Here, bioassays designed to monitor the development of a fungal pathogen, Colletotrichum fioriniae, in the presence of blueberry or cranberry floral extracts on glass coverslips are described. Water-, chloroform-, and field rainwater- based floral extraction techniques are detailed as well as insight into how this information can be applied.
To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral extraction methods and coverslip bioassays were developed utilizing Colletotrichum fioriniae. In blueberry and cranberry, this pathogen is optimally controlled by applying fungicides during the bloom period because of the role flowers play in the initial stages of infection. The protocol detailed here describes how floral extracts (FE) were obtained using water-, chloroform-, and field rainwater-based methods for later use in corresponding glass coverslip bioassays. Each FE served to provide a different set of information: response of C. fioriniae to mobilized floral chemical cues in water (water-based), pathogen response to flower and fruit surface waxes (chloroform-based), and field-based monitoring of collected floral rainwater, moving in vitro observations to an agricultural setting. The FE is broadly described as either water- or chloroform-based, with an appropriate bioassay described to compensate for the inherent differences between these two materials. Rainwater that had run off flowers was collected in unique devices for each crop, alluding to the flexibility and application of this approach for other crop systems. The bioassays are quick, inexpensive, simple, and provide the ability to generate spatiotemporal and site-specific information about the presence of stimulatory floral compounds from various sources. This information will ultimately better inform disease management strategies, as FE decrease the time needed for infection to occur, thus providing insight into changing risks for pathogen infection over the growing season.
Colletotrichum fioriniae causes a fruit rot of both Highbush Blueberry (Vaccinium corymbosum L.) and the large American Cranberry (V. macrocarpon Aiton)1,2. This pathogen was recently delineated from the C. acutatum species complex3,4,5,6 and is a causal agent of blueberry anthracnose and a member of the cranberry fruit rot complex, in addition to causing numerous other plant diseases worldwide7. C. fioriniae has a latent, hemibiotrophic lifestyle8, with infections occurring during bloom and symptom development not becoming apparent until the fruit are in final stages of maturation9. In blueberry and cranberry, fruit rot is only adequately controlled with fungicide applications made during the bloom period. The pathogen overwinters in dormant blueberry floral bud scales10 and sporulates during bloom. Conidia are moved throughout the canopy via rain-splash dispersal11,12 and inoculum buildup has been strongly correlated to the bloom period13. Response of Colletotrichum species to host flowers is not unique to Vaccinium, as flowers are important components of citrus post bloom fruit drop (PFD)14 as well as strawberry anthracnose15, in both cases causing the pathogen to sporulate. All of these cases highlight the need for effective methods to evaluate the temporal dynamics of floral chemical cues on C. fioriniae and other pathogens that infect during bloom. The insights provided by the methods described here are becoming increasingly more valuable.
This protocol details methods of floral extract (FE) procurement and guides the evaluation of C. fioriniae responses to FE via glass coverslip bioassays15,16. The floral extraction techniques are broken into two main types; water-based extractions (active-FE, passive (pass-FE), and field rainwater-based (rw-FE)), and chloroform-based (ch-FE)17 extractions. The water-based extractions allow for inspection of water mobilized floral chemical cues. These mobilized cues are likely important components of the infection court, since FE greatly increases the speed of infection16, in addition to providing the moisture required for the infection to occur. Additionally, they represent a more natural condition as floral stimulation can be washed throughout the canopy during wetting-events as previously observed in blueberry and other crop systems14,16. Chloroform-based floral extractions (ch-FE) also provide valuable information pertaining to pathogen response to host surface waxes17,18, elucidating the early growth stages of conidia once deposited onto susceptible host organs (i.e. flowers, ovaries and developing fruit). Pathogen response to seasonal changes in host surface waxes can also be monitored using this protocol. Accordingly, the bioassays are tailored to working with either water-based FE or chloroform-based FE to mitigate the inherent differences between these two materials.
The data generated from the bioassays revealed that water-based extractions stimulate higher levels of secondary conidiation than chloroform-based extractions where there was a definitive appressorial response, therefore implicating multiple compounds present in the FE. Interestingly, both of these growth responses were observed when using rainwater that had run off of blueberry and cranberry flowers, indicating multiple stimulatory compounds can be washed from the surface of flowers. Thus, monitoring for floral stimulation will provide insight into the probability of pathogen success in an agricultural system.
The ultimate goal of this protocol is to provide a methodology for generating baseline biological information on fungal plant pathogens in response to floral chemical cues, as well as initiating methodologies that can utilize this floral information to aid in site-specific disease management and decision-making processes.
The bioassays detecting the C. fioriniae response to floral extracts (FEs) were developed for the blueberry and cranberry fruit rot pathosystems but can be readily adapted to other horticultural crops. The protocol detailed above has been valuable in acquiring many important data sets including, but not limited to: FE effects on multiple isolates of numerous pathogens, time-course information pertaining to fungal growth stages in the presence of various FEs, comparison of extraction techniques, inspection of individual chemicals on C. fioriniae growth and differentiation, evaluation of individual flower organ extracts, effects of temperature on C. fioriniae while in the presence of FE, effects of phenology dependent wax extractions, and floral rainwater effects. Through the use of these techniques, data generated has also provided a much clearer understanding of C. fioriniae life stages and partially elucidates why the bloom period is so critical to the control of many fruit rotting pathogens.
