to 10 cells, but this is only an estimate: ∗ A nucleotide is 330g/mole in size, in dsDNA that equals 660g/mole or 660Da. ∗ The amount of Da (or g/mole) per genome equivalent (or genome molecule) is 660g/mole multiplied with the genomesize. ∗ The complete genome of P. cichorii has not been sequenced yet, but the average genome size of other Pseudomonas species is ± 6000000bp (35). If one 9 applies the formula, the net result is: 660 x 6000000 = 3,96 x 10 .

∗ If one applies the formula, 1g = NA (Avogadro’s number)/ units (Dalton), on this example: 1g = 6,02214 x 1023/ 3,96 x 109 = 1,52 x1014

∗ In this case 100ng, which equals 2µL OD1 that was always used in this study, is then 1,52 x 107 (100ng = 1,52 x 107). 7 7 ∗ To actually use 10 cells or 1 x 10 , one should add 65,79ng or 1,316µL OD1.

Whether a detection limit of 102 cells (1,52 x 102 genomic equivalents per PCR reaction) is applicable for the detection of P. cichorii in lettuce samples in the field is still a subject for further research.

Figure 32:Amplification curves of a dilution series of P. cichorii LMG 2162T

Figure 33: Melting curves of a dilution series of P. cichorii LMG 2162T

Figure 34: Amplification curves of a dilution series of P. cichorii R-25254

Figure 35: Melting curves of a dilution series of P. cichorii R-25254

eee) Figure 36: Amplification curves of a dilution series of P. cichorii R‐31877

fff) Figure 37: Melting curves of a dilution series of P. cichorii R‐31877 b) Reproducibility 60

The run to test the analytical sensitivity was performed in triplicate to verify the reproducibility. Below, reproducibility, standard curves, average and standard deviation are discussed for the three strains in the three consecutive runs. The standard deviation and the error bars are measures for reproducibility. As can be observed in Fig. 38, 39 and 40, error bars are small, the standard deviations are low and the standard curves are straight lines crossing all intercepts. Thus, one can conclude that the results are reproducible (Table 24, 25 and 26).

ggg) Table 24: Reproducibility for P. cichorii LMG 2162T

LMG 2162T Dilution Run 1 Run 2 Run 3 Average St. deviation 107 13.3 13,69 14,77 13,92 0,425 106 17 17,65 19 17,88333 0,558333 105 20.94 21,47 23,04 21,81667 0,611667 104 24.21 24,7 25,98 24,96333 0,508333 103 28.86 28,02 29,14 28,67333 0,326667 102 31.95 30,88 29,99 30,94 0,505

hhh) Figure 38: Analytical sensitivity of the SYBR Green assay determined with serial dilutions of P. cichorii LMG T 2162 genomic DNA using primer couple F2‐R1. The standard curve is the linear regression line through the data point on a plot of Ct values versus the logarithm of sample concentration. Slope, Y‐intercept and mean squared error of the standard curve are given. iii) Table 25: Reproducibility for P. cichorii R‐25254

R-25254 Dilution Run 1 Run 2 Run 3 Average St. deviation 107 14,64 15,04 16,45 15,37667 0,715556 106 18,18 18,64 20,06 18,96 0,733333 105 21,86 21,67 23,25 22,26 0,66 104 24,95 24,92 26,01 25,29333 0,477778 103 28,51 29,06 29,11 28,89333 0,255556 102 31,66 28,02 31,57 30,41667 1,597778

jjj) Figure 39: Analytical sensitivity of the SYBR Green assay determined with serial dilutions of P. cichorii R‐25254 genomic DNA using primer couple F2‐R1. The standard curve is the linear regression line through the data point on a plot of Ct values versus the logarithm of sample concentration. Slope, Y‐intercept and mean squared error of the standard curve are given.

