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.

<ol>
	<li>Pszcz&#243;&#322;kowska, A., Okorski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+report+of+anthracnose+disease+caused+by+Colletotrichum+fioriniae+on+blueberry+in+western+Poland.">First report of anthracnose disease caused by Colletotrichum fioriniae on blueberry in western Poland.</a> Plant Disease. 100, 2167 (2016).</li><li>Oudemans, P. V., Caruso, F. L., Stretch, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranberry+fruit+rot+in+the+northeast:+a+complex+disease.">Cranberry fruit rot in the northeast: a complex disease.</a> Plant Disease. 82, 1176-1184 (1998).</li><li>Damm, U., Cannon, P. F., Woudenberg, J. H. C., Crous, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Colletotrichum+acutatum+species+complex.">The Colletotrichum acutatum species complex.</a> Studies in Mycology. 73, 37-113 (2012).</li><li>Marcelino, J., Giordano, R., Gouli, S., Gouli, V., Parker, B. L., Skinner, M., TeBeest, D., Cesnik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colletotrichum+acutatum+var.+fioriniae+(teleomorph:+Glomerella+acutata+var.+flioriniae+var.+nov.)+infection+of+a+scale+insect.">Colletotrichum acutatum var. fioriniae (teleomorph: Glomerella acutata var. flioriniae var. nov.) infection of a scale insect.</a> Mycologia. 100, 353-374 (2008).</li><li>Pennycook, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colletotrichum+fioriniae+comb.+&amp;+stat.+nov.,+resolving+a+nomenclatural+muddle.">Colletotrichum fioriniae comb. &amp; stat. nov., resolving a nomenclatural muddle.</a> Mycotaxon. 132, 149-154 (2017).</li><li>Shivas, R. G., Tan, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+taxonomic+re-assessment+of+Colletotrichum+acutatum,+introducing+C.+fioriniae+comb.+et+stat.+nov+and+C.+simmondsii+sp+nov.">A taxonomic re-assessment of Colletotrichum acutatum, introducing C. fioriniae comb. et stat. nov and C. simmondsii sp nov.</a> Fungal Diversity. 39, 111-122 (2009).</li><li>Wharton, P. S., Di&#233;guez-Uribeondo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+Colletotrichum+acutatum.">The biology of Colletotrichum acutatum.</a> Anales del Jard&#237;n Bot&#225;nico de Madrid. 61, 3-22 (2004).</li><li>Prusky, D., Alkan, N., Mengiste, T., Fluhr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quiescent+and+necrotrophic+lifestyle+choice+during+postharvest+disease+development.">Quiescent and necrotrophic lifestyle choice during postharvest disease development.</a> Annual Review of Phytopathology. 51, 155-176 (2013).</li><li>Milholland, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+surface-topography+and+rain+intensity+on+splash+dispersal+of+Colletotrichum+acutatum.">Effects of surface-topography and rain intensity on splash dispersal of Colletotrichum acutatum.</a> Phytopathology. 80, 1115-1120 (1990).</li><li>Wharton, P. S., Dickman, J. S., Schilder, A. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+spore+release+by+Colletotrichum+acutatum+in+Michigan+blueberry+fields.">Timing of spore release by Colletotrichum acutatum in Michigan blueberry fields.</a> Phytopathology. 92, 86 (2002).</li><li>MacKenzie, S. J., Peres, N. A., Timmer, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+citrus+leaves+and+secondary+conidiation+response+to+citrus+flower+extracts+by+non-postbloom+fruit+drop+strains+of+Colletotrichum+acutatum.">Colonization of citrus leaves and secondary conidiation response to citrus flower extracts by non-postbloom fruit drop strains of Colletotrichum acutatum.</a> Tropical Plant Pathology. 35, 333-342 (2010).</li><li>Leandro, L. F. S., Gleason, M. L., Nutter, F. W., Wegulo, S. N., Dixon, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strawberry+plant+extracts+stimulate+secondary+conidiation+by+Colletotrichum+acutatum+on+symptomless+leaves.">Strawberry plant extracts stimulate secondary conidiation by Colletotrichum acutatum on symptomless leaves.</a> Phytopathology. 93, 1285-1291 (2003).</li><li>Waller, T. J., Vaiciunas, J., Constantelos, C., Oudemans, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+the+blueberry+floral+extracts+influence+secondary+conidiation+and+appressorial+formation+of+Colletotrichum+fioriniae.">Evidence the blueberry floral extracts influence secondary conidiation and appressorial formation of Colletotrichum fioriniae.</a> Phytopathology. 108, 561-567 (2017).</li><li>Gager, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The influence of cranberry floral wax on appressorium formation in Colletotrichum fioriniae. , (2015).</li><li>Podila, G. K., Rogers, L. M., Kolattukudy, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+signals+from+avocado+surface+wax+trigger+germination+and+appressorium+formation+in+Colletotrichum+gloeosporioides.">Chemical signals from avocado surface wax trigger germination and appressorium formation in Colletotrichum gloeosporioides.</a> Plant Physiology. 103, 267-272 (1993).</li><li>Polashock, J. J., Caruso, F. L., Oudemans, P. V., McManus, P. S., Crouch, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+North+American+cranberry+fruit+rot+fungal+community:+a+systematic+overview+using+morphological+and+phylogenetic+affinities.">The North American cranberry fruit rot fungal community: a systematic overview using morphological and phylogenetic affinities.</a> Plant Pathology. 58, 1116-1127 (2009).</li><li>Miller, P. 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The authors have nothing to disclose.

