Some Molecular Techniques Based on Polymerase Chain Reaction in Detection of Plant Pathogen Fungi

Öz

Fungi are an important group of plant pathogens and cause devastating losses in cultivated plants. Accurate and early detection of plant pathogens is the first important step for reducing yield losses caused by the pathogens and developing the effective disease control methods. The characteristics used in classical identification of fungal pathogens are very variable and these methods based on the morphological characteristics are time consuming and require taxonomical expertise. Thus, many molecular techniques have been developed for the identification of determination of plant pathogens and used widely in surveys, epidemiological studies, plant quarantine, seed certification, breeding programs, and fungicide resistance. The aim of this study was to provide detailed information about routinely used developed molecular techniques based on PCR (loop mediated isothermal amplification, magnetic capture hybridization, fluorescent in situ hybridization, next genaration sequencing, Real-time PCR i.e.) in identification and determination of plant pathogen fungi. These results will contribute to the more effective use of these molecular methods in phytopathological studies and the development of innovative methods for reducing economic losses caused by plant pathogenic fungi.

Anahtar Kelimeler

• Fungal pathogens
• LAMP
• (MCH)-PCR
• molecular identification
• Real time PCR

Bitki Patojeni Fungusların Tespitinde Polimeraz Zincir Reaksiyonu’na Dayalı Bazı Moleküler Teknikler

Öz

Kültür bitkilerinde ciddi kayıplara neden olan hastalık etmenleri içerisinde bitki patojeni funguslar önemli bir grubu oluşturmaktadır. Bu patojenlere karşı etkin mücadele yöntemlerinin geliştirilmesi ve neden oldukları ürün kayıplarının en aza indirilmesi için doğru ve hızlı bir şekilde tespit edilmesi en önemli adımdır. Bu kapsamda fungal patojenlerin klasik tespitinde kullanılan morfolojik karakterlere dayalı yöntemler değişkenlik göstermekle birlikte uzun zaman almakta ve taksonomik açıdan deneyimli personel gerektirmektedir. Bu nedenle bitki patojenlerinin tespiti için çok sayıda moleküler teknik geliştirilmiş ve epidemiyolojik çalışmalarda, karantina uygulamalarında, tohum sertifikasyonunda, ıslah programlarında ve fungisit direnci tespitinde yaygın olarak kullanılmaktadır. Bu çalışmada da bitki patojeni fungusların tespitinde yaygın olarak kullanılan polimeraz zincir reaksiyonuna dayalı bazı moleküler teknikler (Loop aracılı izotermal amlifikasyon, manyetik yakalama hibridizasyon, floresan in situ hibridizasyon, yeni nesil dizileme, Real Time PCR) hakkında bilgi verilmesi amaçlanmıştır. Bitki patojeni fungusların neden olduğu ekonomik kayıpları azaltmak amacıyla fitopatolojik çalışmalarda moleküler yöntemlerin daha etkin kullanılmasına katkı sağlayacağı düşünülmektedir.

Anahtar Kelimeler

• Fungal patojenler
• LAMP
• (MCH)-PCR
• moleküler tespit
• Real time PCR

Kaynakça

1. Adams IP, Glover RH, Monger WA, Mumford R, Jackeviciene E, Navalinskiene M, Boonham N, 2009. Next‐Generation Sequencing and Metagenomic Analysis: A Universal Diagnostic Tool in Plant Virology. Molecular Plant Pathology, 10 (4): 537-545.
2. Amann R, Glöckner FO, Neef A, 1997. Modern Methods in Subsurface Microbiology: in Situ Identification of Microorganisms with Nucleic Acid Probes. FEMS Microbiology Reviews, 20 (3-4): 191-200. https://doi.org/10.1111/j.1574-6976.1997.tb00308.x.
3. Amann RI, 1995. In Situ Identification of Micro-Organisms by Whole Cell Hybridization with rRNA-Targeted Nucleic Acid Probes. In: Akkermans A.D.L., Van Elsas J.D., De Bruijn F.J. (eds) Molecular Microbial Ecology Manual. 331-345. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-0351-0_23.
4. Amann, R, Fuchs BM, 2008. Single-cell Identification in Microbial Communities by Improved Fluorescence in Situ Hybridization Techniques. Nature Reviews Microbiology, 6 (5): 339-348.
5. Anonim, 2014. Real-time PCR Handbook, Life Technologies, https://www.gene-quantification.de/real-time-pcr-handbook-life-technologies-update-flr.pdf. (Erişim Tarihi: 29.01.2020).
6. Anonim, 2016. Real-Time PCR Applications Guide. https://www.bio-rad.com/webroot/web/pdf/lsr/literatüre /Bulletin_5279.pdf. (Erişim Tarihi: 29.02.2020).
7. Anonim, 2020a. Scorpions® Primers and Probes. https://www.biosyn.com/scorpions-primers.aspx (Erişim Tarihi: 28.03.2020).
8. Anonim, 2020b. Dual-Labeled Probes. https://www.sigmaaldrich.com/technical-documents/articles/biology/ dual-labeled-probes.html (Erişim Tarihi: 27.03.2020).

