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

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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 2 3 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 5 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) 6 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 7 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.
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    Wild apple-associated fungi and bacteria compete to colonize the larval gut of an invasive wood-borer Agrilus mali in Tianshan forests Tohir Bozorov ( [email protected] ) Xinjiang Institute of Ecology and Geography https://orcid.org/0000-0002-8925-6533 Zokir Toshmatov Plant Genetics Research Unit: Genetique Quantitative et Evolution Le Moulon Gulnaz Kahar Xinjiang Institute of Ecology and Geography Daoyuan Zhang Xinjiang Institute of Ecology and Geography Hua Shao Xinjiang Institute of Ecology and Geography Yusufjon Gafforov Institute of Botany, Academy of Sciences of Uzbekistan Research Keywords: Agrilus mali, larval gut microbiota, Pseudomonas synxantha, invasive insect, wild apple, 16S rRNA and ITS sequencing Posted Date: March 16th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-287915/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/17 Abstract Background: The gut microora of insects plays important roles throughout their lives. Different foods and geographic locations change gut bacterial communities. The invasive wood-borer Agrilus mali causes extensive mortality of wild apple, Malus sieversii, which is considered a progenitor of all cultivated apples, in Tianshan forests. Recent analysis showed that the gut microbiota of larvae collected from Tianshan forests showed rich bacterial diversity but the absence of fungal species. In this study, we explored the antagonistic ability of gut bacteria to address this absence of fungi in the larval gut. Results: The results demonstrated that gut bacteria were able to selectively inhibit wild apple tree-associated fungi. However, Pseudomonas synxantha showed strong antagonistic ability, producing antifungal compounds. Using different analytical methods, such as column chromatography, mass spectrometry, HPLC and NMR, an antifungal compound, phenazine-1-carboxylic acid (PCA), was identied.
  • (12) United States Patent (10) Patent No.: US 7476,532 B2 Schneider Et Al

    (12) United States Patent (10) Patent No.: US 7476,532 B2 Schneider Et Al

    USOO7476532B2 (12) United States Patent (10) Patent No.: US 7476,532 B2 Schneider et al. (45) Date of Patent: Jan. 13, 2009 (54) MANNITOL INDUCED PROMOTER Makrides, S.C., "Strategies for achieving high-level expression of SYSTEMIS IN BACTERAL, HOST CELLS genes in Escherichia coli,” Microbiol. Rev. 60(3):512-538 (Sep. 1996). (75) Inventors: J. Carrie Schneider, San Diego, CA Sánchez-Romero, J., and De Lorenzo, V., "Genetic engineering of nonpathogenic Pseudomonas strains as biocatalysts for industrial (US); Bettina Rosner, San Diego, CA and environmental process.” in Manual of Industrial Microbiology (US) and Biotechnology, Demain, A, and Davies, J., eds. (ASM Press, Washington, D.C., 1999), pp. 460-474. (73) Assignee: Dow Global Technologies Inc., Schneider J.C., et al., “Auxotrophic markers pyrF and proC can Midland, MI (US) replace antibiotic markers on protein production plasmids in high cell-density Pseudomonas fluorescens fermentation.” Biotechnol. (*) Notice: Subject to any disclaimer, the term of this Prog., 21(2):343-8 (Mar.-Apr. 2005). patent is extended or adjusted under 35 Schweizer, H.P.. "Vectors to express foreign genes and techniques to U.S.C. 154(b) by 0 days. monitor gene expression in Pseudomonads. Curr: Opin. Biotechnol., 12(5):439-445 (Oct. 2001). (21) Appl. No.: 11/447,553 Slater, R., and Williams, R. “The expression of foreign DNA in bacteria.” in Molecular Biology and Biotechnology, Walker, J., and (22) Filed: Jun. 6, 2006 Rapley, R., eds. (The Royal Society of Chemistry, Cambridge, UK, 2000), pp. 125-154. (65) Prior Publication Data Stevens, R.C., “Design of high-throughput methods of protein pro duction for structural biology.” Structure, 8(9):R177-R185 (Sep.