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.

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.

<table><tbody><tr><td>0.22 &amp;micro;m pore size, acetate sterilizing filter</td><td>VWR</td><td>101102-280</td><td>Blueberry floral extract (FE) clarification&amp;nbsp;</td></tr><tr><td>200-1000 &amp;micro;l pipette with tips</td><td>-</td><td>-</td><td>Equipment, any make within range will be adequate</td></tr><tr><td>40-200 &amp;micro;l pipette with tips</td><td>-</td><td>-</td><td>Equipment, any make within range will be adequate</td></tr><tr><td>5-40 &amp;micro;l pipette with tips</td><td>-</td><td>-</td><td>Equipment, any make within range will be adequate</td></tr><tr><td>Air spray gun disposable paint spray cup with connection adapter&amp;nbsp;</td><td>Harbor Freight</td><td>97098</td><td>Blueberry rainwater (rw-)FE collection</td></tr><tr><td>Autoclave</td><td>Amsco</td><td>3011</td><td>Equipment, media preparation</td></tr><tr><td>Bar mesh matting (plastic mesh sheet)</td><td>Winco</td><td>BL-240</td><td>Passive (pass)-FE collection</td></tr><tr><td>Benchtop timer</td><td>Fisher Scientific</td><td>06-662-47</td><td>Equipment, FE preparation</td></tr><tr><td>Black pressure/vacuum hose</td><td>VWR</td><td>62994-795</td><td>Vacuum filter component</td></tr><tr><td>Buchner funnel</td><td>Coors USA</td><td>60240</td><td>Vacuum filter component, accepts 55 mm filter paper disks</td></tr><tr><td>Bunsen burner&amp;nbsp;&amp;nbsp;</td><td>-</td><td>-</td><td>Equipment</td></tr><tr><td>Calcium carbonate</td><td>Fisher Scientific</td><td>C64-500</td><td>Media component</td></tr><tr><td>Centrifuge</td><td>Sorvall&amp;nbsp;</td><td>RC 5B Plus</td><td>Equipment</td></tr><tr><td>Centrifuge tubes (15 ml)</td><td>Fisher Scientific</td><td>05-527-90</td><td>Equipment</td></tr><tr><td>Centrifuge tubes (50 ml)</td><td>VWR</td><td>10025-694</td><td>Equipment, rw-FE collection</td></tr><tr><td>Cheesecloth (grade 50)</td><td>Fisher Scientific</td><td>AS240</td><td>Equipment, FE preparation</td></tr><tr><td>Chloroform</td><td>VWR</td><td>JT9175-3</td><td>Chemical, trichloromethane: assay grade, &amp;ge; 99% pure, for molecular biology, peroxide-free</td></tr><tr><td>Corn Meal Agar (CMA)</td><td>Fisher Scientific</td><td>B11132</td><td>&amp;nbsp;Pre-mix media, isolate storage on slants</td></tr><tr><td>Cotton-blue stain</td><td>Sigma-Aldrich</td><td>61335</td><td>Lactophenol cotton-blue stain</td></tr><tr><td>Difco Agar</td><td>VWR</td><td>90004-032</td><td>Media component</td></tr><tr><td>Drill-press</td><td>Delta</td><td>-</td><td>Equipment, rw-FE collection</td></tr><tr><td>EASYpure LF Ultrapure water</td><td>Barnstead</td><td>D738</td><td>Equipment, deionized water source</td></tr><tr><td>Ethanol (95%)</td><td>-</td><td>-</td><td>Chemical</td></tr><tr><td>Filter flask (500 ml)</td><td>Pyrex</td><td>No. 5340</td><td>Vacuum filter component</td></tr><tr><td>Fume hood</td><td>Hamilton</td><td>-</td><td>Equipment, chloroform usage</td></tr><tr><td>Funnel (7 X 7 cm)&amp;nbsp;</td><td>VWR</td><td>60820-110</td><td>Cranberry rw-FE collection, FE preparation</td></tr><tr><td>Generic glass slide&amp;nbsp;</td><td>Fisher Scientific</td><td>22-038-101</td><td>Bioassay conductance</td></tr><tr><td>Generic plastic pump spray bottle</td><td>VWR</td><td>16126-454</td><td>pass-FE collection, at least 250 ml capacity</td></tr><tr><td>Glass cell culture tubes</td><td>-</td><td>-</td><td>Storage of ch-FE</td></tr><tr><td>Glass coverslips (22 x 22 mm)</td><td>Fisher Scientific</td><td>12-542B</td><td>Bioassay conductance</td></tr><tr><td>Glass Van Tieghem cells (hand cut glass tubes)</td><td>-</td><td>-</td><td>Chloroform (ch)-FE bioassay, (8 mm OD 6 mm ID)</td></tr><tr><td>Glass-pipette (1-100 &amp;micro;l)</td><td>Hamilton Co. Inc.