Laboratory of diagnostics in plant pathology

A.Y. 2021/2022
Overall hours
Learning objectives
The main objective of the course is to supply the information requested for performing the diagnosis of the plant diseases. Based on the topics described during the lessons and the laboratory trainings, the student will optimize his capability to select the work strategy suitable to formulate the diagnostic report.
Expected learning outcomes
Identification of main symptom patters of plant diseases caused by fungi, bacteria, phytoplasmas, viruses, and viroids; (ii) competence in challenging the diagnostic process steps: field surveys, sample collection and preparation for the laboratory analyses; (iii) Utilization of diagnostic tools employed for pathogen detection and identification (biological and traditional methods, serological methods, molecular analyses); (iv) employment of the criteria utilized for evaluating the diagnostic tools (efficiency, specificity, sensitivity); (v) interpretation of obtained data analyses and statement of the diagnostic report.
Course syllabus and organization

Single session

Lesson period
Second semester
Course syllabus
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.

Unit 2:
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
Prerequisites for admission
It is recommended that the students have a knowledge of the plant pathology basics. In fact, it is requested the knowledge of the main features of the plant pathogens (not accurately explained during this course) for understanding the topics illustrated throughout the lessons.
Teaching methods
Lessons and exercises conducted both in classroom and in the laboratory/greenhouse. Technical visit.
Teaching Resources
Powerpoint slides showed during the course and supplied scientific papers.
Assessment methods and Criteria
The exam will be carried out as oral exam. Therefore, the students have to know the topics described in both the modules during the lessons and the laboratory trainings.
AGR/12 - PLANT PATHOLOGY - University credits: 6
Field activity: 16 hours
Laboratories: 16 hours
Lessons: 32 hours
Monday from 10 to 12 am
Building 21070 (former Patologia vegetale)
On appointment by email (in case of health restrictions on Microsoft Teams)
Second flor - Building 7, via Celoria 2, Milan