kkk) lll) Table 26: Reproducibility for P. cichorii R‐31877

R-31877 Dilution Run 1 Run 2 Run 3 Average St. deviation 107 14,74 14,29 15,46 14,83 0,42 106 18,24 18,15 19,22 18,53667 0,455556 105 22,16 21,83 23,04 22,34333 0,464444 104 25,44 25,67 26,47 25,86 0,406667 103 28,56 28,88 30,14 29,19333 0,631111 102 31,9 32,53 33,25 32,56 0,46

mmm) Figure 40: Analytical sensitivity of the SYBR Green assay determined with serial dilutions of P. cichorii R‐31877 genomic DNA using primer couple F2‐R1. The standard curve is the linear regression line through the data point on a plot of Ct values versus the logarithm of sample concentration. Slope, Y‐intercept and mean squared error of the standard curve are given. c) Efficiency Estimation of the efficiency of the reaction via classical ‘calibration dilution curve and slope calculation’ can be calculated by the following equation: E = 10(-1/SLOPE)-1 The efficiency of the PCR should be as close as possible to 100% meaning doubling of the amplicon at each cycle. This corresponds to a slope of 3.1 to 3.6 in the Ct versus dilution standard curve. A number of variables can affect the efficiency of the PCR. These factors can include length of the amplicon, presence of inhibitors, secondary structure and primer design. If efficiency is < 0.90, the real-time PCR should probably be further optimized. (36, 37) The results for the efficiencies for the runs for analytical sensitivity can be found in Table 27. The efficiency for the assay was calculated separately for P. cichorii LMG 2162T and two P. cichorii strains isolated from lettuce, R-25254 and R-31877. Although for R-31877, the efficiency was only 91,64%, the result for efficiency were overall satisfactory. nnn) Table 27: Efficiencies of runs for analytical sensitivity

Strain Slope Efficiency LMG 2162T 3.4462 95,298% R-25254 3.0867 111,191% R-31877 3.5468 91,64%

Analytical specificity (Pcichrc 2007-04-27B, Pcichrc 2007-05-03, Pcichrc 2007-05-7A, Pcichrc 2007-05-8B) As mentioned in the previous paragraph, the sensitivity of any assay has to be examined carefully. This is also true for the specificity. The primers F2-R1 must detect the correct organisms in the presence of non-target organisms, in other words: there should not be an effect of non-target organisms in a multi-component DNA sample. To determine the assay’s specificity, real-time PCR experiments were performed on a serial 7 2 tenfold dilutions (10 Æ 10 , the lower limit of the working range) of DNA OD1 of P. cichorii LMG 2162T (DNA A) and DNA B. DNA B was successively: P. mediterranea LMG 23075 T, P. poae LMG 21465T and DNA from approximately 20g of a lettuce head. During testing, it seemed that 107 genome equivalents of non-target DNA resulted in a reduction of end-fluorescence. That is why a fourth assay (Pcichrc 2007-05-8B) was performed with a dilution series of P. poae LMG 21465T and P. mediterranea LMG 23075T, 8 7 starting from 10 cells (equal to OD10) instead of 10 cells (equal to OD1). P. poae LMG 21465T, P. mediterranea LMG 23075T and the DNA isolated from lettuce showed similar results for al dilutions (Addendum J). Here only 108, 107 and 102 for strain P. mediterranea LMG 23075T are shown (Fig. 41 and 42). For strains P. mediterranea LMG 23075T and P. poae LMG 21456T, 107 genome equivalents of non-target DNA resulted in a reduction of end-fluorescence, but it had no effect on the Ct values. For 108 genome equivalents this concentration-effect was even more pronounced. For the DNA isolated from lettuce, the concentration-effect could not be visualised, because the starting dilution only 6 contained 10 genome equivalents. The melting curves of a dilution of 102 and 108 of P. cichorii LMG 2162T, exhibited no effect of the excess of non-target DNA, P. mediterranea LMG 23075T, although this strain was chosen because it showed a different melting peak than P. cichorii (Fig.43).

Figure 41: Analytical specificity for strain P. mediterranea LMG 23075T at a dilution of 108 and 107 (amplification curves)

Figure 42: Analytical specificity for strain P. mediterranea LMG 23075T at a dilution of 102 (amplification curves)

ooo) Figure 43: Analytical specificity for strain P. mediterranea LMG 23075T at a dilution of 102 and 108 In conclusion, an OD of 1 might by the highest concentration to get an accurate detection of P. cichorii. In the LightCycler System, the accumulation of amplicons can be monitored using either SYBR Green I dye, which binds to any double stranded DNA or a sequence-specific fluorogenic probe. The main goal of this study the main interest was to detect the presence of P. cichorii and to make a start for its quantification. Therefore, it was decided to start with the simpler and less expensive SYBR Green I format with Tm analysis of the PCR product The possibility that a fluorogenic probe might have a higher sensitivity, was also considered, hence the development of a TaqMan probe (see paragraph 5.2.3).