</td><td>#710</td><td>ch-FE bioassay</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td>Lactophenol cotton-blue stain</td></tr><tr><td>Hemocytometer</td><td>Bright-Line</td><td>5971R10</td><td>Equipment</td></tr><tr><td>Lactic acid</td><td>Sigma-Aldrich</td><td>W261106</td><td>Lactophenol cotton-blue stain</td></tr><tr><td>Laminar flow hood</td><td>Labconco</td><td>3730400</td><td>Equipment, sterile work environment</td></tr><tr><td>Metal probe (generic)&amp;nbsp;</td><td>-</td><td>-</td><td>Equipment&amp;nbsp;</td></tr><tr><td>Microcentrifuge tubes (2 ml)</td><td>Fisher Scientific</td><td>05-408-138</td><td>Aqueous treatment mixture storage and preparation</td></tr><tr><td>Microscope, Leica DMLB</td><td>Leica</td><td>020-519.010</td><td>Equipment</td></tr><tr><td>Mortar (ceramic)</td><td>Coors USA</td><td>60313</td><td>Vacuum filter component</td></tr><tr><td>Nitrile gloves&amp;nbsp;</td><td>Fisher Scientific</td><td>19-130-1597D</td><td>Flower collection</td></tr><tr><td>Paper disks (cut paper towels)</td><td>Office Basics</td><td>KCC01510</td><td>humidity control in bioassay</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td>Plastic paraffin film&amp;nbsp;</td></tr><tr><td>Pestle (ceramic)</td><td>Coors USA</td><td>60314</td><td>Vacuum filter component</td></tr><tr><td>Phenol crystals</td><td>Fisher Scientific</td><td>A92-100</td><td>Lactophenol cotton-blue stain</td></tr><tr><td>Plastic bags (~100 mm X 152 mm)</td><td>Uline</td><td>S1294</td><td>Equipment, flower refrigeration</td></tr><tr><td>Plastic cell culture dishes (9 cm diameter)&amp;nbsp;</td><td>Fisher Scientific</td><td>FB0875712</td><td>(Petri dish), bioassay conductance</td></tr><tr><td>Polytetrafluoroethylene (PTFE) lined caps&amp;nbsp;</td><td>VWR</td><td>60927-228</td><td>Storage of ch-FE</td></tr><tr><td>Pyrex beakers (100 ml)</td><td>Pyrex</td><td>No. 1000</td><td>Preparation of ch-FE</td></tr><tr><td>Pyrex bread-pan</td><td>-</td><td>-</td><td>pass-FE collection</td></tr><tr><td>Pyrex graduated cylinder</td><td>-</td><td>-</td><td>Equipment, FE preparation</td></tr><tr><td>Sealed plastic container (30 mm X 13 mm X 7 mm)&amp;nbsp;</td><td>-</td><td>-</td><td>Bioassay conductance</td></tr><tr><td>Sharp-pointed dissecting scissors</td><td>Fisher Scientific</td><td>8940</td><td>Equipment, to cut cheese-cloth and paper disks</td></tr><tr><td>Stainless steel mesh strainer</td><td>VWR</td><td>470149-756</td><td>Preparation of ch-FE</td></tr><tr><td>Step drill bit (step-bit)</td><td>Dewalt</td><td>-</td><td>Equipment, rw-FE collection</td></tr><tr><td>Sterile loop (combi-loop)</td><td>Fisher Scientific</td><td>22-363-602&amp;nbsp;</td><td>Culture preparation</td></tr><tr><td>Telephone wire (internal wires)</td><td>-</td><td>-</td><td>Blueberry rw-FE collection</td></tr><tr><td>Test tube basket&amp;nbsp;</td><td>VWR</td><td>470137-792</td><td>Readily available substitution for plastic mesh [strawberry] basket&amp;nbsp;</td></tr><tr><td>V8 Juice</td><td>Campbell&#039;s Soup Company</td><td>-</td><td>Fungal media component</td></tr><tr><td>Vintage plastic mesh [strawberry] baskets</td><td>Donation</td><td>-</td><td>pass-FE collection, can substitute for test tube basket (470137-792)</td></tr><tr><td>Vortex Genie (Vortex)</td><td>Fisher Scientific</td><td>12-812</td><td>Spore suspension preparation</td></tr><tr><td>Whatman No. 1 Qualitative 55 mm circles</td><td>Whatman</td><td>1001-055</td><td>Vacuum filter component</td></tr><tr><td>White plastic twist ties (100 mm)</td><td>Uline</td><td>S-566W</td><td>Cranberry rw-FE collection</td></tr></tbody></table>

