Aplikasi Teknik Molekuler untuk Deteksi & Identifikasi Patogen Tumbuhan/Bakteri

1. Aplikasi Teknik Molekuler untuk Deteksi & Identifikasi Patogen Tumbuhan/Bakteri Oleh Irda Safni

2. General approach for bacterial identification Different identification techniques • Physicalmethods • Based on the characterization of proteome of the bacteria • Geneticalmethods • Based on the characterization of specificsgenes of the bacteria • Biochemicalmethods • Based on the characterization of metabolicpathways of the bacteria To identify unknown bacteria , results are compared to databases

3. Struktur Sel Bakteri

4. Metabolism of the bacteria Bacteria are living cells who : - consume nutrients (carbohydrates, proteins…) - reject metabolic waste. Bacteria Enzymes Metabolic waste Nutrients Biochemical techniques for identifying bacteria are based on the characterization of enzymes and metabolic waste

5. Application to the identification: Physical / morpological method Gram stain Gram Negative Gram Positive

6. DindingSel Gram Negatif & Gram Positif

7. Application to the identification : Biochemical Methods Example : research the ability to use glucose Bacteria inoculation • Yellow color : • Acid pH • Production of acidic waste by bacteria • Proves the presence of enzymes which allow the use of glucose as nutrient by bacteria • Bacteria « glucose + » incubation Medium + glucose + pH indicator : -green color for pH = 7 -yellow color for acid pH (pH <7)

8. The API system The most used API system… API Strep  Identification of Streptococcus species API Staph  Identification of Staphylococcus species API 20NE  Identification of Non Enterobacteria (Pseudomonas for example) API 20E  Identification of Enterobacteria

9. The API system API 20 E after incubation…Positive results for all tests : API 20 E after incubation…Negative results for all tests :

10. The API system Example of results for bacteria to test : 3-Expression of results by software : In this example we have a very good identification of bacteria Salmonella spp Name of the identified bacteria Quality identification

11. E E E E Y Y Y Y Application to the identification : Biochemical Methods Enzyme-Linked Immunosorbent Assay (ELISA): immunological detection is a test that uses antibodies and color change to identify a substance. A. Bind sample to the support (commonly plastic or a membrane) B. Add primary antibody; wash C. Add secondary antibody-enzyme conjugate; wash D. Add substrate enzyme linked secondary antibody bound primary antibody antigenic site Y Y colorless substrate Y Y Target molecule colored product i i i i i i i i i i i i i i i Support

12. Molecular Methods Used For Detection of Plant Pathogens • Polymerase Chain Reaction • Molecular hybridization • Molecular markers • Nucleic acid sequence/Probes • Micro-arrays

13. Polymerase Chain Reaction • PCR is an in vitro method of nucleic acid synthesis by which a particular segment of DNA can be specifically replicated. • Invented by Karry Mullis(1987) • PCR is an ingenious new tool for molecular biology for identification of plant pathogens.

14. PCR Principle: The double stranded DNA of interest is denaturedto separate into two individual strands each strand is then allowed to hybridize with a primer(renaturation). The primer template duplex is used forDNA synthesis (the enzyme DNA polymerase). These three steps denaturation, renaturation and synthesis are repeated again and again to generate multiple forms of target DNA.

16. Steps in PCR: I.Denaturation of DNA: • Primers will only attach to end subsequently elongate the Developing nucleic acid chain on a template of single stranded (ss) DNA • Thus if the target sequences is double stranded (ds)DNA, the strand need to be split apart from each other to produce ssDNA. This is performed by heating the material to 90-96◦C a process termed as denaturation. • The step is of 4 min in the first cycle of PCR but of only 2 min in subsequent cycles.

17. II. Annealing of primers: • The primers are then attached to the ends of the segment to be amplified this process is called annealing. • This takes place at a temperature range of 37-50◦C.

18. III. Polymerization: • After the primers are attached they “kick-start the polymerization which then elongates the developing nucleic acid chain between them. • Bases are successfully added on to the developing new nucleic acid chain according to the sequence of the segment, which is being detected, producing a new chain consisting of a complementary sequence to that of the target segment.

19. IV. Amplification: • The new ds DNA need to be split apart again to yield two ss DNA strands by reheating the mixture to 95˚C i.e. denaturation, and the cycle is repeated. • Each cycle resulting in a logarithmic increase in the amount of DNA which is amplified. • Thus, within 20 cycles a million fold amplification of the starting amount of nucleic acid can be achieved. • The cyclical variation of temperature of the reaction is carried out by an automated thermal cycler.