5.2.2. ROCHE‐ AND SIGMA‐ KIT FOR REAL‐TIME PCR

During the course of this study, two SYBR Green I kits were used, a kit purchased from Sigma (SYBR® Green JumpStart TM Taq ReadyMixTM) and a kit purchased from Roche (LightCycler® Faststart DNA Master SYBR Green I with Hot Start). At the start of the development of the assay the Sigma-kit was used. It functioned well up to the moment the runs for analytical sensitivity were performed. To test analytical sensitivity, three runs were carried out subsequently on the same day. The first run (Pcichrc 2007-04- 19A) exhibited the expected results. Although all parameters and products used were the same, the second run and third run exhibited aberrant results (Pcichrc 2007-04-19B and Pcichrc 2007-04-19C). The cause of failure of the Sigma-kit was unknown. In order to try to solve the problem some parameters were changed, such as using new enzyme or MgCl2 and preparing new primer solutions. The problem however did not disappear and the Sigma-kit was replaced by the Roche-kit. The Roche kit seemed stable and was used in subsequent runs.

5.2.3. DEVELOPMENT AND PRELIMINARY TESTING OF A TAQMAN PROBE

(Pcichrc 2007-03-09, Pcichrc 2007-04-17, Pcichrc 2007-04-19D, Pcichrc 2007-04-20B) A TaqMan probe was developed during this study. A reverse probe (Rprobe) and a forward probe (Fprobe) were both tested as primer to be sure of the proper binding to the target. The

F-probe was tested in combination with primer R1 and the R-probe was tested with primer F1, before final ordering the sequence as TaqMan probe. Reverse probe

In a first step, a reverse probe Rprobe0 was tested in combination with primer F1. It gave a positive signal for P. cichorii LMG 2162T in gradient PCR and in regular PCR for all P. cichorii strains. In one of two similar real-time PCR assays however, not all P. cichorii strains were detected (Fig. 44, the other strains tested in run Pcichrc 2007-03-09 can be found in

Addendum K). Hence, Rprobe0 was adapted into a new, degenerate Rprobe based on sequence info and tested in combination with primer F1 (Fig. 45).

Figure 44: Amplification curves of F1-Rprobe0 for five P. cichorii strains that were detected(Pcichrc 2007-03-09)

ppp) Figure 45: Amplification curves for F1‐Rprobe for P. cichorii (Pcichrc 2007‐04‐17)

F1-Rprobe was tested again (Fig. 46) with extra P. cichorii strains that gave high Ct-values (± 30) in a previous run (data not shown). The Ct values of those strains dropped (10.97-16.07).

qqq) Figure 46: Amplification curves for F1‐Rprobe with additional P. cichorii strains (Pcichrc 2007‐04‐19D) Forward probe

At 60°C the combination of F1-Rprobe seemed to work better than the combination Fprobe-

R1, since the end fluorescence for F1-Rprobe reached a value of ± 45 in comparison to one of

± 27 for Fprobe-R1 and since the amplification curves were flattened out (Fig. 45 and 47).

Fprobe-R1 was tested again (Fig. 48) at 65°C, as the Tm of Fprobe is 64,9°C. Whereas in run Pcichrc 2007-04-17, a lot of the Ct values were >30, in run Pcichrc 2007-04-20B Ct values ranged between 10.99 and 13.99.

rrr) Figure 47: Amplification curves for Fprobe‐R1 for P. cichorii strains (Pcichrc 2007‐04‐17)

sss) Figure 48: Amplification curves for Fprobe‐R1 at 65°C for 8 P. cichorii strains

6. CONCLUDING REMARKS For the detection of P. cichorii, the causal agent of midrib rot in lettuce, a real-time PCR assay was developed and optimised. The use of primer-pair F2-R1 in a concentration of 0.5µM and a MgCl2 concentration between 2 and 2.5mM revealed the most optimal results. A final PCR program can be found in Table 28. ttt) Table 28: Lightcycler amplification and melting curve protocol for real‐time PCR detection of P. cichorii

Program No. of Target temp Hold time Temp Transition Fluorescence cycles (°C) (s) rate (°C/s) acquisition mode Polymerase activation 1 95 600 20 None Three-step PCR 45 Denaturation 95 10 20 None Amplification 62 20 20 None Extension 72 15 20 None Three-step melting curve 1 Denaturation 95 0 20 None Holding 65 15 20 None Melting 95 0 0.1 Continuous Cooling 1 40 30 20 None