9. Anonim, 2020c. Fluorescence in Situ hybridization (FISH). https://www.genome.gov/genetics-glossary/ Fluorescence-In-Situ-Hybridization (Erişim Tarihi: 29.03.2020).
10. Anonim, 2020d. Illumina dye sequencing. https://en.wikipedia.org/wiki/Illumina_dye_sequencing (Erişim Tarihi: 25.02.2020).
11. Anonim, 2020e. https://www.ncbi.nlm.nih.gov/ (Erişim Tarihi: 25.02.2020).
12. Anonim, 2020f. Transcriptome and analysis of gene transcription. https://slideplayer.com/slide/5880309/ (Erişim Tarihi: 25.02.2020).
13. Anonim 2021. Oxford Nanopore Technologies. https://nanoporetech.com/applications/dna-nanopore-sequencing (Erişim Tarihi: 01.04.2021).
14. Arnoldi J, Schlüter C, Duchrow M, Hübner L, Ernst M, Teske A, Flad HD, Gerdes J, Böttger EC, 1992. Species-Specific Assessment of Mycobacterium Leprae in Skin Biopsies by in Situ Hybridization and Polymerase Chain Reaction. Laboratory İnvestigation; a Journal of Technical Methods and Pathology, 66 (5): 618-623.
15. Barba M, Czosnek H, Hadidi A, 2014. Historical Perspective, Development and Applications of Next-Generation Sequencing in Plant Virology. Viruses 6: 106–36.
16. Bayraktar H, Özer G, Aydoğan A, Palacıoğlu G, 2016. Determination of Ascochyta Blight Disease in Chickpea Using Real-Time PCR. Journal of Plant Diseases and Protection, 123 (3): 109-117.
17. Bilodeau GJ, Lévesque CA, De Cock Awam, Duchaine C, Brière S, Uribe P, Martin FN Hamelin RC, 2007. Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using Taqman, SYBR Green, Molecular Beacons. Phytopathology, 97 (5): 632-642.
18. Bonants PJ, van Gent-Pelzer MP, Hooftman R, Cooke DE, Guy DC, Duncan JM, 2004. A Combination of Baiting and Different PCR Formats, İncluding Measurement of Real-Time Quantitative Fluorescence, for the Detection of Phytophthora fragariae in Strawberry Plants. European Journal of Plant Pathology, 110 (7): 689-702.
19. Bumgarner R, 2013. Overview of DNA Microarrays: Types, Applications, and Their Future. Current Protocols in Molecular Biology, 101 (1): 22-1.
20. Cantu D, Govindarajulu M, Kozik A, 2011. Next-Generation Sequencing Provides Rapid Access to the Genome of Puccinia striiformis f. sp. tritici, The Causal Agent of Wheat Stripe Rust. PLoS ONE 6 (8): e24230.
21. Capote N, Aguado A, Pastrana AM, Sánchez-Torres P, 2012. Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance, 151-202.
22. Chen X, Ma L, Qiang S, Ma D, 2016. Development of A Loop-Mediated Isothermal Amplification Method for the Rapid Diagnosis of Ascochyta rabiei L. in Chickpeas. Scientific Reports, 6.
23. Deschamps S, Campbell MA, 2010. Utilization of Next-generation Sequencing Platforms in Plant Genomics and Genetic Variant Discovery. Molecular Breeding, 25 (4): 553-570.
24. Didenko VV, 2001. DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications. Biotechniques, 31 (5): 1106.
25. Duan Y, Ge C, Zhang X, Wang J, Zhou M, 2014. A Rapid Detection Method for the Plant Pathogen Sclerotinia sclerotiorum based on Loop-Mediated Isothermal Amplification (LAMP). Australasian Plant Pathology, 43 (1): 61-66.
26. Egan AN, Schlueter J, Spooner DM, 2012. Applications of Next-Generation Sequencing in Plant Biology.
27. Ellison MA, McMahon MB, Bonde MR, Palmer CL, Luster DG, 2016. In Situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections. Plant Methods, 12 (1): 37.
28. El-Metwally S, Osama OM, Mohamed H, 2014. Next Generation Sequencing Technologies and Challenges in Sequence Assembly. Springer No:7, s. XII-118, New York-USA.
29. Fredslund J, Lange M, 2007. Primique: Automatic Design of Specific PCR Primers for Each Sequence in a Family. BMC Bioinformatics, 8 (1): 369.
30. Fu S, Qu G, Guo S, 2011. Applications of Loop-Mediated Isothermal DNA Amplification. Applied Biochemistry and Biotechnology, 163: 845–50.
31. Fukuta S, Iida T, Mizukami Y, 2003. Detection of Japanese Yam Mosaic virus by RT-LAMP. Archives of Virology, 148: 1713–20.
