Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

1. Fungal Isolates and Spore Suspensions

1. Isolate Colletotrichum fioriniae from a naturally infected host¹⁹. Then, store the clean cultures on corn meal agar (CMA) slants. Place plug of culture (from CMA slant) onto a standard plastic cell culture dish (9 cm diameter) containing either clarified V8 juice agar (cV8A) (modified from Miller 1955), or non-clarified V8 juice agar (V8A)²⁰. When colonies begin to sporulate, streak conidia (orange conidial masses) onto another cV8A or V8A containing cell culture dish (with a standard sterile loop) to produce a high-density sporulating culture.
   NOTE: Any protocol steps with fungi should be performed in a Laminar flow hood to reduce the possibility of contaminating fungal cultures and/or bioassays.
2. After 7 days of growth, using a standard sterile loop gather a small amount of conidia from the high-density culture (by lightly touching the loop to the conidial mass) and stir this into a 15 mL centrifuge tube containing 10 mL of sterile deionized water (SDW). Vortex this sample for 10 s, then using a standard pipette plunge up and down numerous times to further mix the sample.
3. Then, place a drop of the vortexed sample onto the hemocytometer and estimate spore concentrations. Count 5-10 fields on the hemocytometer, obtain the average, and multiply average by the appropriate dilution factor (i.e. 10,000), thus obtaining the conidial concentration per mL SDW.
4. Then using a volume (V)/concentration (C) equation (V₁●[C₁]=V₂●[C₂]) adjust (with SDW) to 1.0 X 10⁵ conidia per mL of SDW with a 5 mL final volume. This is referred to as the spore suspension and is only made immediately prior to use in either bioassay.

2. Active, Water-based Floral Extracts (active -FE)^15,16

Note: See Supplemental Figure 1, Supplemental Figure 2, Supplemental Figure 3, and Supplemental Movie 1.

1. Carefully hand-collect blueberry (April-May) and cranberry (June-July) flowers during their respective peak blooms in the field (Supplemental Figure 1). Wear and change nitrile gloves between sampling locations (cultivars/varieties/physical locations) to inhibit human skin oil contamination as well as cross contamination between floral varieties. Place either blueberry or cranberry flowers (from a single source) into plastic bags (fill a 100 x 150 mm bag) and immediately refrigerate at 4 ° C once flowers are collected.
   NOTE: The active- extraction is modified from Leandro et al. (2003)¹⁶.
2. Prior to extraction, remove any deteriorated, damaged, diseased flowers, then using healthy flowers remove the ovaries, sepals and peduncles with curved forceps (45˚ tweezers) and discard. Use only the remaining organs (corolla, stigma, style and stamen) for active- extractions. Cut cheesecloth (150 x 150 mm) and place within a 7 x 7 mm funnel, place into a clean 50 mL centrifuge tube, and set aside.
3. Combine 1 part processed flowers to 9 parts SDW (1 wt: 9 vol ratio) in a mortar. Gently grind with a pestle for 30 s (take care to not completely pulverize the samples). Strain resulting pulp through prepared cheesecloth/funnel and collect in centrifuge tube (50 mL). Clean or use new mortar/pestle between each extraction with 95% EtOH and warm running water.
4. Prepare a vacuum filtration apparatus: gather Buchner funnel, vacuum filter Erlenmeyer flask (up to 1,000 mL capacity), 55 mm circle filter paper, and vacuum hose/source. Add filter paper to an appropriate sized Buchner funnel and place atop the flask, then connect apparatus to a water/ suction source via vacuum hose and set aside.
5. Further clarify the blueberry floral extracts by centrifuging for 10 min at 8,055 x g. Pour off supernatant into prepared vacuum filter apparatus, turn on vacuum source, filter supernatant, then pour flask contents into a new centrifuge tube (50 mL). Clean all apparatus components between each filtration with 95% EtOH and warm running water. Further filter through a syringe adapted with a 0.22 µm pore size, acetate sterilizing, filter into a new centrifuge tube (50 mL).
   1. For the cranberry floral extracts, only vacuum filter through filter paper and pour flask contents into a new centrifuge tube (50 mL).
6. Resulting preparation is referred to as active– floral extract (active-FE). Store all water-based floral extracts (active-, passive-, rainwater collections) at -20° C in 5-50 mL aliquots until experimental use. Repeat extractions with multiple samples (at least 3 extractions per sample type) to provide replicates.