colletotrichum fioriniae development in water and chloroform-based blueberry and cranberry floral extracts

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.

The results presented here are a few examples of the many assays that can be performed using this methodology. Figure 1 is an illustrated guide to the water-based FE bioassay, and is supplemented by Figure 2 which follows on to the chloroform-based FE bioassay. Figure 3 provides a visual guide to what can be expected upon microscopic evaluation of C. fioriniae at 24 h, in both water- and chloroform-based bioassays (compared SDW controls). Figure 4 details a 24 h time-course study with C. fioriniae in the presence of the cranberry variety &#39;Stevens&#39; ch-FE, and gives visual reference to an import result of this research: FE decreased the time needed to form infection structures compared to SDW. Figure 5 provides an example of data collected from a coverslip bioassay using cranberry floral rainwater runoff (CB &#34;Flower&#34; rw-FE). Figure 6 represents another important result: floral ovary ch-FE was much more stimulatory than fruit ch-FE, indicating the importance of bloom in the lifecycle of C. fioriniae. The supplemental photos and movies provide important visuals of the flowers used in the extractions and floral rainwater collection devices/deployment, in addition to movies that visualize the active- and passive- extraction (water-based) processes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58880/58880fig1.jpg" /> 
Figure 1: General overview of the water-based floral extract (FE) bioassay. This assay was utilized for water-based floral extracts with both blueberry and cranberry flowers: active-floral extracts (active-FE), passive-floral extracts (pass-FE) and floral rainwater runoff (rw-FE). The -FE portion typically constitutes the experimental/variable factor. Conversely the -FE portion can remain constant and time points/hours post-inoculation can be evaluated. Preference has been made towards 4 field analysis at 200X magnification. Abbreviations: Sterile deionized water, SDW; Area per field of view, A. <a href="https://www.jove.com/files/ftp_upload/58880/58880fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58880/58880fig2.jpg" /> 
Figure 2: General overview of the chloroform-based floral extract (ch-FE) bioassay. This assay was utilized for both blueberry and cranberry (flowers, ovaries and fruit). Only one type of aqueous treatment mixture was used in this assay, 1 part spore suspension to 2 parts SDW (to keep conidial concentration consistent due to ch-FE evaporation). This assay can be used to compare multiple ch-FE (waxes from various plant surfaces), or multiple time points/hours post-inoculation using a single ch-FE. Abbreviations: Sterile deionized water, SDW; Van Tieghem [glass] cells, VanT. cell. <a href="https://www.jove.com/files/ftp_upload/58880/58880fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58880/58880fig3.jpg" /> 
Figure 3: Visual comparison of Colletotrichum fioriniae in the presence of water-based FE and ch-FE. In this assay blueberry &#39;Bluecrop&#39; (active-FE, water-based) (fungal isolate: BB#10) and cranberry &#39;Stevens&#39; chloroform-based (ch-FE) (fungal isolate: CB-PMAP182) floral extracts were compared to SDW controls. A dramatic increase in secondary conidiation (rings) and appressorium formation (arrowheads) were observed when comparing conidia in the presence of SDW (control) (A) to active-FE (B) at 24h post-inoculation. However, secondary conidiation was not as apparent when comparing the chloroform bioassay SDW control (C) to ch-FE (D); rather, C. fioriniae growth shifted towards appressorial formation. Shown is a common response to each extraction type, water-based and chloroform-based, regardless of host/floral species described. <a href="https://www.jove.com/files/ftp_upload/58880/58880fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58880/58880fig4.jpg" /> 
