PHENOTYPING PROTOCOLS FOR TOLERANCE TO IMPORTANT CHERRY DISEASES. Fruit Growing Institute, Plovdiv; Department of Plant Protection

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1 STSM Scientific Report: Subject: PHENOTYPING PROTOCOLS FOR TOLERANCE TO IMPORTANT CHERRY DISEASES STSM Reference number: COST-STSM-FA Period: to COST Action number: FA1104 STSM Applicant: Diyana Vasileva Panayotova Fruit Growing Institute, Plovdiv; Department of Plant Protection 12 Ostromila Str., 4004 Plovdiv, Bulgaria Purpose of the STSM Objectives of the STSM: - Studies on methods for field evaluation of important cherry diseases; - Introduction to methods of testing of cherry cultivars susceptibility to diseases - Training on different approaches for screening of the susceptibility of some sweet cherry cultivars to diseases - Comparing methods and results for screening of sweet cherry cultivars for susceptibility to diseases Work Plan: - Field evaluation of important cherry diseases - Sample collection and preparation - Protocols for pathogens isolation and identification - Training on different methods for diagnostic of pathogenicity and virulence

2 - Training on different approaches for screening of the susceptibility of some cherry cultivars to diseases Description of the work carried out during the STSM Activities during the time of the STSM : I was involved in : monitoring of orchards, collection of samples, isolation and culture of the pathogens, methods for diagnostic and methods for pathogenicity and virulence testing. 1. Collection of samples Samples are collected at experimental fields of The Research Institute of Pomology In the experimental orchards are grown different varieties of sweet cherries and sour cherries at different rootstock. During the visit I took part in the collection of plant samples with symptoms of bacterial canker, bitter and brown rot caused by Pseudomonas syringae, Colletotrichum spp and Monilinia spp, respectively. Picture 1 Experimental fields of The Research Institute of Pomology Poland orchards in private farms I took part in the weekly days of monitoring of the laboratory staff. During each day of monitoring we visited about 10 private farms and/or

3 orchards for diagnostic and collection of samples. The purpose was collection of samples and monitoring of the most economically important diseases in the region and the country. The field work with the team of Laboratory of Phytopathology was very important for me to improve my knowledge and experience in field evaluation of important cherry diseases. Picture 2. Sour cherry fruits with symptoms of bitter rot caused by Colletotrichum spp. and brown rot caused by Monilinia spp. collected during monitoring of orchards 2. Isolation and culture of the pathogens on artificial culture media Another important part of my training was studying of laboratory methods for isolation and identification of different pathogens. Fungal isolates We isolated Colletotrichum spp. and Monilinia sp. collected from cherry and sour cherry orchards. Pure cultures were isolated on PDA medium and identified using morphological traits of the fungal growth, typical for each pathogen.

4 Picture 3/4 Colonies of Monilinia spp on growing media Bacterial isolates We worked mostly with isolates of Pseudomonas spp., cultured on media King B and NAS (nutrient agar with saccharose) to obtain a pure culture. Picture 5 Pure culture of Pseudomonas spp. 3. Methods for diagnostic of pathogenicity and virulence The work on the evaluation of the aggressiveness of isolated strains of cherry and sour cherry fungal diseases as well as studies of sensitivity of different varieties of sweet and sour cherries was the important part of my training. All tests were based on cultivation on different cultural media. Before inoculation, cherries are decontaminated with 70% ethanol for 1 minute and washed in sterile water.

5 Picture 6/7 Test for virulence of different strains of Monilinia spp For bacterial diseases we used different tests such as hypersensitivity reaction of tobacco, nitrate reductase test, soft rot of potato tissue, fluorescent pigment, arginine dihydrolase (Picture 8 13). Picture 8/9 Hypersensitivity reaction of tobacco Picture 10 Nitrate reductase Picture 11 Soft rot of potato tissue Picture 12 Fluorescent pigment Picture13 Arginine dihydrolase Table 1 Tests for the identification of bacteria

6 Strain Arginine Gelatin Fluorescent pigment Soft rot of potato Kovacs Oxidase Nitrate reductase Hypersensitivity reaction 6M M A E Molecular methods I was involved in : electrophoresis methods, PCR and Real Time PCR Identification of fungal species using PCR from all fungal isolates DNA was extracted and tested in PCR with primers CaInt2/ITS4 - specific for Colletotrichum acutatum and CgInt/ITS4 specific for C. gloeosporioides. PCR identification of Pseudomonas syringae pv. syringae DNA was extracted using Genomic Mini kit (A&A Biotechnology, Gdynia, Poland. DNA was amplified by Polymerase Chain Reaction (PCR) using specific primers for the detection of P. syringae pv. syringae. DNA purity and concentration was tested on NanoDrop. Additional knowledge During a visit to the experimental fields of the institute I had an opportunity to take part in the monitoring of apple, pear and apricot orchards as well. I had the opportunity to see tests for biocontrol agents against fire blight, different methods of Erwinia amylovora virulence testing and test for resistance of apple scab to chemical agents. For me, it was very important to get acquainted with some innovative approaches for combating the most economically important diseases of these fruit species.

7 Acknowledgments The knowledge and information collected during my short-term scientific mission helped me a lot to improve my professional qualification and experience in innovative methods for monitoring and the diagnosis of diseases as well as phenotypic tests of tolerance of sweet cherry cultivars to diseases. The STSM surely advanced my scientific career and it helped me to build a network of international scientific co-operatorsi want to thank my supervisor Dr. Joanna Puławska for her help and her efforts to make my STSM extremely fruitful and interesting. I want to express and my gratitude to the COST FA1104 for financial support of my STSM.

8 dr. hab. Joanna Puławska, professor IO Research Institute of Horticulture, Pomology Division Konstytucji 3 Maja 1/ Skierniewice Poland RESEARCH INSTITUTE OF HORTICULTURE Skierniewice, August 13 th, 2014 Ph: Fax: joanna.pulawska@inhort.pl - COST 1104 Chair, Dr. José Quero Garcia - COST 1104 STSM Coordinator, Dr. Francesco Paolo Marra - Diyana Vasileva Panayotova - To whom it may concern COST-STSM-FA Final Report Approval Dear COST 1104 Chair and STSM coordinator, To whom it may concern, As a host of COST-STSM-FA , I confirm that Diyana Vasileva Panayotova has fulfilled her one month-long short-term scientific mission in the Department of Plant Protection at the Research Institute of Horticulture, Skierniewice, Poland. Yours sincerely, dr. hab. Joanna Puławska, professor IO Research Institute of Horticulture, Skierniewice, POLAND, Konstytucji 3 Maja 1/3 Str. phone: , fax: , io@inhort.pl,