3. Passive , Water-based Floral Extracts ( pass -FE) ¹⁶

Note: See Supplemental Movie 2.

1. Hand collect whole blueberry flowers (50 g) as above, and refrigerate after collecting. Prior to extraction, remove any deteriorated, damaged, diseased flowers and remove only the peduncles from intact flower. Refrigerate prepared samples until the passive- extraction system, described below, is in place.
2. Rinse all components prior to use with 95% ethanol (EtOH) and warm running water to prevent contamination (plastic mesh sheet, two plastic mesh baskets, glass bread pan, and pump mist bottle). Also prepare a 4 layer cheesecloth/funnel/50 mL centrifuge tube for each extraction as described above (in step 2.2), and set aside.
3. Prepare the sieve: place a plastic mesh sheet (aka clear bar matting) into one plastic mesh basket (114 x 102 mm). Place a second, identical, mesh basket upside down (inverted) into a glass bread-pan (127 x 229 mm) (to avoid flowers sitting in SDW) and place the mesh sheet containing basket atop. Add 50 g of prepared flowers into the sieve.
   NOTE: Alternatively, small test tube [cleansing] baskets can be used in place of the originally used plastic mesh baskets (old, green, strawberry mesh pint baskets are often difficult to source).
4. Evenly mist flowers with 250 mL using a pump mist bottle and capture runoff in the glass bread-pan. Then strain filtrate through the prepared cheesecloth/funnel into a clean centrifuge tube. Resulting preparation is referred to as passive– floral extract (pass-FE); store as per above (step 2.6).
5. Clean all components between each extraction with 95% EtOH and warm running water. Repeat extractions with multiple samples to provide replicates (at least 3 per sample type).

4. Chloroform-based Floral Extracts (ch-FE) ¹⁷

1. Collect blueberry and cranberry flowers as per above (step 2.1), and prepare flowers for extraction (step 2.2, without cheesecloth/funnel preparation). Keep prepared flowers refrigerated until prior to use.
   1. Any work performed with chloroform must be performed in a fume hood for safety reasons. This includes preparation of materials / glassware, extraction procedures, and bioassay conductance (steps using ch-FE).
2. Clean all components with 95% EtOH twice, then twice with chloroform to prevent contamination for each extraction: threaded glass culture tubes, 2 glass beakers, small stainless steel screen, and a [glass] graduate cylinder. Set aside to dry upside-down. Rinse polytetrafluoroethylene (PTFE) lined caps twice with 95% EtOH only (chloroform will damage the outer materials of the cap) and set aside to dry.
3. Combine 1 part processed flowers to 9 parts chloroform (1 wt: 9 vol ratio) in a beaker (flowers then chloroform), gently swirl for 30 s, and strain through a stainless steel screen into the second beaker. Pour ch-FE from the second beaker into the glass culture tube (10 – 15 mL) and affix the PTFE cap. Wrap the cap with parafilm to prevent evaporation.
4. This preparation is the chloroform-based floral extract (ch-FE). Store sample in darkness (to reduce light degradation) at 4 ° C until experimental use. Repeat extractions with multiple samples to provide replicates (at least 3 per sample type).

5. Collection of Rainwater from Blueberry Flowers (BB rw-FE)¹⁶

NOTE: The blueberry floral rainwater collection device consists of an air spray gun disposable paint spray cup with connection adapter (cup: female thread, adapter: male to male thread), 50 mL centrifuge tubes (polypropylene), parafilm, and plastic coated wires (standard telephone wire, individual internal wire strand contents).