Figure 4: Time-course study (24 h) with Colletotrichum fioriniae in the presence of ch-FE. In this assay, an SDW control (A-D) and cranberry &#39;Stevens&#39; ch-FE (E-F) were visually inspected at 0, 6, 12, and 24 h post-inoculation (an example of variable time points instead of comparing multiple FE). Appressorium formation (arrowheads) began at 6 h in the ch-FE and steadily increased throughout subsequent time points. This results eludes to an important factor of pathogen biology during the bloom period: flowers reduce the time needed to form infection structures. <a href="https://www.jove.com/files/ftp_upload/58880/58880fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58880/58880fig5.jpg" /> 
Figure 5: Graphical display of data collected using rw-FE in a bioassay. Rainwater run off of cranberry flowers (CB &#34;Flower&#34; rw-FE) and virgin rainwater that had not touched any cranberry plant tissues (&#34;Ground&#34; rw-FE) from a single wetting-event plus a standard active, cranberry water-based floral extract (CB active-FE) (positive control) and SDW (negative control) were subjected to a water-based coverslip bioassay and evaluated for C. fioriniae growth. CB &#34;Flower&#34; rw-FE had the same level of secondary conidiation and appressorium formation as the standard CB active-FE at 24 h post-inoculation, indicating that the collection devices were effective in capturing floral stimulants released during a wetting-event. Total conidia is comprised of primary (deposited), conidia and newly formed secondary conidia. Letters indicate significant differences at p &#60; 0.05 according to Fischer&#39;s Least Significant Difference test (LSD); uppercase, total conidia; lowercase, appressoria. <a href="https://www.jove.com/files/ftp_upload/58880/58880fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58880/58880fig6.jpg" /> 
Figure 6: Cranberry phenology based ch-FE bioassay, visual inspection. Disease management for fruit rotting fungi often involves bloom time fungicide applications. Here, cranberry chloroform-based extracts (ch-FE) from multiple growth stages of cranberry (&#39;Stevens&#39;) were visually evaluated for the effect of surface waxes on C. fioriniae at 24 h post-inoculation. Ovaries collected in June (A), immature fruit collected in July and August (B, C), harvested fruit collected in October (D), and an SDW control (E) were inspected for appressorial formation (arrowheads). Ovary ch-FE had the greatest magnitude of appressorial formation, indicating that this plant phenology (bloom) is critically important to the lifecycle of C. fioriniae. <a href="https://www.jove.com/files/ftp_upload/58880/58880fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/58880/58880supfig1.jpg" /> 
Supplemental Figure 1: Blueberry inflorescence. Blueberry flowers were collected for extractions during full bloom (April-May in New Jersey, USA) (shown &#39;Bluecrop&#39;). Note the overlap of corollas/ovaries from adjacent flowers and the overall architecture of the inflorescence compared to Supplemental Figure 2 (cranberry upright). <a href="https://www.jove.com/files/ftp_upload/58880/58880supfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 2" src="/files/ftp_upload/58880/58880supfig2.jpg" /> 