32. Garrido C, Acero FGF, Carbú M, Rodriguez VEG, Liniero E, Cantoral JM, 2012. Molecular Microbiology Applied to the Study of Phytopathogenic Fungi. Biochemistry, Genetics and Molecular Biology. Rijeka, InTech, 139-156.
33. Garrido C, Carbu M, Acreo FJ, Boonham N, Coyler A, Cantoral JM, Budge G, 2009. Development of Protocols for Detection of Colletotrichum acutatum and Monitoring of Strawberry Anthracnose Using Real-Time PCR. Plant Pathology, 58: 43–51.
34. Ha Y, Fessehaie A, Ling KS, Wechter WP, Keinath AP, Walcott RR, 2009. Simultaneous Detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in Cucurbit Seedlots Using Magnetic Capture Hybridization And Real-time Polymerase Chain Reaction. Phytopathology, 99 (6): 666-678.
35. Hardinge P, Murray JA, 2019. Reduced False Positives and Improved Reporting of Loop-Mediated Isothermal Amplification Using Quenched Fluorescent Primers. Scientific Reports, 9 (1): 1-13.
36. Hawksworth DL, 2001. The Magnitude of Fungal Diversity: the 1.5 Million Species Estimate Revisited. Mycological Research, 105 (12): 1422-1432.
37. Kempf VA, Trebesius K, Autenrieth IB, 2000. Fluorescent in Situ Hybridization Allows Rapid İdentification of Microorganisms in Blood Cultures. Journal of Clinical Microbiology, 38 (2): 830-838.
38. König S, Schwenkbier L, Pollok S, Riedel M, Wagner S, Popp J, Weber K, Werres S, 2015. Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab‐on‐a‐chip DNA hybridization array. Plant Pathology, 64 (5): 1176-1189.
39. Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R. 2009. Complete Viral Genome Sequence and Discovery of Novel Viruses by Deep Sequencing of Small RNAs: a Generic Method for Diagnosis, Discovery and Sequencing of Viruses. Virology. 25;388(1):1-7. doi: 10.1016/j.virol.2009.03.024. Epub 2009 Apr 23. PMID: 19394993.
40. Kristensen R, Berdal KG, Holst‐Jensen A, 2007. Simultaneous Detection and Identification of Trichothecene and Moniliformin producing Fusarium Species Based on Multiplex SNP Analysis. Journal of Applied Microbiology, 102 (4): 1071-1081.
41. Kuang T, Chang L, Peng X, Hu X, Gallego-Perez D, 2017. Molecular Beacon Nano-sensors for Probing Living Cancer Cells. Trends in Biotechnology, 35 (4): 347-359.
42. Levene MJ, Korlach J, Turner SW, Foquet M, Craighead HG, Webb WW, 2003. Zero-mode waveguides for single-molecule analysis at high concentrations. Science, 299 (5607): 682-686.
43. Li AY, Crone M, Adams PJ, Fenwick SG, Hardy GE, Williams N, 2014. The Microscopic Examination of Phytophthora cinnamomi in Plant Tissues Using Fluorescent in Situ Hybridization. Journal of Phytopathology, 162 (11-12): 747-757.
44. Liebe S, Christ DS, Ehricht R, Varrelmann M, 2015. Development of a DNA Microarray-based Assay for the Detection of Sugar Beet Root Rot Pathogens. Phytopathology, 106 (1): 76-86.
45. Lievens B, Claes L, Vanachter AC, Cammue BP, Thomma BP, 2006. Detecting Single Nucleotide Polymorphisms Using DNA Arrays for Plant Pathogen Diagnosis. FEMS Microbiology Letters, 255 (1): 129-139.
46. Martin FN, Tooley PW, Blomquist C, 2004. Molecular Detection of Phytophthora ramorum, The Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered From Diseased Plant Material. Phytopathology, 94 (6): 621-631.
47. Mirmajlessi SM, Loit E, Maend M, Mansouripour SM, 2015. Real-Time PCR Applied to Study on Plant Pathogens: Potential Applications in Diagnosis–A Review. Plant Protection Science, 51 (4): 177-190.
48. Mullis K, Faloona F, Scharf S, Saiki RK, Horn GT, Erlich H, 1986. Specific Enzymatic Amplification of DNA in vitro: the Polymerase Chain Reaction. In Cold Spring Harbor Symposia on Quantitative Biology, 51: 263-273.
49. Mumford R, Boonham N, Tomlinson J, Barker I, 2006. Advances in Molecular Phytodiagnostics – New Solutions for Old Problems. European Journal of Plant Pathology, 116: 1–19.