Initially, all flowers were processed identically to the active-FE, but the extraction process has moved towards using whole flowers. Floral dissection was time consuming and had very little effect on the bioactivity of the resulting FEs. However, individual floral organs can and have been evaluated using this protocol, but great care must be taken to not completely macerate the floral tissues (Supplemental Movie 1, with precautions detailed in step 2.3), as this may result in released fungi-toxic/static compounds into the FE that could distort the microscopic evaluations. Less invasive extractions such as pass-FE (Supplemental Movie 2) and rw-FE are now more favorable due to their ease of acquisition. Additionally, these extraction techniques require only vacuum filtration to acquire biologically active floral chemical cues.
The flowers utilized in all extractions were typically refrigerated for 0-3 days prior to extract preparation. A challenge of this protocol is time management of FE turnover (field collection through storage of extracts). This was exacerbated by numerous samples from multiple sources and dates. Frozen flowers have not been evaluated to any real extent, as thawed flowers appear deteriorated and discolored. However, once the water-based FEs have been prepared, repeated freezing and thawing has shown no effect on the bioactivity of the FE, so as long as the FE are quickly refrozen after bioassay preparation (viable 3 year old FE).
Chloroform-based extraction enables the investigation of pathogen responses to three-dimensional floral/fruit surface waxes in a two-dimensional plane via ch-FE evaporation on glass coverslips. However, it is unlikely that the actual crystalline structures of waxes deposited from the ch-FE are identical to the surface from which they were collected. Meaning, supplemental techniques should be implemented if fungal response to specific wax structures in vivo are the main focus of investigation. Chloroform-based extracts need more storage maintenance than the water-based extractions. In addition to keeping the ch-FE extracts in the dark, the PTFE lined cell culture tube caps and parafilm sealing wrap need to be regularly checked for evaporative leaks and replaced as necessary.
The concept of monitoring floral rainwater runoff is rooted in the idea of advancing site-specific disease monitoring tools. The rainwater collection devices can be adapted to many other plant architectures, so long as the collection device captures rainwater that has run off of flowers. This approach provides information on whether or not floral stimulation is present in the field at any given time and can be monitored throughout the season. Alternatively, collection devices can be deployed at multiple canopy locations to determine how far floral cues have been washed during any given wetting-event. In future experiments, rw-FE will dictate when fungicide applications should begin and when they can safely end. Additionally, by monitoring phenology dependent wax extractions (protocol section 9), the importance of the bloom period to pathogen biology has become even more evident. That section was also included to demonstrate the flexibility of these bioassays, providing methods that allow for side-by-side comparison of host surface waxes that are temporally separated. The data generated using the floral extraction techniques and bioassays represent tangible indicators of pathogen stimulation, specific chemical classes important to pathogen biology, and targets for future control strategies.
The authors have nothing to disclose.
We thank the William S. Haines, Sr. Endowed Cranberry Research Fund and the New Jersey Blueberry and Cranberry Research Council, Inc. for support. We also thank Jennifer Vaiciunas (guidance and floral preparations), Christine Constantelos (fungal culture and floral preparations), David Jones (floral preparations and extractions), Langley Oudemans (floral preparations, filming/photography), Jesse Lynch (floral preparations), Roxanne Tumnalis (general support), and numerous student/summer interns.