20. V. Detection of target DNA sequence: • Amplification of DNA can be readily demonstrated by conventional DNA detection techniques such as nucleic acid hybridization or by agarose gel electrophoresis.

21. Advantages: • The major advantage of PCR is sensitivity (one molecule of nucleic acid within 1,00,000 cells). • It can detect infections at an early stage. • Useful in detection of non-replicating virus. • PCR tests are more rapid (result within 24-48 hours).

22. Disadvantages: • False positive results due to contamination from operator, residual matter in testing utensils or air contamination can result in false positive reaction. • Reagents used are still very expensive. • Useful only for those pathogens for which primers have been specifically designed.

23. PCR and other amplification methods are extremely sensitive and very specific. For accurate test interpretation, use proper controls. • Positive control: positive template • Negative control: negative template • Amplification control: constantly encounter template unrelated to target • Reagent blank: no template present

24. Application of PCR technique in plant pathology: • Diagnosis and quantification of diseases • Study of pathogen mating type • Production of virus free material. • Field surveys to assess the incidence and geographic distribution of plant pathogens • Domestic and international plant quarantine programmes. • .

25. Detection of mixed virus infections • Analysis of virus distribution in different plant tissues. • Identification of alternative host plant • Germplasm screening programmes. • Evaluation of levels of resistance of cultivars. • Determination of virus

26. Techniques: RT-PCR • “Gold standard molecular method” for detection of viruses. • An RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA). • The technique consists of two parts: 1) The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) 2) The amplification of a specific cDNA by PCR.

27. Schematic diagram of RT-PCR procedure

28. Many viroids like Apple dimple fruit viroid (ADFVd),Pear blister canker viroid (PBCVd), Peach latent mosaic viroid (PLMVd) have been detected by this method (Shamloul etal.,2002).It also applied in detection of viruses like CLRV, ACLSV, Apple mosaic virus and TomRV.The sensitivity afforded tends to be similar to ELISA or hybridization techniques (Olmos et al., 2005)

29. Bio-PCR • BIO-PCR involves an enrichment technique prior to extraction and amplification of DNA of the target bacterium (Schaad et al.,1995). • The intent of the method is to achieve target bacterium and suppress the growth of non target bacterium. • Normally the success of the method is wholly dependent on the availability of a reliable selective medium.

30. Application: • Detection of Pseudomonas savastanoi pv. savastanoi by PCR was enhanced by pre-enrichment in either non selective King’s B medium or on a semi-selective medium design for Pseudomonas savastanoipv. savastanoi by PCR(Penyalver et al;2000). • Song et al. (2004) describe the development of nutrient enrichment medium for Acidovorax avenae subsp. avenae on rice seeds.

31. Advantages: • BIO-PCR increases the sensitivity of detection. • It avoids the possibility of detecting dead bacterial cells. • Limitations: • Quantification of bacterial population cannot be readily done. • If selective medium is lacking sensitivity of detection is lacking.

32. Nested PCR • Sensitivity and specificity problems associated with conventional PCR and RT-PCR can be reduced by using nested PCR-based methods. • It involves the introduction of a second round of amplication using the amplicon of the first PCR reaction as template for the second. • Some authors proposed single-tube nested-PCR protocols for the bacteria E. amylovora (Llop et al., 2000), for Pseudomonas savastanoi pv. savastanoi (Bertolini et al., 2003), and some viruses (Yourno, 1992).

33. Bertolini et al., (2003) developed a nested PCR test for Pseudomonas savastanoi pv. savastanoi using external primers that amplified at 620C and internal primers that amplified at 500C. • A simple device based on the use of a compartmentalized Eppendorf tube, which enables RT reaction and nested PCR to be carried out in a single tube and in one-manipulation, has also been described for detection of Citrus tristeza virus (CTV) (Olmos et al., 1999 and 2003). • Coupling nested-PCR variants with squashed or printed samples on paper membranes has allowed the detection of RNA targets from several viruses in plant material and in individual insect vectors(Cambra et al., 2006a; Moreno et al., 2007).

34. Advantages: • Sensitivity is increased by two orders of magnitude reaching about 102 bacterial cells/ml of extract. • Limitations: • Need to accurately establish the ratio between external and internal primers • Use of limiting amounts of external primers to avoid interference during the second amplification.