50. Nie XZ, 2005. Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y. Plant Disease, 89: 605–10.
51. Niessen L, Vogel RF, 2010. Detection of Fusarium graminearum DNA Using A Loop-Mediated Isothermal Amplification (LAMP) Assay. International Journal of Food Microbiology, 140: 183–91.
52. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T, 2000. Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Research, 28 (12): e63-e63. doi: 10.1093/nar/28.12.e63. PMID: 10871386; PMCID: PMC102748.
53. Okubara PA, Schroeder KL, Paulitz TC, 2005. Real-Time Polymerase Chain Reaction: Applications to Studies on Soilborne Pathogens. Canadian Journal of Plant Pathology, 27 (3): 300-313.
54. Okuda M, Matsumoto M, Tanaka Y, Subandiyah S, Iwanami T, 2005. Characterization of the Tufb-Sece-Nusg-Rplkajl-Rpob Gene Cluster of the Citrus Greening Organism and Detection by Loop-Mediated Isothermal Amplification. Plant Disease, 89: 705–11.
55. Pollock J, Glendinning L, Wisedchanwet T, Watson M, 2018. The Madness of Microbiome: Attempting to Find Consensus “Best Practice” for 16s Microbiome Studies. Applied and Environmental Microbiology, 84(7). Ririe KM, Rasmussen RP, Wittwer CT, 1997. Product Differentiation by Analysis of DNA Melting Curves During the Polymerase Chain Reaction. Analytical Biochemistry, 245(2): 154-160.
56. Rodriguez D, Longo AV, Zamudio KR, 2012. Magnetic Capture Hybridization of Batrachochytrium dendrobatidis Genomic DNA. Journal of Microbiological Methods, 90 (3): 156-159.
57. Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M, Hoon J, 2011. An Integrated Semiconductor Device Enablingnon-Optical Genome Sequencing. Nature, 475 (7356): 348-352.
58. Schena L, Li Destri Nicosia MG, Sanzani SM, Faedda R, Ippolito A, Cacciola SO, 2013. Development of Quantitative PCR Detection Methods for Phytopathogenic Fungi and Oomycetes. Journal of Plant Pathology, 7-24.
59. Shendure J, Ji H, 2008. Next-generation DNA Sequencing. Nature Biotechnology, 26 (10): 1135-1145.
60. Tomita N, Mori Y, Kanda H, Notomi T, 2008. Loop-mediated Isothermal Amplification (LAMP) of Gene Sequences and Simple Visual Detection of Products. Nature Protocols, 3 (5): 877-882.
61. Varshney RK, Nayak SN, May GD, Jackson SA, 2009. Next-Generation Sequencing Technologies and Their Implications for Crop Genetics and Breeding. Trends in Biotechnology, 27: 522–30.
62. Voelkerding KV, Dames SA, Durtschi JD, 2009. Next-generation sequencing: from basic research to diagnostics. Clinical chemistry, 55 (4): 641-658.
63. Wagner M, Horn M, Daims H, 2003. Fluorescence In Situ Hybridisation for the İdentification and Characterisation of Prokaryotes. Current Opinion in Microbiology, 6 (3): 302-309.
64. Walcott RR, Gitaitis RD, Langston DB, 2004. Detection of Botrytis aclada in Onion Seed Using Magnetic Capture Hybridization and The Polymerase Chain Reaction. Seed Science and Technology, 32 (2): 425-438.
65. Yang YP, Corley N, Garcia-Heras J, 2001. Reverse Dot-Blot Hybridization As an Improved Tool for the Molecular Diagnosis of Point Mutations in Congenital Adrenal Hyperplasia Caused By 21-Hydroxylase Deficiency. Molecular Diagnostics, 6: 193–199.
66. Yao X, Li P, Xu J, Zhang M, Ren R, Liu G, Yang X, 2016. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay. Frontiers in Microbiology, 7.
67. Zhang Y, Coyne MY, Will SG, Levenson CH, Kawasaki ES, 1991. Single-Base Mutational Analysis of Cancer And Genetic Diseases Using Membrane Bound Modified Oligonucleotides. Nucleic Acids Research, 19: 3929–3933.
68. Zouhar M, Mazáková J, Prokinová E, Váňová M, Ryšánek P, 2010. Quantification of Tilletia caries and Tilletia controversa Mycelium in Wheat Apical Meristem by Real-Time PCR. Plant Protection Science, 46 (3): 107-115.
69. Zwirglmaier K, 2005. Fluorescence in Situ Hybridisation (FISH)–The Next Generation. FEMS Microbiology Letters, 246 (2): 151-158.