1. Select multiple locations within a blueberry bush to capture rainwater run off of flowers prior to creating collection devices. These include directly under inflorescences (flower) to the very base of the bush (crown). Record diameter of the stems (ranging from 1-5 cm) at selected locations, as this will dictate the size of holes used for device attachment, described below.
2. For each selected location create a collection device. First drill a hole at the bottom of a spray cup (where the cup curves towards the threaded opening) to the corresponding stem diameter, with a step-bit attached to a drill press. Then, cut a straight line from the top of thehole to the mouth of the spray cup. Drill 4 equidistant holes (large enough to thread the plastic coated wire), at the mouth of the spray cup, and attach one end of the plastic coated wires leaving one end free.
3. Drill a hole large enough to thread the paint sprayer cup adapter into a 50 mL centrifuge cap lid. Seal adapter threads with parafilm to prevent leaking. Attach this to both the centrifuge cap and threaded portion of the sprayer cup. Attach the mated 50 mL centrifuge tube.
4. Repeat steps 5.3-5.4 to create multiple devices as per selected locations, a minimum of 4 collection devices per sampling location.
5. Deploy devices at selected locations by flexing sprayer cups to fit onto stems. Orient the cut-side of the spray cup upwards using the plastic-coated wires attached to other stems (to insure water passing over flowers is captured). Affix parafilm any openings that could leak rainwater. (Supplemental Figure 3, Supplemental Figure 4, Supplemental Figure 5, and Supplemental Figure 6).
6. Label collection tubes with deployment date / time. After a rain-event, remove and replace the bottom (tube) portion of the centrifuge tube (label date / time of collection). Bring runoff collections (referred to as blueberry rainwater floral extract (BB rw-FE)) inside and vacuum filter (filtration described in steps 2.4-2.5). Store as above (step 2.6), until used in a water-based bioassay.

6. Collection of Rainwater from Cranberry Flowers (CB rw-FE)

1. The floral rainwater collection device in cranberry consists of a 7 X 7 cm polypropylene funnel, 50 mL centrifuge tubes (polypropylene), parafilm, and 4 standard, plastic coated twist ties (per device).
2. First, heat puncture 8 equidistant holes (diameter of twist ties) around the mouth of the funnel using a metal probe. Insert twist ties into 4 holes. Attach the other ends to that holes' opposite location, forming a neat crossing pattern. Wrap funnel down-stem with parafilm and set aside.
3. Drill a hole large enough to insert the funnel down-stem into a 50 mL centrifuge cap with a step-bit. Insert prepared funnel into the centrifuge cap. Repeat steps 6.2-6.3 to create multiple devices, a minimum of 4 collection devices per sampling location.
4. Deploy labeled (date/time) devices into selected cranberry bogs. Neatly tuck two flower bearing inflorescences (known as uprights) under the crossed plastic twist ties. Then vertically orient the device by piercing the centrifuge tube into the cranberry canopy (Supplemental Figures 7-8, Supplemental Movie 3).
5. After rainfall or overhead irrigation remove and replace the bottom (tube) portion of the centrifuge tube (label date / time of collection). Bring runoff (referred to as cranberry rainwater floral extract (CB rw-FE)) inside and vacuum filter (filtration described in steps 2.4-2.5). Store as above (step 2.6), until used in a water-based bioassay.

7. Bioassay using Water Based Floral Extracts^15,16 (active-FE, pass-FE, rw-FE)

Note: See Figure 1.

1. This bioassay is prepared in a Laminar flow hood. Prepare materials: triple rinse glass coverslips with 95% EtOH and air dry, then put aside. Cut paper towel disks to the internal diameter of a standard plastic cell culture dishes (9 cm). Place 2 layers of paper disks within culture dishes and soak with 2 mL SDW.
2. Prepare at least 5 mL of a 1.0 X 10⁵ conidia per mL of SDW spore suspension (C. fioriniae) as per above (step 1.2-1.4), set aside. Next, mix equal volumes of SDW and water-based floral extract in 2 mL microcentrifuge tubes. Then, add an equal volume of the spore suspension to the prepared 2 mL microcentrifuge tubes (SDW plus FE); the resulting preparation is referred to as an aqueous treatment mixture. For the control, omit FE portion and replace with SDW, to keep conidial concentrations consistent.
   NOTE: portion size is dependent on number of replicates and time-points. Typically, portions do not exceed 500 µL. Once conidia and FE are combined the bioassay has begun, 0 h post-inoculation.
3. Place the pre-cleaned glass coverslips on top of the soaked paper towels within the cell culture dish. Place a 40 µL droplet of aqueous treatment mixture onto the center of a coverslip.Repeat for desired treatments, including the control, close the cell culture dish. Repeat in separate cell culture dishes for replications (at least 3) and time points (each dish is for 1 time point). Once all treatments and replicates have been dispensed, place all replicated cell culture dishes into a sealed plastic container (30 x 13 x 7 mm) and incubate at 25° C in the dark .
4. At predetermined time-points, add 10 µL of a fixative like lactophenol cotton-blue (20.0 g phenol crystals, 20.0 mL 2.5% lactic acid, 40.0 mL glycerol, 0.05 g cotton blue) to the droplets, stopping growth and semi-preserving the mount.
   1. Wait 2 h to collect the 0 h time point as this allows conidial adhesion to the glass surface but is not long enough for conidial germination. For all other time points, add fixative at corresponding time.
5. Once a fixative has been added, carefully invert the coverslips, placing them droplet side down on a glass microscope slide to facilitate microscopic examination (1 coverslip per slide). When all coverslips are on the glass microscope slides, leave them to settle and partially dry in the flow hood for 20 min.
6. Count all conidia (total conidia) present on the coverslip (primary conidia, germinated conidia, and newly formed secondary conidia) as well as appressoria at either 400X magnification (counting 16 fields) or 200X (counting 4 fields), totaling an area of 3.808 mm². Replicate entire bioassay for statistical analysis. For assays using water-based FE, analyze data as described in Waller et al. 2017¹⁶, typically displaying as mean count/mm² (total conidia or appressoria).

