This protocol provides an inexpensive and adaptable system to evaluate the temporal dynamics of fluorochemical cues on Colletotrichum fiorniae and other plant pathogens that infect term bloom. Novel floor rainwater collection devices enable the user to monitor natural stimulants and conditions affecting pathogen development in a near real time site-specific manner. Begin this procedure with a culture of Colletotrichum fiorniae from a naturally infected host as described in the text protocol.
When colonies begin to sporulate, streak conidia onto another V 8 Juice agar containing cell culture dish to produce a high-density sporulating culture. After seven days of growth, use a standard sterile loop to gather a small amount of conidia from the high-density culture by lightly touching the loop to the conidial mass. Stir this into a 15 milliliter centrifuge tube containing 10 milliliters of sterile deionized water.
Vortex this sample for 10 seconds. Using a standard pipette, plunge up and down numerous times to further mix the sample. Then place a drop of the vortexed sample onto the hemocytometer and estimate spore concentrations.
Adjust the concentration to 0.1 million conidia per milliliter of sterile distilled water with a five milliliter final volume. This is referred to as the spore suspension and is only made immediately prior to use in either bioassay. To perform chloroform-based floral extraction, clean all components with 95%ethanol twice.
Then clean the components twice with chloroform to prevent contamination for each extraction. Do not clean the PTFE lined caps with chloroform as it will damage them. After cleaning, set the components aside to dry upside down.
Combine one part processed flowers to nine parts chloroform in a beaker by adding flowers and then chloroform. Gently swirl for 30 seconds and strain through a stainless steel screen into the second beaker. Pour the chloroform-based extract from the second beaker into the glass culture tube and affix the PTFE cap.
Wrap the cap with parafilm to prevent evaporation. Store the resulting chloroform-based floral extract in darkness to reduce light degradation at four degrees Celsius until experimental use. Repeat extractions with multiple samples to provide replicates.
To create a collection device for each selected location, use a step bit attached to a drill press to drill a hole at the bottom of a spray cup. Then cut a straight line from the top of the hole to the mouth of the spray cup. Now drill four equidistant holes large enough to thread the plastic coated wire at the mouth of the spray cup.
Attach one end of the plastic coated wires leaving one end free. Drill a hole large enough to thread the paint spray or cup adapter into a 50 milliliter centrifuge cap lid. Seal the adapter threads with parafilm to prevent leaking.
Attach this to both the centrifuge cap and threaded portion of the sprayer cup. Then attach the mated 50 millimeter centrifuge tube. Create four collection devices per sampling location.
Deploy devices at selected locations by flexing sprayer cups to fit onto stems. Orient the cut side of the sprayer cup upwards using the plastic coated wires attached to other stems. This ensures water passing over the flowers is captured.
To collect rainwater from cranberry flowers, first heat puncture eight equidistant holes around the mouth of the funnel using a metal probe. Insert twist ties into four of the holes. Attach the other ends to that hole's opposite location forming a neat crossing pattern.
Wrap the funnel down steam with parafilm and set aside. Drill a hole large enough to insert the funnel down stem into a 50 milliliter centrifuge cap with a step bit. Insert the prepared funnel into the centrifuge cap.
After rainfall or overhead irrigation, remove and replace the bottom tube portion of the centrifuge tube. The rainwater collection tubes should be quickly changed after wetting events as they are prone to degradation from natural contaminants mobilized in the runoff such as bacteria and other fungi. Place two layers of paper discs within culture dishes and soak with two milliliters of sterile distilled water.
Next, mix equal volumes of sterile distilled water and water-based floral extract in two milliliter microcentrifuge tubes. Then add an equal volume of the spore suspension to the prepared two milliliter microcentrifuge tubes. The resulting preparation is referred to as an aqueous treatment mixture.
For the control, omit the floral extract portion and replace with sterile distilled water to keep the conidial concentrations consistent. Now place pre-cleaned glass coverslips on top of the soaked paper towels within the cell culture dish. Place a 40 microliter droplet of aqueous treatment mixture onto the center of a coverslip.
Repeat this for desired treatments including the control. Then close the cell culture dish. Once all treatments and replicates have been dispensed, place all replicated cell culture dishes into a sealed plastic container and incubate at 25 degrees Celsius in the dark.
At pre-determined time points, add 10 microliters of a fixative to the droplets stopping growth and semi-preserving the mount. Once a fixative has been added, carefully invert the coverslips placing each of them droplet side down on a glass microscope slide to facilitate microscopic examination. When all coverslips are on the glass microscope slides, leave them to settle and partially dry in the flow hood for 20 minutes.
Count all conidia present on the coverslip including primary conidia, germinated conidia, and newly formed secondary conidia, as well as appressoria. Work at either 400 times magnification or 200 times totaling an area of 3.808 square millimeters. In a laminar flow hood, add two equal volumes of sterile distilled water and one volume of spore suspension to a two milliliter microcentrifuge tube.
Set this aqueous treatment mixture aside for chloroform-based floral extract bioassays. In a fume hood, place a Van T Cell onto a glass coverslip within the prepared plastic cell culture dish. Dispense 33 microliters of desired chloroform-based floral extract into the center of the cell and allow to dry.
Do not touch the walls of the cell. For the control treatment, add virgin chloroform. Once the chloroform-based floral extract has dried, dispense 99 microliters of the prepared aqueous treatment mixture with a standard pipette into the center of the Van T Cell.
Then close all cell culture dishes and incubate. At pre-determined time points, add 15 microliters of a fixative to the Van T Cell and let it sit for at least five minutes to ensure adequate fungal staining. After that time, carefully remove the Van T Cell.
Perform coverslip inversion and data acquisition steps as before. Upon microscopic evaluation of C.fiorniae at 24 hours post inoculation, comparison of conidia in the presence of sterile distilled water versus water-based floral extract reveals a dramatic increase in secondary conidiation and appressorium formation. However, secondary conidiation was not as apparent when comparing the chloroform bioassay sterile distilled water control to chloroform-based extracts.
Rather, C.fiorniae growth shifted toward appressorial formation. In this assay, a sterile distilled water control and cranberry chloroform-based floral extractions were visually inspected at zero, six, 12, and 24 hours post inoculation. Appressorium formation began at six hours in the chloroform-based floral extract and steadily increased throughout subsequent time points.
This result supports the finding that flowers reduce the time needed to form infection structures. Disease management for fruit rotting fungi often involves bloom time fungicide applications. Here, cranberry chloroform-based extracts from multiple growth stages of cranberry were visually evaluated for the effective surface waxes on C.fiorniae at 24 hours post inoculation.
Ovaries collected in June, immature fruit collected in July, and in August, harvested fruit collected in October, and a sterile distilled water control were inspected for appressorial formation. Ovary chloroform-based extracts had the greatest magnitude of appressorial formation indicating the importance of bloom in the lifecycle of C.fiorniae. The pathogen is the most important component of this protocol.
Fungi should be moved to fresh media routinely to retain consistency between trials. Any work with chloroform must be performed in a fume hood for safety reasons. This includes preparation of materials and glassware, extraction procedures, and conductance of the chloroform-based bioassay.
Combinations of time points, pathogens, floral rainwater collections, or host extractions can be utilized to evaluate various host-pathogen interactions. The rainwater collection devices and associated bioassays will enable others to work with host stimulants mobilized during rain events in numerous path systems.