Roche claims that PCR results with the LightCycler Instrument could be available within 20- 60 minutes (39). In this study, once DNA was extracted from suitable specimens and reaction mixtures were completed, the assay allowed amplification and detection to take place about 1h. So, the LightCycler approach simplified the workflow by automation. It also reduced the assay’s turn around time compared to conventional PCR with analysis on agarose-gel. In addition, since amplification in the LightCycler Instrument is performed in a closed system, the chance of product contamination frequently associated with post-PCR amplicon manipulation is definitely decreased. SYBR Green I real-time PCR was successfully used to detect and quantitate P. cichorii targeting the hrcRST genes. This SYBR Green I real-time PCR was easier to develop than a probe-based assay. The disadvantage was that SYBR Green I dye could bind to any dsDNA, including primer-dimers. Primer-dimer formation was observed in almost all real-time PCR runs. If primer-dimers are formed, the fluorescence signal from the primer-dimer amplicons may superimpose on that of the desired target. Specificity though could be improved by melting curve analysis (MCA) of the target DNA and of the primer-dimers. To detect primer-dimer formation MCA was performed, as discussed above. Since primer- dimer products are shorter than the target product, they melt at lower temperature and their presence is easily recognised. So, fluorescence from SYBR Green I binding to primer-dimers did not pose a problem due to the lower Tm, which was several degrees below that of the

specific product. To avoid this problem in the future, development of a real-time PCR assay on the hrcRST genes with a TaqMan probe might be an alternative. A further reason to develop a TaqMan probe assay, is that it will probably lower the detection-limit to < 152 (1.52 x 102) genome equivalents. The real-time PCR SYBR Green I assay developed in this study, showed a high specificity and reproducibility. The usability of this SYBR Green I assay in the field is not clear yet, because further research on a lower detection limit still is necessary. The detection limit might depend on the sample. Especially in two cases: (1) when infection in the lettuce heads is latent and therefore, not visible and (2) when the sample is taken from water. A small amount of P. cichorii DNA present in a large amount of water, might easily be missed when a water sample is taken. Although a water sample can be pre-cultured on a liquid semi-selective medium, the possibility of quantification and determination of specificity is lost in that way. Pre- concentrating the water sample could also be an alternative to subsequently extract more DNA.

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LIST WITH USED ABBREVIATIONS

BCCM-LMG Laboratorium voor Microbiologie Gent Ct treshold Cycle Cp crossing point dNTPs deoxyNucleotideTriPhosphates ds double stranded DNA DeoxyriboNucleic Acid EDTA Ethyleen Di-amine Tetra –Acetate EtBr EthidiumBromide FRET Fluorescence Resonance Energy Transfer GES Guanidinium-thiocyanate-EDTA-Sarkosyl Hrp Hypersensitive response and pathogenicity Hrc Hypersensitive response and conserved IWT Instituut voor de aanmoediging van innovatie door Wetenschap en Technologie in Vlaanderen LED Light Emitting Diode MQ milliQ MCA Melting Curve Analysis NCPPB National collection of Plant-Pathogenic Bacteria OD Optical Density PAIs Pathogenicity Islands PCR Polymerase Chain Reaction rRNA ribosomal RiboNucleic Acid RS ReSupension buffer Ss single stranded Taq Thermus aquaticus TSA Tryptone Soy Agar TE Tris-EDTA bufffer TAE Tris-Acetic EDTA TBE Tris-Borate EDTA UV Ultra Violet

ADDENDUM

Addenda

Addendum A List of bacterial strains used in this study Addendum B Results gradient PCR Addendum C Real-time PCR Mastermixes used in this study

Addendum D Optimisation MgCl2 concentration for F1-R1: strains other than LMG 2162T and LMG 8401

Addendum E Optimisation MgCl2 concentration for F2-R1: strains other than LMG 2162T and LMG 8401 Addendum F Optimisation annealing temperature at 0,25µM; 0,75µM and 1,00µM Addendum G Further optimisation primer concentrations Addendum H Non P. cichorii strains used in the exclusivity assays Addendum I P. cichorii strains used in the inclusivity assay Addendum J Analytical specificity, all dilutions of the three tested strains

