We are going to conduct two experiments in order to test the gray mold fungus, Botrytis Cenerea for its resistance and sensitivity to the fungicide Fenhexamid. This video covers part one and another video covers part two. In part one we're going to test the fungus in auger plates mended with various concentrations of the fungicide. In part two, we'll test to see if the fungus can actually overcome the presence of the chemical on live plants and cause disease or if the fungicide protects plants from botrytis. We'll be using two different isolates of botrytis. One is labeled B16 and the other is labeled B1728. On part one on auger, this is a very artificial way of testing a fungus's resistance to a fungicide. By trial and error, you find out the amount of fungicide that will completely stop the growth of the fungus, and that's called the EC100 or effective concentration that slows growth 100% as compared to growth on no fungicide. Then you make delusions of that amount of fungicide to get a range of inhibition so that you can find the amount that slows growth 50% as compared to growth in the absence of fungicide. That concentration is called the EC50 or effective concentration 50. The EC50 and the EC100 are usually characteristic of a particular isolate. The concentrations you use in auger plates are very different from those actually sprayed on a plant in order to control the particular pathogen. Many times the concentration used on a plant is 10 times or more the amount that slows growth in an auger plate but sometimes the EC50 can give you an indication as to whether a fungus is resistant or sensitive to a fungicide on plants. Resistance means that the fungus can overcome the fungicide on a plant and cause a disease even though the fungicide is present. However there are some cases where a fungus can actually grow on a high concentration of fungicide and appear to be resistant to that fungicide in auger, but it cannot overcome the label rate of the chemical sprayed on a plant. And so then you really can't say that it's resistant. So we need to test both situations. We'll test it in auger to give us a rough guess as to whether it's resistant, and then we'll test it again on plants treated with a label rate of chemical and then make a final assessment as to whether one or both of the botrytis isolates are resistant to Fenhexamid. Now there are written details for both parts of the experiment in the lab report resources folder of ANGEL. In part one we will use auger with no fungicide and auger with Fenhexamid fungicide at concentrations of .002, .02, .2 and 2 micrograms of Fenhexamid per milliliter of auger. Micrograms per mil is the same as parts per million, or PPM. We calculated how much fungicide needed to go in the auger. We melted the auger, applied the fungicide, mixed it in, and then poured the plates in to let it solidify. In the meantime, we grew B16 and B1728 separately in plates, and then used a sterile cork borer to cut plugs of the growing fungi from the plates. Then we used a sterile needle and transferred a plug of inoculum and put it fungicide down on the edge of the auger plate in order to start the test. We label the plate with the isolate number and the fungicide concentration and date. Once the plates were inoculated we put them into a plastic bag upisde down. If you incubate them right side up, you get a lot of condensation on the lid of the plate and then it's difficult to see the fungal growth. So then we incubated the plates for one day and then we used a marker and marked the curved edge of the inoculum block and we used a straight line to mark the edge of the fungal growth. We did that for each plate. Now you can measure the amount of growth in our virtual experiment by clicking and dragging the ruler, and drag the ruler over and measure the difference between those two lines. Record that amount of growth for day one for each plate. The photos are the same size so that you can use that single ruler to measure the distance on each plate. There's an excel spreadsheet provided for you in the lab report resources folder where you can enter the data for each plate. Now if you look at the B16 photographs for day one, it looks like B16 grew more on the .002 micrograms per mil plate than on the plate with no fungicide. In fact that's exactly what happened. That's a phenomenon called hormesis. In hormesis, a small amount of a toxic material can actually stimulate the growth of a particular organism. Now you're very familiar with hormesis if you've ever taken any kind of a medicine, because in most cases medicines are very toxic chemicals if they're taken in high amounts. But they can be very helpful if taken in low amounts. So for example, where as one asprin is good for you, 100 asprin probably is not good for you. Now hormesis does not occur in every isolate or to every chemical. But this is a good example here. After two days of incubation, we mark the plates again where the fungus had grown out from the inoculum block, and you need to measure all the growth on all those plates, and record the data in the spreadsheet. Finally after three days of incubation we mark the plates and you need to measure the growth from the edge of the inoculum block to the edge of the growth after three days on each plate and record that information in the spreadsheet. Now you do that for both isolates B16 and then do it for isolate B1728. One way to look at the data is to graph the millimeters of growth on each plate VS the days after inoculation for each isolate and then draw a line for each concentration of fungicide. Now here this is just an example, and it's not based on any data from the experiment. It's just to illustrate one way of looking at the data. So using your measurements, you can create a graph like