0.22 µm pore size, acetate sterilizing filter | VWR | 101102-280 | Blueberry floral extract (FE) clarification |
200-1000 µl pipette with tips | – | – | Equipment, any make within range will be adequate |
40-200 µl pipette with tips | – | – | Equipment, any make within range will be adequate |
5-40 µl pipette with tips | – | – | Equipment, any make within range will be adequate |
Air spray gun disposable paint spray cup with connection adapter | Harbor Freight | 97098 | Blueberry rainwater (rw-)FE collection |
Autoclave | Amsco | 3011 | Equipment, media preparation |
Bar mesh matting (plastic mesh sheet) | Winco | BL-240 | Passive (pass)-FE collection |
Benchtop timer | Fisher Scientific | 06-662-47 | Equipment, FE preparation |
Black pressure/vacuum hose | VWR | 62994-795 | Vacuum filter component |
Buchner funnel | Coors USA | 60240 | Vacuum filter component, accepts 55 mm filter paper disks |
Bunsen burner | – | – | Equipment |
Calcium carbonate | Fisher Scientific | C64-500 | Media component |
Centrifuge | Sorvall | RC 5B Plus | Equipment |
Centrifuge tubes (15 ml) | Fisher Scientific | 05-527-90 | Equipment |
Centrifuge tubes (50 ml) | VWR | 10025-694 | Equipment, rw-FE collection |
Cheesecloth (grade 50) | Fisher Scientific | AS240 | Equipment, FE preparation |
Chloroform | VWR | JT9175-3 | Chemical, trichloromethane: assay grade, ≥ 99% pure, for molecular biology, peroxide-free |
Corn Meal Agar (CMA) | Fisher Scientific | B11132 | Pre-mix media, isolate storage on slants |
Cotton-blue stain | Sigma-Aldrich | 61335 | Lactophenol cotton-blue stain |
Curved forceps (45˚) | Fisher Scientific | 10-270 | Equipment, flower processing and coverslip inversion |
Difco Agar | VWR | 90004-032 | Media component |
Drill-press | Delta | – | Equipment, rw-FE collection |
EASYpure LF Ultrapure water | Barnstead | D738 | Equipment, deionized water source |
Ethanol (95%) | – | – | Chemical |
Filter flask (500 ml) | Pyrex | No. 5340 | Vacuum filter component |
Freezer (set to -20˚ C) | – | – | Equipment, storage of active-FE, pass-FE, rw-FE |
Fume hood | Hamilton | – | Equipment, chloroform usage |
Funnel (7 X 7 cm) | VWR | 60820-110 | Cranberry rw-FE collection, FE preparation |
Generic glass slide | Fisher Scientific | 22-038-101 | Bioassay conductance |
Generic plastic pump spray bottle | VWR | 16126-454 | pass-FE collection, at least 250 ml capacity |
Glass cell culture tubes | – | – | Storage of ch-FE |
Glass coverslips (22 x 22 mm) | Fisher Scientific | 12-542B | Bioassay conductance |
Glass Van Tieghem cells (hand cut glass tubes) | – | – | Chloroform (ch)-FE bioassay, (8 mm OD 6 mm ID) |
Glass-pipette (1-100 µl) | Hamilton Co. Inc. | #710 | ch-FE bioassay |
Glycerol | Sigma-Aldrich | G5516 | Lactophenol cotton-blue stain |
Hemocytometer | Bright-Line | 5971R10 | Equipment |
Incubator (set to 25˚ C, dark) | Percival | 50036 | Equipment, bioassay conductance |
Lactic acid | Sigma-Aldrich | W261106 | Lactophenol cotton-blue stain |
Laminar flow hood | Labconco | 3730400 | Equipment, sterile work environment |
Metal probe (generic) | – | – | Equipment |
Microcentrifuge tubes (2 ml) | Fisher Scientific | 05-408-138 | Aqueous treatment mixture storage and preparation |
Microscope, Leica DMLB | Leica | 020-519.010 | Equipment |
Mortar (ceramic) | Coors USA | 60313 | Vacuum filter component |
Nitrile gloves | Fisher Scientific | 19-130-1597D | Flower collection |
Paper disks (cut paper towels) | Office Basics | KCC01510 | humidity control in bioassay |
Parafilm | Bemis | PM-996 | Plastic paraffin film |
Pestle (ceramic) | Coors USA | 60314 | Vacuum filter component |
Phenol crystals | Fisher Scientific | A92-100 | Lactophenol cotton-blue stain |
Plastic bags (~100 mm X 152 mm) | Uline | S1294 | Equipment, flower refrigeration |
Plastic cell culture dishes (9 cm diameter) | Fisher Scientific | FB0875712 | (Petri dish), bioassay conductance |
Polytetrafluoroethylene (PTFE) lined caps | VWR | 60927-228 | Storage of ch-FE |
Pyrex beakers (100 ml) | Pyrex | No. 1000 | Preparation of ch-FE |
Pyrex bread-pan | – | – | pass-FE collection |
Pyrex graduated cylinder | – | – | Equipment, FE preparation |
Refrigerator (set to 4˚ C) | – | – | Equipment, storage of ch-FE |
Sealed plastic container (30 mm X 13 mm X 7 mm) | – | – | Bioassay conductance |
Sharp-pointed dissecting scissors | Fisher Scientific | 8940 | Equipment, to cut cheese-cloth and paper disks |
Stainless steel mesh strainer | VWR | 470149-756 | Preparation of ch-FE |
Step drill bit (step-bit) | Dewalt | – | Equipment, rw-FE collection |
Sterile loop (combi-loop) | Fisher Scientific | 22-363-602 | Culture preparation |
Telephone wire (internal wires) | – | – | Blueberry rw-FE collection |
Test tube basket | VWR | 470137-792 | Readily available substitution for plastic mesh [strawberry] basket |
V8 Juice | Campbell's Soup Company | – | Fungal media component |
Vintage plastic mesh [strawberry] baskets | Donation | – | pass-FE collection, can substitute for test tube basket (470137-792) |
Vortex Genie (Vortex) | Fisher Scientific | 12-812 | Spore suspension preparation |
Whatman No. 1 Qualitative 55 mm circles | Whatman | 1001-055 | Vacuum filter component |
White plastic twist ties (100 mm) | Uline | S-566W | Cranberry rw-FE collection |