Addendum K TaqMan probe, real-time PCR with F1-Rprobe0

Addendum A: List of bacterial strains used in this work

Species Code Origin or reference Pseudomonas abietaniphila LMG 20220T BCCM-LMG collectiona Pseudomonas aeruginosa LMG 1242T BCCM-LMG collection Pseudomonas agarici LMG 2112T BCCM-LMG collection Pseudomonas alcaligenes LMG 1224 T BCCM-LMG collection Pseudomonas alcaliphila LMG 23134 T BCCM-LMG collection Pseudomonas amygdali LMG 13184 T BCCM-LMG collection Pseudomonas anguilliseptica LMG 21629 T BCCM-LMG collection Pseudomonas antarctica LMG 22709 T BCCM-LMG collection Pseudomonas argentinensis LMG 22563 T BCCM-LMG collection Pseudomonas asplenii LMG 21749 T BCCM-LMG collection Pseudomonas aurantiaca LMG 21630 T BCCM-LMG collection Pseudomonas azotoformans LMG 21611 T BCCM-LMG collection Pseudomonas balearica LMG 18376 T BCCM-LMG collection Pseudomonas beteli LMG 978 T BCCM-LMG collection Pseudomonas borbori LMG 23199 T BCCM-LMG collection Pseudomonas boreopolis LMG 979 T BCCM-LMG collection Pseudomonas brassicacearum LMG 21623 T BCCM-LMG collection Pseudomonas brenneri LMG 23068 T BCCM-LMG collection Pseudomonas cannabina LMG 5096 T BCCM-LMG collection Pseudomonas caricapapayae LMG 2152 T BCCM-LMG collection Pseudomonas chloritidismutans LMG 23064 T BCCM-LMG collection Pseudomonas cichorii LMG 2162 T BCCM-LMG collection LMG 2164 BCCM-LMG collection LMG 2165 BCCM-LMG collection LMG 2166 BCCM-LMG collection LMG 1248 BCCM-LMG collection LMG 2163 BCCM-LMG collection LMG 5002 BCCM-LMG collection LMG 5034 BCCM-LMG collection LMG 5035 BCCM-LMG collection LMG 5052 BCCM-LMG collection LMG 5053 BCCM-LMG collection LMG 5054 BCCM-LMG collection LMG 5055 BCCM-LMG collection LMG 5056 BCCM-LMG collection LMG 5869 BCCM-LMG collection LMG 5868 BCCM-LMG collection LMG 8401 BCCM-LMG collection LMG 6771 BCCM-LMG collection R-25295 ILVO-PGBb R-35806 or 83-1 Articlec R-25315 IWT project 030848d R-26430 IWT project 030848d R-26451 IWT project 030848d R-26431 IWT project 030848d R-25254 IWT project 030848d R-27168 IWT project 030848d R-28087 IWT project 030848d

