As 2024 comes to a close, the Strawberry Center Pathology Lab reflects on all of the projects, grants, and student successes this year. One of the highlights is our ongoing diagnostic plant service provided to California strawberry growers at no fee, funded by the California Strawberry Commission. As of November 15, we’ve received 185 plant samples, the largest amount since the inception of the service in 2014 (Fig. 1).

As our diagnostic service continues to grow, we have worked to optimize our methods to ensure we provide accurate results with a quick turnaround. This blog post details how to submit a sample, our methods, and how they compare to other labs.

How to submit a sample

Any strawberry grower can submit a plant sample to our lab for free. We request that your sample consist of 4 to 6 plants that are symptomatic, but not fully necrotic (dead). Samples should come in a sealed plastic bag with the completed submission form attached (https://strawberry.calpoly.edu/strawberry-disease-information-form). Samples can be dropped off or shipped to our office (Cal Poly Strawberry Center, 1 Grand Ave, Building 83, STE 1B, San Luis Obispo, CA 93407). We suggest that you ship samples early in the week to ensure they arrive before Friday. If you are dropping off samples, we request that you avoid doing this Thursday after business hours or on Friday to ensure that we process the samples prior to the weekend.

What happens when you submit a sample

When we receive a plant sample, we do our best to process it the same day. Utilizing sterile techniques, 1-inch sections of petioles (stems) and 1/2-inch pieces of roots are removed from each plant for plating on artificial media. The crown tissue of each plant is then removed and sliced in half (Fig. 2). Twenty, 1 cm squares of crown tissue are then removed for plating. In addition, one 1 cm square of crown from each plant is placed into a tube that will eventually undergo a crude DNA extraction for molecular analysis. Cal Poly student research assistants are trained to look for signs and symptoms throughout the process, including lesions on petioles, swelling or galls on roots, and dark tissue in plant crowns.

Following processing, the different plant material (crown, petiole, root) is plated on semi-selective media. Media is the jelly-like substance in a Petri dish that has different nutrients to promote growth of a particular pathogen, as well as antibiotics that prevent the growth of unwanted bacteria or fungi. While the plating process is relatively short, the growth of pathogens in certain media can take up to 10 days. Unfortunately, there is no shortcut here. Our goal is to give any potential pathogen the perfect home with all the food it needs, now we must wait.

To provide preliminary results within a few days of receiving the sample, we utilize two different molecular techniques. The first is called Recombinase Polymerase Amplification (RPA). This is a commonly used molecular assay to detect the four main strawberry soilborne pathogens (Fusarium oxysporum f. sp. fragariae race 1, Macrophomina phaseolina, Verticillium dahliae, and Phytophthora spp.), published by Dr. Frank Martin’s lab of the USDA-ARS Salinas. In the process of RPA, primers and probes with predesigned nucleotide sequences, bind to plant and pathogen DNA with the complementary sequence, causing amplification (Fig. 3)

We have recently adopted a secondary molecular assay called High Resolution Melting (HRM) that was developed by pathologists at the University of Florida Gulf Coast Research and Education Center. Different from other molecular assays such as RPA, HRM allows us to screen for multiple pathogens at the same time (Fig. 4). This reduces the amount of time it can take to provide growers and PCAs with results. Because this assay is new to our lab, we still verify results using traditional plating and other molecular methods. This ensures our final diagnosis is as accurate as possible, verified with three different methods.

What are other labs doing?

There are numerous other labs in California that provide a fee-for-service diagnosis. While methods of many private labs remain proprietary, there are industry-wide standards for molecular diagnosis and plating. Some labs provide a diagnosis based entirely on molecular assays such as qPCR and RPA while others plate as a preliminary screening.

The factors that vary among different methods include time, price, and accuracy. Results of plating can take almost two weeks in some cases, but it is much cheaper than any molecular method. Molecular methods can be rapid but take a trained scientist to run them and come at a much higher cost per sample. At the Cal Poly Strawberry Center Pathology Lab, we want to ‘cover all of our bases’, so we continue to conduct both traditional plating and molecular assays to give the most accurate diagnosis possible.

Questions about our service and how to submit?

Feel free to contact Dr. Shashika Hewavitharana at shewavit@calpoly.edu or Samantha Simard at ssimard@calpoly.edu.