Instructor Notes

1. This laboratory does not require cultures of the pathogen and fits well into a classroom situation with weekly scheduled laboratories. A period of 14 days is required for collecting data, as 7 days may be insufficient for growth of Thielaviopsis basicola. Pansies are sold as transplants in the fall and in the spring in many regions, and experience with this laboratory exercise indicates that this pathogen is associated with pansies sold at many nurseries or retail outlets. However, instructors may want to sample pansies from retail outlets as they plan the experiment. This laboratory exercise is amenable to statistical analysis; for example, multiple containers of plants purchased from multiple sources may be included as part of the laboratory exercise.
2. Selectivity of the modified TB-CEN medium includes the use of carrot juice, a nutrient source favorable for growth of T. basicola. The antibiotics streptomycin sulfate, chlortetracycline, and penicillin G are added to inhibit bacteria; penicillin G has been added to the medium as originally published. Nystatin is an antimycotic antibiotic added to inhibit many fungi and etridiazole is a fungicide added to inhibit oomycetes. Etridiazole is available in retail outlets by the trade names Koban, Terrazole, Truban, Terraflo, and Ethazol. It can also be purchased on-line through Sigma Aldrich (CAS 2593-15-9) and ULTRA Scientific Analytical Solutions (CAT# ULPST-1770). Based on preliminary assays, canned carrot juice does not work as well as fresh carrot juice; if using canned juice, double the amount of juice. To demonstrate the selectivity of TB-CEN medium, each group should also select one of the assayed soil or potting media samples and plate the sample on a nonselective medium using the dilution-pour plate technique. Potato dextrose agar (PDA) is a good option to observe the fungal and bacterial density and diversity present in a soil or potting medium.
3. Try to sample pansies purchased from different retail outlets for comparison; for example, select plants from large retail stores as well as those from local nurseries. From the landscape areas, choose flower beds that have a history of growing pansies. Also, sample from landscape areas near these annual flower beds. For example, sampling soil from turfgrass or other beds in a location away from water flow patterns might permit students to see if T. basicola is common in soils to the area or if it was introduced.
4. Alternative materials. Sterile water may be substituted for sterile water agar which is used to keep spores suspended longer. If a wrist action shaker is not available, shake the samples for 15 seconds at 3-minute intervals over 15 minutes. In case pipettors are not available, plastic 1.5 ml Pasteur pipettes can be used. Soil or potting medium at this concentrated dilution may clog the tip during pipetting. Cutting off the end of the plastic tip to increase the orifice diameter may help. The organic matter in the suspension from the potting medium may also be filtered out using cheese cloth or a sieve. For the baiting technique, Whatman filter papers can be replaced by paper towels. Plastic containers with lids (e.g., Rubbermaid-type containers) can also be used as a substitute for Petri plates for the baiting technique.
5. Magnifications under a stereomicroscope will help to reveal the symptoms of black root rot as discolored or blackened areas on the roots. Diseased pansies may not have severe symptoms. To clearly see chlamydospore chains, prepare a squash mount on a microscope slide and examine at a magnification of >100X (10X eyepiece and >10X objective).
6. Soil populations are often calculated on a weight basis (colony forming units (CFU)/g soil oven dry weight). In this study, organic potting media (low density substrate) and soil (high density substrate) were compared on a volume basis. To convert these samples to a weight basis, record the weight of a known volume of soil or potting media (wet weight), oven dry the samples for 48 h at 105 to 110°C, and weigh the samples again (dry weight). Calculate the water in the sample using the formulas below:

   1. Proportion H2O = (wet soil weight – dry soil weight)/dry soil weight 2. Dry soil weight assayed = wet soil weight assayed/(1 + proportion of H2O in sample)

7. Recording number of colonies of T. basicola. Colonies can be confirmed as T. basicola by examining a few colonies that appear to be T. basicola under a microscope and looking for chlamydospore chains or endoconidia. If a student group’s dilution plates contain a high density of colonies, the Petri plate can be divided into four quadrants or areas (see figure below). Just any arbitrarily selected area would be a fair estimate of the total number of colonies in the Petri plate (e.g., if quadrant 1 = 96 colonies, then 96 x 4 = 384 total colonies per plate). Previous experience with samples indicates a 1/100 or 1/1000 dilution rather than a 1/10 is better for counting colonies for heavily infested samples.
   
    
After colony counting, the soil population of the pathogen can be expressed on a weight or volume basis as colony forming units (CFU) per gram (g) of soil or CFU per cm3 of soil. Calculate CFU/cm3 of soil using the formula:

CFU/cm3 of soil= average number of colonies per plate x dilution factor