R-29002 IWT project 030848d R-31877 IWT project 030848d Pseudomonas citronellolis LMG 18378 T BCCM-LMG collection Pseudomonas congelans LMG 21466 T BCCM-LMG collection Pseudomonas coronafaciens LMG 13190 T BCCM-LMG collection Pseudomonas corrugate LMG 2172T BCCM-LMG collection Pseudomonas constantini LMG 22119 T BCCM-LMG collection Pseudomonas extremorientalis LMG 19695 T BCCM-LMG collection Pseudomonas flavescens LMG 18387 T BCCM-LMG collection Pseudomonas fluorescens LMG 1794 T BCCM-LMG collection Pseudomonas fragi LMG 2191 T BCCM-LMG collection Pseudomonas LMG 19851 T BCCM-LMG collection frederiksbergensis Pseudomonas fulva LMG 11722 T BCCM-LMG collection Pseudomonas fuscovaginae LMG 2158 T BCCM-LMG collection Pseudomonas geniculata LMG 2195 T BCCM-LMG collection Pseudomonas gessardii LMG 21604 T BCCM-LMG collection Pseudomonas graminis LMG 21661 T BCCM-LMG collection Pseudomonas hibiscicola LMG 980 T BCCM-LMG collection Pseudomonas indica LMG 23066 T BCCM-LMG collection Pseudomonas jessenii LMG 21605 T BCCM-LMG collection Pseudomonas jinjuensis LMG 21316 T BCCM-LMG collection Pseudomonas kilonensis LMG 21624 T BCCM-LMG collection Pseudomonas koreensis LMG 21318 T BCCM-LMG collection Pseudomonas libanensis LMG 21606 T BCCM-LMG collection Pseudomonas lini LMG 21625 T BCCM-LMG collection Pseudomonas lutea LMG 21974 T BCCM-LMG collection Pseudomonas luteola LMG 7041 T BCCM-LMG collection Pseudomonas mandelii LMG 21607 T BCCM-LMG collection Pseudomonas mediterranea LMG 23075 T BCCM-LMG collection Pseudomonas mendocina LMG 1223 T BCCM-LMG collection Pseudomonas mephitica LMG 21632 T BCCM-LMG collection Pseudomonas migulae LMG 21608 T BCCM-LMG collection Pseudomonas monteilii LMG 21609 T BCCM-LMG collection Pseudomonas mosselii LMG 21539 T BCCM-LMG collection Pseudomonas mucidolens LMG 2223 T BCCM-LMG collection Pseudomonas nitroreducens LMG 21614 T BCCM-LMG collection Pseudomonas oleovorans LMG 2229 T BCCM-LMG collection Pseudomonas orientalis LMG 23660 T BCCM-LMG collection Pseudomonas oryzihabitans LMG 7040 T BCCM-LMG collection Pseudomonas palleroniana LMG 23076 T BCCM-LMG collection Pseudomonas peli LMG 23201T BCCM-LMG collection Pseudomonas pertucinogena LMG 1874 T BCCM-LMG collection Pseudomonas pictorum LMG 981 T BCCM-LMG collection Pseudomonas plecoglossicida LMG 21750 T BCCM-LMG collection Pseudomonas poae LMG 21465 T BCCM-LMG collection Pseudomonas LMG 1225 T BCCM-LMG collection pseudoalcaligenes Pseudomonas psychrotolerans LMG 21977 T BCCM-LMG collection Pseudomonas putida LMG 2257 T BCCM-LMG collection Pseudomonas resinovorans LMG 2274 T BCCM-LMG collection Pseudomonas rhizosphaera LMG 21640 T BCCM-LMG collection Pseudomonas rhodesiae LMG 17764 T BCCM-LMG collection

Pseudomonas salomonii LMG 22120 T BCCM-LMG collection Pseudomonas savastanoi LMG 2209 T BCCM-LMG collection Pseudomonas straminea LMG 21615 T BCCM-LMG collection Pseudomonas stutzeri LMG 11199 T BCCM-LMG collection Pseudomonas syringae LMG 1247 T BCCM-LMG collection Pseudomonas teatrolens LMG 2336 T BCCM-LMG collection Pseudomonas tremae LMG 22121 T BCCM-LMG collection Pseudomonas trivialis LMG 21464 T BCCM-LMG collection Pseudomonas tolaasii LMG 2342 T BCCM-LMG collection Pseudomonas umsongensis LMG 21317 T BCCM-LMG collection Pseudomonas vancouverensis LMG 20222 T BCCM-LMG collection Pseudomonas viridiflava LMG 2352 T BCCM-LMG collection R-35807 or LP23.1a Articlec R-35808 or ME3.1b Articlec R-35809 or PNA3.3a Articlec R-35810 or RMX23.1a Articlec R-35811 or RMX3.1b Articlec a. BCCM-LMG bacteria collection: Belgian Coordinated Collections of Micro-organisms-Laboratorium voor Microbiologie Gent, Belgium b. ILVO-PGB: Instituut voor Landbouw en Visserijonderzoek-Plant en Gewas Bescherming, Merelbeke, Belgium c. Araki et al.(2006). Presence/absence polymorphism for alternative pathogenicity islands in Pseudomonas viridiflava, a pathogen of Arabidopsis. PNAS 103 (15): 5887-5892 d. Calus A, Maes M, Höfte M, De Vos P (2004-2006). Karakterisering, ecologie en epidemiologie van pseudomonaden bij bladgroenten. Jaarverslag 2