this for B16 and another graph for B1728, plotting the growth of those isolates at each fungicide concentration against the days after inoculation. And you can do that with the excel spreadsheet. And if you're not sure how to do that, if you look in the written directions, again those directions are in the lab report resources folder. I explain exactly how you can take that data and use excel to create the graph. Another way to analyze the data is to compare the amount of growth on a particular concentration of fungicide VS the amount of growth of that isolate on an auger plate with no fungicide. That's called the percent relative growth. So the percent relative growth equals the growth at a fungicide concentration divided by the growth on zero fungicide times 100. So, for day 3, calculate that number for each isolate and each fungicide concentration. Again, record those numbers and the percent relative growth calculations in the excel spreadsheet that was provided. In the percentages shown here, these are just an example and they're not based on any actual measurements. So you fill in the excel spreadsheet table with your measurements. In this example, you can estimate that the EC50 falls somewhere between .002 and .02 micrograms per mil of Fenhexamid. So you should do that for your two isolates based on your data and estimate the EC50 for where it falls for each of those two isoles. Then you need to answer a couple questions. So, based on this experiment, which isolate do you think is more sensitive to the fungicide? In other words, which one is inhibited the most? Or which one appears to be resistant? Do you think that one of the isolates should be said to be resistant of Fenhexamid? Meaning it's not not inhibited significantly by Fenhexamid. Now as I said in the beginning, it's one thing for a fungus to be inhibited by a chemical in auger, but it's quite something different on plant tissue, so before we can really say that an isolate is truly resistant to a fungicide, you need to test that isolate to see if it can cause disease on a plant that's been treated with a chemical. The plate tests that we've done here give some indication of resistance, but it actually has to be verified on plants using the label rate of fungicide. That's what we're going to do in part two of this lab experiment./nB Student Name: Date: Days C Day 1 D Day 2 E F Isolate B16 Growth vs days after inoculation for each Fenhexamid Concentration (µg/ml) G Botrytis Fungicide Resistance Report Record Use the tables below to record measurements of the TOTAL growth from the rounded agar disk edge to the edge of the newly black-marked outward mycelial growth for each day (not incremental daily growth). Start measurements from the curved disk edge each time. Notes: Only place numerical data in the cells or the graphs will not show. Measure in millimeters, not centimeters. 0 (µg/ml) 0.002 (µg/ml) 0.02 (μg/ml) 0.2 (µg/ml) H 2.0 (μg/ml) 1.20 1.00 0.80 0.00 0.40 I 0.20 K Graph B16: (Place your B16 Graph below) Chart Title/nA Days Day 2 Day 3 Days Day 1 Day 2 Isolate 1728 Growth vs days after inoculation for each Fenhexamid Concentration (µg/ml) Day 3 TAL 0 (μg/ml) I 0.002 (μg/ml) 0.02 (μg/ml) Calculate Relative Growth 0 (µg/ml) 0.002 (µg/ml) 0.02 (μg/ml) I 0.2 (μg/ml) 0.2 (µg/ml) 2.0 (μg/ml) .I 2.0 (μg/ml) H 0.20 0.00 1.20 1.00 0.80 0.00 0.40 0.20 0.00 I Day 2 Day 1 ➡0(μg/ml) -0.002 (8/ml) -0.02 (μg/ml) -0.2 (μg/ml) Day 1 Graph B17: (Place your B17 Graph below) Chart Title - (u/ml) -0.002 (ug/ml) Day 2 -0.02 (µg/ml) K -0.2 (μg/ml) Day 3 2.0 (μg/ml) Day 3 -2.0(μg/ml)/nCalculate Relative Growth At the end of the incubation (Day 3), calculate the relative growth % on each fungicide concentration as compared to growth on agar lacking fungicide. Relative growth= Fenhexamid Relative concentration growth % for B16 (μg/ml = ppm) 0.002 D Use the % relative growth @ 0.002 concentration as the starting point for EC100. To determine the EC50s divide the EC100 value by 1/2 (50%) for each isolate. Where does this number fall on your calculated relative growth %'s for each isolate? 0.02 growth in mm on Day 3 on a fungicide growth in mm Day 3 on 0 fungicide NOTE: EC50 answers may fall above, below, or between fenhexamid concentrations. Follow where the data leads. Looking across at the corresponding feneximide ppm concentrations, at what µg/ml or ppm does your EC50 correllate? 0.2 12 Relative growth % for B1728 x 100 At which Fenheximid concentration in the Relative growth % values at left do the values fall below 50% for each of the isolates? for B1729 Space to Perform Relative Growth Calculations for B16 here if desired: 0.002 H 0.02 0.2 2 Space to Perform Relative Growth Calculations for B1728 here if desired: 0.002 teren valias may fall hefween hun finnicide concentratione or Shelowlahovel the lovele facted OP 2 0.02 0.2 2 EC50 (Effective Concentration) = concentration that slows growth by 50% as compared to no growth on agar free of fungicide. Between what 2 concentrations is the EC50 for each isolate? L M N O P/n3 QUESTIONS: D Part 1 Experiment Video Questions 1. Based on the agar plate test, which isolate do you think is sensitive to fenhexamid (growth is slowed significantly by the fungicide)? 2. Based on the agar plate test, which of the isolates might be resistant to fenhexamid (growth is NOT slowed significantly by the fungicide)? Part 2 Experiment Video Question 3. Based on the plant inoculation experiment, which isolate is resistant to fenhexamid? Or, asked another way... Which isolate overcomes the presence of fenhexamid on the plants and causes disease? Note: Be certain to understand the difference between a fungus being sensitive or resistant to a fungicide. This will be a test question. M N O P

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