The short course wound up on Friday, September 12 with a half-day of insect pest ID practice.  Dr. Luis Cañas and Nuris Acosta presented a final case study on the insect group Thysanoptera: Thripidae and Diptera: Tephritidae, Agromyzidae.  Participants made great use of the digital microscopes provided to them to identify key morphological characteristics.  

We finished the course with a “post”- test, an anonymous follow-up clicker survey to assess knowledge changes among the participants. Participants indicated that they now have a better understanding of classical, molecular and digital tools to diagnose plant diseases and identify insect pests than they had 2 weeks ago.

After lunch, OARDC Director Dr. Steve Slack presented course completion certificates to all of the participants.

Stay tuned for updates on next year’s short course.

Day 10 was a very busy day, beginning with a lecture on nematode pest identification by Ellie Walsh, a senior Ph.D. student in Dr. Chris Taylor‘s lab.  Dr. Taylor was also on hand to answer questions after the lecture and during the nematode identification lab that followed.  Grad students Tim Frey, Krystel Navarro and Rachel Medina, and staff member Theresa Miller led the participants in exercises to practice extraction, staining and identification of plant pathogenic nematodes including root knot nematode (Meloidogyne) from tomato and bloat nematode (Ditylenchus) from leek.

In the afternoon Hemiptera:Homoptera took center stage as Dr. Cañas led a case study session on identification of these pests, using microscopy and internet tools to key in on diagnostic morphological characteristics.

Finally, Dr. Pierce Paul and postdoc Dr. Jorge Salgado conducted a case study on mycotoxin contamination of cereals, including practice with a test kit to identify and quantify these toxic compounds.  Moldy  wheat seeds infected with Fusarium graminearum were tested for deoxynivalenol (DON; vomitoxin) using Reveal Q+,  a commercial kit.  Non-moldy grain was also tested.

It’s been a week since plants were inoculated as part of an exercise to identify three maize viruses.  Participants evaluated symptoms on inoculated plants and conducted tissue blot immunoassays (TBIAs).  After setting up the TBIAs, they worked with Dr. Fiorella Cisneros-Carter and Tea Meulia to search for virus particles in leaf dips from inoculated maize plants by transmission electron microscopy.

In the afternoon, participants completed the TBIAs and finalized virus identification in their unknowns.  Some extra time in the afternoon was allotted to practice sequence alignment and BLAST searches for pathogen/pest identification using available software.  They also purified internal transcribed ribosomal DNA amplified last week from DNA extracted in Ghana from a putative Choanephora cucurbitarum isolate.   The purified DNA was submitted to the OSU-OARDC Molecular and Cellular Imaging Center (MCIC) for sequencing.

On Monday the short course participants traveled to Reynoldsburg, OH with Drs. Cañas and Miller to tour the Ohio Department of Agriculture Plant Health Division.  Nancy Taylor, Director of OSU’s C. Wayne Ellett Plant and Pest Diagnostic Clinic and the Ohio Plant Diagnostic Network, provided a tour of the modern diagnostic lab facilities. We then heard a lecture from Dan Kenny, State Plant Regulatory Officer and Assistant Chief, Plant Health Division, who described the work of ODA in safeguarding plant health in Ohio.  After the tour and discussion, the group had lunch on the OSU campus and toured the Schottenstein Center.

Ohio has a number of claims to fame, from “Cradle of Presidents” (eight US presidents from Ohio), to the home of the Wright brothers, the first American to orbit the earth (Senator John Glen) and the first person to walk on the moon (Neil Armstrong).  It is also the home of the Rock and Roll Hall of Fame and Museum, sited on the shore of Lake Erie.  None of our participants have ever been to the Rock Hall, but all have  an appreciation for music.  We visited the Hall on Saturday, a rather rainy day, for a unique American experience.  Lunch in Little Italy on Cleveland’s east side capped the day.

Day 5 began with a lecture by OSU Ph.D. student Nagendra Subedi on the theory and practice of Real-Time PCR.  Real-time PCR technology has moved from research labs into diagnostic applications for numerous plant pathogens.   The technique requires less time and labor to perform than traditional PCR, and allows quantitation of the diagnostic target in a sample.   DNA was extracted on Day 4 from tomato plants suspected to be infected with Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial canker.

Bacterial canker of tomato – note typical marginal necrosis.

“Bird’s-eye” lesions typical of bacterial canker on tomato fruit.

DNA extracts were tested for Cmm by both conventional (endpoint) and Real-Time PCR (TaqMan).  Results were positive for all suspected samples by RT- PCR, a few were missed in endpoint PCR.  Participants also tested the putative Cmm samples with DNAble tests kits – DNAble kits are based on isothermal DNA amplification technology.

Dr. Sally Miller presented a case study for detection and diagnosis of  Ralstonia solanacearum, a potentially devastating disease of many crops in the subtropics and tropics.  All of the diagnostic techniques in practice in the short course so far are used in identification of this pathogen.

Day 4 of the Diagnostics Short Course is mostly about PCR – but with a good dose of mite identification included for good measure.  Dr. Fulya Baysal-Gurel started the day with a lecture on classical PCR methods and isothermal DNA amplification.  We will talk about Real-Time PCR on Day 5.  After Fulya’s lecture, the group learned all about mite identification from one of the world experts – Dr. Cal Welbourne, Florida Department of Agriculture & Consumer Services, Division of Plant Industry via videoconference.  In the afternoon, Dr. Andy Michel presented the latest information on the use of PCR to identify insect pests.

The remainder of the afternoon was dedicated to a hands-on PCR lab.  During the lab, participants extracted DNA from tomato plants suspected to be infected with Clavibacter michiganensis subsp. michiganensis (Cmm).  Known cultures of CMM were also prepared for testing.  These samples will be tested by endpoint PCR and Real-Time PCR.

The third day of the Diagnostics Short Course was devoted to the diagnosis of plant diseases caused by bacteria.  Dr. Sally Miller presented a lecture on groups of bacterial pathogens most commonly infecting plants, and approaches to identify them.  The lecture was followed up with a lab for the remainder of the day.  Participants gained practical experience in identifying symptoms of bacterial diseases, culturing bacteria on various general and semi-selective media, and conducting biochemical tests such as the gram reaction and the oxidase test, and plant based tests including tobacco hypersensitive reaction (HR) and potato and pepper soft rot assays.   For diagnosis of bacterial diseases of tomato, participants are directed to OSU fact sheets detailing diagnostic procedures for bacterial spot, bacterial speck, bacterial canker and pith necrosis. 

Day 2 – Introduction to diagnosis of plant diseases caused by fungi and oomycetes.  Dr. Anne Dorrance, OSU soybean pathologist and expert on Pythium, Phytophthora and many fungal pathogens, presented a primer on identification of these groups of plant pathogens, focusing on key features of each major group used in identification. She also talked about the process of diagnosis in the laboratory and the tools needed to induce fungal and oomycete growth and sporulation.

In the lab, participants used a simplified diagnostic key to identify fungal cultures, used microscopy to identify morphological diagnostic structures, conducted an ELISA assay for Phytophthora in field samples.   A video describing  double antibody sandwich ELISA  was shown to describe the principles.   Participants also isolated pathogens from plant tissues.  Ph.D. student Nagendra Subedi and Dr. Fulya Baysal-Gurel led the exercises.

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