Addendum C: Real-time PCR Mastermixes used in this study

Mix A Mix L 10µL E+ 10µL E+ 2µL F (10µM) 2µL F (2.5µM) 2µL R (10µM) 2µL R (2.5µM) 4µL MgCl2 (25mM) 2µL MgCl2 (25mM) 2µL MQ Mix B Mix M 10µL E+ 10µL E+ 2µL F (10µM) 2µL F (5µM) 2µL R (10µM) 2µL R (5µM) 1µL MgCl2 (10mM) 2µL MgCl2 (25mM) 3µL MQ 2µL MQ Mix C Mix N 10µL E+ 10µL E+ 2µL F (10µM) 2µL F (7.5µM) 2µL R (10µM) 2µL R (7.5µM) 3µL MgCl2 (10mM) 2µL MgCl2 (25mM) 1µL MQ 2µL MQ Mix D Mix O 10µL E+ 10µL E+ 2µL F (10µM) 2µL F (10µM) 2µL R (10µM) 2µL R (10µM) 2µL MgCl2 (25mM) 2µL MgCl2 (25mM) 2µL MQ 2µL MQ Mix E Mix P 10µL E+ 10µL E+ 2µL F (10µM) 2µL F (1.0µM) 2µL R (10µM) 2µL R (1.0µM) 2.8µL MgCl2 (25mM) 4µL MgCl2 (10mM) 1.2µL MQ Mix F Mix Q 10µL E+ 10µL E+ 2µL F (10µM) 2µL F (1.5µM) 2µL R (10µM) 2µL R (1.5µM) 4µL MgCl2 (10mM) 4µL MgCl2 (10mM) Mix G Mix R 10µL E+ 10µL E+ 2µL F (2.5µM) 2µL F (2.0µM) 2µL R (2.5µM) 2µL R (2.0µM) 4µL MgCl2 (10mM) 4µL MgCl2 (10mM) Mix H Mix S 10µL E+ 10µL E+ 2µL F (5µM) 2µL F (2.5µM) 2µL R (5µM) 2µL R (2.5µM) 4µL MgCl2 (10mM) 4µL MgCl2 (10mM) Mix I Mix T(Roche) 10µL E+ 2µL Mastermix 2µL F (7.5µM) 2µL F (5µM) 2µL R1 (7.5µM) 2µL R (5µM) 4µL MgCl2 (10mM) 1µL MgCl2 (10mM) 11µL MQ Mix J Mix U (Roche) 10µL E+ 2µL Mastermix 2µL F (10µM) 2µL F (5µM) 2µL R (10µM) 2µL R (5µM) 4µL MgCl2 (10mM) 1µL MgCl2 (25mM) 9µL MQ Mix K 10µL E+ 2µL F2 (5µM) 2µL R1 (5µM) 2µL MgCl2 (25mM) 2µL MQ

T Addendum D: Optimisation MgCl2 concentration for F1-R1: strains other than LMG 2162 and LMG 8401

Figure D1: Optimisation MgCl2 concentration : MgCl2 concentration of 0.5mM

Figure D2: Optimisation MgCl2 concentration : MgCl2 concentration of 1,5mM

Figure D3: Optimisation MgCl2 concentration : MgCl2 concentration of 2mM

Figure D4: Optimisation MgCl2 concentration: MgCl2 concentration of 2,5mM

Figure D5: Optimisation MgCl2 concentration: MgCl2 concentration of 3,5mM

Figure D6: Optimisation MgCl2 concentration: MgCl2 concentration of 5mM

T Addendum E: Optimisation MgCl2 concentration for F2-R1: strains other than LMG 2162 and LMG 8401

Figure E1: Optimisation MgCl2 concentration for F2-R1: MgCl2 concentration of 0,5mM

Figure E2: Optimisation MgCl2 concentration for F2-R1: MgCl2 concentration of 1,5mM

Figure E3: Optimisation MgCl2 concentration for F2-R1: MgCl2 concentration of 2,5mM

Figure E3: Optimisation MgCl2 concentration for F2-R1: MgCl2 concentration of 3,5mM

Addendum F: Optimisation annealing temperature

Figure F1: Optimisation annealing temperature: 60°C and 0,25µM primers

Figure F2: Optimisation annealing temperature: 62°C and 0,25µM primers

Figure F3: Optimisation annealing temperature: 64°C and 0,25µM primers

Figure F4: Optimisation annealing temperature: 60°C and 0,75µM primers

Figure F5: Optimisation annealing temperature: 62°C and 0,75µM primers

Figure F6: Optimisation annealing temperature: 64°C and 0,75µM primers

Figure F7: Optimisation annealing temperature: 60°C and 1,00µM primers

Figure F8: Optimisation annealing temperature: 62°C and 1,00µM primers 87

Figure F9: Optimisation annealing temperature: 64°C and 1,00µM primers Addendum G: Final optimisation primer concentrations

