Laboratory of Diagnostics in Plant Pathology

Basic concepts in diagnostic of plant diseases will be described. In particular, main symptom patters of plant diseases associated with fungi, bacteria, phytoplasmas, viruses and viroids will be illustrated.
Main steps of diagnostic process will be explained: preparation of specialized examination (preliminary circumstantial consultation of literature); overview of the case study; investigation on the examined area and its agronomical management; anamnesis through acquisition of documented and testimonial information; the collecting activity [choice of the significant sample: selection of the sampling date and the collected tissue in terms of quantity and typology (leaf, root, etc...)]; storage, transport to laboratory and conservation of fresh plant samples; sample preparation for microscopy analyses; based on observed symptoms and possibly associated pathogen, choice of appropriate sample preparation method for serological assays (preparation for ELISA assays) and/or molecular analyses (nucleic acid extraction); criteria to select the suitable technique(s) for pathogen detection (this topic will be discussed more accurately in the module 2 of the course); evaluation of the analytical process; data interpretation and diagnostic report formulation.
Diagnostic report delivery and its relationships with the compliance of legislation regulating the phytosanitary sector will be described.
Diagnostic techniques
Serological diagnosis: Polyclonal and monoclonal antibodies, main laboratory tests (ELISA, lateral Flow)
Molecular diagnosis
Isolation and purification of nucleic acids (i) genomic DNA from bacterial cells, fungal mycelium and plant tissues (ii) RNA from plant tissues, from bacterial cells and fungal mycelium
Molecular hybridization,
Polymerase chain reaction (PCR) principles and techniques (PCR, Hot start PCR, RT-PCR, semi-nested and nested -PCR, Cooperational-PCR, Multiplex-PCR, immunocapture (IC) -PCR), real-time PCR (qualitative and quantitative), digital PCR
Validation of the identity of PCR products (RFLP, sequencing),
Advantages and disadvantages of PCR
DNA microarray.
Target genes (for viruses, fungi and bacteria) for molecular diagnosis and primer design. Primer design.
NGS next generation sequencing: use for the diagnosis of pathogens

TRAININGS: Elements determining the decisions made during the diagnostic process and to formulate the diagnostic report will be analyzed in applied experiences on field surveys focused on case studies.
Laboratory tests (ELISA, PCR) will be carried out to determine the presence of pathogens in plant samples