8. Bioassay using Chloroform-based Floral Extracts (ch-FE)¹⁷

Note: See Figure 2.

1. Prepare materials and cell culture dishes as per above (steps 7.1). Additionally, rinse an equal number of Van Tieghem cells (VanT. cells) (8 mm OD, 6 mm ID) to coverslips, as well as a glass pipette (1 mL with 1 µl increments) within a fume hood, (twice with 95% EtOH then twice with chloroform), and set aside.
2. In laminar flow hood, using a 2 mL microcentrifuge tube add two equal volumes (at least 500 µL portions) of SDW and 1 volume of spore suspension, then set aside. This is the aqueous treatment mixture for ch-FE bioassays.
3. In a fume hood, place a VanT. cell onto a glass coverslip within the prepared plastic cell culture dish. Dispense (with glass pipette) 33 µL of desired ch-FE into the center of the Van T. cell (do not touch the walls of the VanT. Cell; for the control treatment, add virgin chloroform), and allow to dry. Repeat in separate cell culture dishes for replications (at least 3).
4. Once ch-FE has dried, dispense 99 µL of the prepared aqueous treatment mixture with a standard pipette into the center of the VanT. cell, then close all cell culture dishes. Once the aqueous treatment mixture has come in contact with the dried ch-FE treatments, the bioassay has begun, 0 h post-inoculation.
5. Once all treatments and replicates have been dispensed, place all (closed) cell culture dishes into a sealed plastic container (30 x 13 x 7 mm) and incubate at 25° C in the dark.
6. At predetermined time-points, add 15 µL of a fixative (lactophenol cotton-blue) to the VanT. cell and let sit for at least 5 min to insure adequate fungal staining. After that time, carefully remove the VanT. cell and follow coverslip inversion and data acquisition steps above (steps 7.5-7.6). For data analysis, follow methods described in Gager 2015¹⁷, typically displaying data as appressorium formation, which is the ratio of total conidia to appressoria counted in the area observed (3.808 mm²).

9. Cranberry Phenology-based Extractions¹⁷

1. Hand collect cranberry flowers (in June; 100 g), immature fruit (twice; July and August; 200 g), and mature cranberry fruit at harvest (October; 200 g), place into appropriately sized plastic bags and refrigerate at 4 ° C immediately after collection. Use the contamination precautions outlined above (step 2.1).
   NOTE: There will be extra plant material collected at each time but these amounts guard against extracting obviously fungal infected ovaries and fruit (showing symptoms and signs of disease).
2. Prior to extraction, remove any deteriorated, damaged, diseased flowers, then using only healthy flowers, remove the sepals, peduncles, corollas, stigmas, styles and stamens with curved forceps (45 ° tweezers) and discard all but the ovaries. Once ovaries are collected, perform the chloroform-based extraction at a 1 wt: 9 vol ratio (detailed in steps 4.2-4.4, using only ovaries). Store samples until all other extractions are complete, i.e. until bioassay conductance.
3. Once fruit are collected, perform a chloroform-based extraction (steps 4.2-4.4, using collected fruit instead of flowers), but add 10 g of fruit to 90 mL of chloroform and once extracted, allow the solution to evaporate to 9 mL (resulting in a 10 g: 9 mL wt: vol solution).
4. Make fruit extractions immediately after collecting in July, August, and October. Store as per above (step 4.4) until all extractions are collected. After the last chloroform-based fruit extraction, subject all penology-based chloroform extracts collected for this assay to the chloroform-based bioassay and analyze accordingly (steps 8.1-8.6).