Figure E1: Optimisation primer concentrations: 0,25µM

Figure E2: Optimisation primer concentrations: 0,50µM

Figure E3: Optimisation primer concentrations: 0,75µM

Figure E4: Optimisation primer concentrations: 1,00µM

Addendum H: Non P. cichorii strains used in the exclusivity assay

LMG 2158, LMG 23134, LMG 23068, LMG 23066, LMG 2195 T, LMG 18387 T, LMG 18376, LMG 19695, LMG 19851, LMG 20220, LMG 2172, LMG 13190 T, LMG 1794, LMG 2229, LMG 5096 T, LMG 1225 T, LMG 22709 T, LMG 11199 T, LMG 13184, LMG 21614 T, LMG 23076 T, LMG 18378 T, LMG 1874 T, LMG 981 T, LMG 7040, LMG 23075 T, LMG 2352 T, LMG 2323 T, LMG 21464 T, LMG 11722 T, R-20821, LMG 21539 T, LMG 17764, T T T T T T LMG 21604 , LMG 21605 , LMG 21608 , LMG 20222 , LMG 21606 , LMG 2152 , T T T T LMG 21318 , LMG 23197, LMG 21611 , R-20805, LMG 21465 , LMG21750 , LMG 21609 T, LMG 21632 T, LMG 2158 T, LMG 21974 T, LMG 21626 T, LMG 21977 T, LMG 21661 T, LMG 2191 T, LMG 21624 T, LMG 21316 T, LMG 21317 T, LMG 21466 T, LMG 22120 T, LMG 21607 T, LMG 21749 T, LMG 21623 T, LMG 21629, LMG 21615 T, LMG 21630 T, LMG 2342 T, LMG 22563 T, LMG 23064 T, LMG 2274 T, LMG 2209 T, LMG 22119 T, LMG 22121 T, LMG 1212 T, LMG 2257 T, LMG 1223, LMG 1224 T, LMG 1242 T, LMG 1247 T, LMG 978 T, LMG 979 T, LMG 980 T, LMG 23075 T, LMG 2336 T, LMG 21640 T and LMG 7041.

Addendum I: P. cichorii strains used in the inclusivity assay LMG 1248, LMG 2162T, LMG 2163, LMG 2164, LMG 2165, LMG 2166, LMG 5002, LMG 5034, LMG 5035, LMG 5052, LMG 5053, LMG 5054, LMG 5055, LMG 5056, LMG 5868, LMG 5869, LMG 6771, R-35806, LMG 8401, R-25254, R-25295, R-31877, R-26431, R- 26451, R-25315, R-26430, R-28087, R-29002 and R-27168

Addendum J: Analytical specificity, all dilutions of the three tested strains

Figure J1: Analytical specificity: 107 LMG 2162T in the presence of LMG 23075T

Figure J2: Analytical specificity: 106 LMG 2162T in the presence of LMG 23075T

Figure J3: Analytical specificity: 105 LMG 2162T in the presence of LMG 23075T

Figure J4: Analytical specificity: 104 LMG 2162T in the presence of LMG 23075T

Figure J5: Analytical specificity: 103 LMG 2162T in the presence of LMG 23075T

Figure J6: Analytical specificity: 102 LMG 2162T in the presence of LMG 23075T

Figure J7: Analytical specificity: 107 LMG 2162T in the presence of LMG 21465T

Figure J8: Analytical specificity: 106 LMG 2162T in the presence of LMG 21465T

Figure J9: Analytical specificity: 105 LMG 2162T in the presence of LMG 21465T

Figure J10: Analytical specificity: 104 LMG 2162T in the presence of LMG 21465T

Figure J11: Analytical specificity: 103 LMG 2162T in the presence of LMG 21465T

Figure J12: Analytical specificity: 102 LMG 2162T in the presence of LMG 21465T

Figure J13: Analytical specificity: 106 LMG 2162T in the presence of DNA isolated from lettuce

Figure J14: Analytical specificity: 105 LMG 2162T in the presence of DNA isolated from lettuce

Figure J15: Analytical specificity: 104 LMG 2162T in the presence of DNA isolated from lettuce 95

Figure J16: Analytical specificity: 103 LMG 2162T in the presence of DNA isolated from lettuce

Figure J17: Analytical specificity: 102 LMG 2162T in the presence of DNA isolated from lettuce

Addendum K: TaqMan probe, real-time PCR with F1-Rprobe0