35. Multiplex PCR • This methodology has demonstrated to be a valuable tool for detection and identification purposes(Lopez et al, 2006) • The simultaneous detection of two or more DNA or/and RNA targets can be afforded by duplex or multiplex PCR in a single reaction with several specific primers • In this case the choice of primers for amplification depends upon the desired type or detection

36. Two successful examples are the simultaneous detection of the six major characterized viruses affecting olive trees:CMV, Arabis mosaic virus (ArMV), Olivelatent virus-1 and Olive latent virus-2 (Bertolini et al.,2001)Nine grapevine viruses (ArMV, grapevine fanleaf virus, grapevine virus A, grapevine virus B, rupestris stem pitting-associated virus, grapevine fleck virus, grapevine leafroll-associated virus-1, -2 and -3)(Gambino andGrinbaudo,2006).

37. Scheematic representation of multipex PCR

38. Contoh Aplikasi Multiplex PCR untuk Deteksi Bakteri Ralstonia solanacearum species complex

40. Real Time PCR • An advancement of m-PCR is the development of Real Time PCR. • Real Time PCR is a quantitative procedure, which helps to detect, accumulation of PCR products during the PCR reaction. • PCR products can be monitored using either fluorescent DNA intercalating dyes such as SYBR Green I, or sequence specific probe based assays using TaqMan probes.

41. Taqman R ABI prism 7900 continuously measures PCR product accumulation using Taqman probe Sequence of probe is homologous to an internal target sequence Probe intact Energy transfer between fluoroaphore Fluorescent emission is quenched Probe cleaved by 5’ nuclease activity of Taq polymerase (extension phase) Increase in emission intensity is measured

42. Real Time PCR • For detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers (Van Beckhoven et al., 2002), • Ralstonia solanacearum (Weller et al., 2000; Ozakman and Schaad, 2003), • Erwinia amylovora, the causal agent of fire blight.(Salm and Geider, 2004), • Candidatus Liberibacter asiaticum(Li et al., 2006), • Xanthomonas fastidiosa (Bextine et al., 2005), • Xanthomonas fragariae (Weller et al., 2007)

43. Advantages: • It allows the monitoring of reaction while it is in course, thus avoiding the risk of contamination. • Rapid on-side diagnosis of quarantine pathogen. • It offers faster and more accurate detection assays. • Require fewer reagent and allows additional studies to be performed during detection.

44. Nucleic Acid Hybridization • The Basic Process of Binding a Single Strand of Nucleic Acid (DNA or RNA) to Its Complementary Strand Is Called Nucleic Acid Hybridization. • First utilized in plant pathology to detect Potato spindle tuber viroid (Owens and Diener, 1981)

45. Principle: • Denaturation of the ds DNA can be achieved by exposure to high temperature or alkaline pH. Dissociated strands of DNA can be immobilized on a solid phase support, such as latex, magnetic beads, microtitre plates ,nitrocellulose or nylon based membrane, and then hybridized with single strand, labeled nucleic acid (usually DNA) probe. • The probes will hybridized only with the denatured strand of complementary nucleic acid.

46. DNA-DNA hybridization Strain 1 Heat + Strain 2 0% Homology 100% Homology

47. a) Dot-Blot Hybridization Assay • Samples of denatured nucleic acid from healthy and infected plants are directly spotted on to a prewetted, solid matrics such as a nitrocellulose or nylon based membrane. • Nucleic acids are then firmly immolised on a solid support by baking the membrane in an oven for 2hr. at 80◦C. • To block the remaining Free DNA binding sites, the membrane is incubated several minutes in a sealed plastic bag with prehybridisation solution.

48. Continued…. • After removing the prehybridisation solution, the hybridization solution containing the denatured specific DNA probe is added to the plastic bag containing the sample membrane, and incubate for several hours to allow hybridization between the probe and the target nucleic acid. • The membrane is washed several times to remove the unbound DNA probe following hybridization. • Hybridization between the target nucleic acid immobilized on the membrane and the DNA probe is detected either by autoradiography on X-ray film, or by a calorimetric reaction.

49. Continued…. • The sensitivity of dot-blot hybridization may equal or exceed that of ELISA (Polston et al., 1989). • This technique has been employed for Apple mosaic virus(ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV) (Pallás et al., 1998).

50. Nucleic Acid Hybridization Probe present No probe