Supplemental Figure 2: Cranberry upright. Cranberry flowers were collected for extractions during full bloom (June-July in New Jersey, USA) (shown &#39;Stevens&#39;). Note the varied flower stages on a single cranberry inflorescence (upright), and the hooked, water droplet retaining the shape of the corolla. <a href="https://www.jove.com/files/ftp_upload/58880/58880supfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 3" src="/files/ftp_upload/58880/58880supfig3.jpg" /> 
Supplemental Figure 3: Blueberry rainwater deployment (flower). Completed blueberry floral rainwater collection device, placed directly under a cluster of inflorescences. Note the plastic-coated wire used to vertically orient the device. <a href="https://www.jove.com/files/ftp_upload/58880/58880supfig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 4" src="/files/ftp_upload/58880/58880supfig4.jpg" /> 
Supplemental Figure 4: Blueberry rainwater deployment (stem). Completed blueberry floral rainwater collection device, placed half way down the stem between an inflorescence and the crown of the bush. <a href="https://www.jove.com/files/ftp_upload/58880/58880supfig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 5" src="/files/ftp_upload/58880/58880supfig5.jpg" /> 
Supplemental Figure 5: Blueberry rainwater deployment (crown). Completed blueberry floral rainwater collection device, placed at the base of the bush (crown). Note plastic coated wires can be removed if not necessary. <a href="https://www.jove.com/files/ftp_upload/58880/58880supfig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 6" src="/files/ftp_upload/58880/58880supfig6.jpg" /> 
Supplemental Figure 6: Blueberry rainwater deployment (ground). Completed virgin rainwater collection device, placed adjacent to blueberry bushes. <a href="https://www.jove.com/files/ftp_upload/58880/58880supfig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 7" src="/files/ftp_upload/58880/58880supfig7.jpg" /> 
Supplemental Figure 7: Cranberry rainwater deployment (close-up). Completed cranberry floral rainwater collection device, with two uprights tucked under the neatly crossed wire ties. <a href="https://www.jove.com/files/ftp_upload/58880/58880supfig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 8" src="/files/ftp_upload/58880/58880supfig8.jpg" /> 
Supplemental Figure 8: Cranberry rainwater deployment. Multiple completed cranberry floral rainwater devices deployed in a bog. <a href="https://www.jove.com/files/ftp_upload/58880/58880supfig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Movie 1" src="/files/ftp_upload/58880/58880supmovie1.jpg" /> 
Supplemental Movie 1: Active, water-based floral extracts (active-FE). Supplemental video support following steps 2.3-2.5.1. Blueberry &#39;Bluecrop&#39; flowers were used. <a href="https://www.jove.com/files/ftp_upload/58880/active-FEextraction.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Supplemental Movie 2" src="/files/ftp_upload/58880/58880supmovie2.jpg" /> 
Supplemental Movie 2: Passive, water-based floral extracts (pass-FE). Supplemental video support following steps 3.3-3.4. Blueberry &#39;Bluecrop&#39; flowers were used. <a href="https://www.jove.com/files/ftp_upload/58880/pass-FEextraction.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Supplemental Movie 3" src="/files/ftp_upload/58880/58880supmovie3.jpg" /> 
Supplemental Movie 3: Deployment of cranberry floral rainwater collection devices. Supplemental video support following step 6.4. <a href="https://www.jove.com/files/ftp_upload/58880/rw-FEdeployment.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts at JoVE.com

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

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.

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

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral ...

colletotrichum-fioriniae-development-water-chloroform-based-blueberry

Research

JoVE Journal

Environment

7.8K Views.  Rutgers University. Dit protocol biedt een goedkoop en aanpasbaar systeem om de temporele dynamiek van fluorchemische signalen op Colletotrichum fiorniae en andere plantenpathogenen die term bloei infecteren evalueren. Nieuwe vloer regenwater opvangapparaten stellen de gebruiker in staat om natuurlijke stimulerende middelen en omstandigheden die pathogene ontwikkeling te controleren in een bijna real-time site-specifieke manier. Begin deze procedure met een cultuur van Colletotrichum fiorniae van een natuurlijk ...

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

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

Chemistry

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared spectroscopy is used to predict nondestructively total sugar, total organic acid, and total anthocyanin content in each blueberry. The technique is expected to enable the selection of only delicious blueberries from all harvested ones. The near-infrared absorption spectra of blueberries are measured with the diffuse reflectance mode at the positions not on the calyx. The ingredient contents of a blueberry determined by high-performance liquid chromatography are used to construct models to predict the ingredient contents from observed spectra. Partial least squares regression is used for the construction of the models. It is necessary to properly select the pretreatments for the observed spectra and the wavelength regions of the spectra used for analyses. Validations are necessary for the constructed models to confirm that the ingredient contents are predicted with practical accuracies. Here we present a protocol to construct and validate the models for nondestructive prediction of ingredient contents in blueberries by near-infrared spectroscopy.

We present here a protocol to construct and validate models for nondestructive prediction of total sugar, total organic acid, and total anthocyanin content in individual blueberries by near-infrared spectroscopy.

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared ...

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Developmental Biology

Polyol-Based Microwave-Assisted Extraction for Phenolic and Antioxidant Yields

Microwave-Assisted Extraction of Phenolic Compounds and Antioxidants for Cosmetic Applications Using Polyol-Based Technology

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert behavior with plant bioactive chemicals. This study explores the sustainable extraction of phenolic compounds and natural antioxidants from coffee silverskin using the microwave-assisted extraction (MAE) method with polyol-based solvents: glycerin, propylene glycol (PG), butylene glycol (BG), methylpropanediol (MPD), isopentyldiol (IPD), pentylene glycol, 1,2-hexanediol, and hexylene glycol (HG). A comparative analysis was conducted on conventional and non-conventional solvent extractions, focusing on their impact on the bioactive compounds of MAE, encompassing parameters such as total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities like the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH), the 2,2&#8242;-azino-bis(-3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging assay (ABTS), and the ferric reducing antioxidant power assay (FRAP). The highest values were observed for TPC with aqueous-1,2-hexanediol extraction (52.0 &#177; 3.0 mg GAE/g sample), TFC with aqueous-1,2-hexanediol extraction (20.0 &#177; 1.7 mg QE/g sample), DPPH with aqueous-HG&#160;extraction (13.6 &#177; 0.3 mg TE/g sample), ABTS with aqueous-pentylene glycol extraction (8.2 &#177; 0.1 mg TE/g sample), and FRAP with aqueous-HG extraction (21.1 &#177; 1.3 mg Fe (II) E/g sample). This research aims to advance eco-friendly extraction technology through natural plant components, promoting sustainability by minimizing hazardous chemical use while reducing time and energy consumption, with potential applications in cosmetics.

Author Spotlight: Eco-Friendly Extraction of Bioactive Compounds Using Polyol-Based Microwave-Assisted Techniques

This protocol details the utilization of a polyol-based microwave-assisted extraction method for extracting phenolic compounds and natural antioxidants, representing a practical and environmentally sustainable approach to the development of ready-to-use extracts.

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert ...

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Biology

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Bioengineering

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Optical Clearing of Plant Tissues for Fluorescence Imaging

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In addition, autofluorescence attributed to chlorophyll hampers the observation of fluorescent proteins in plants. For a long time, various clearing reagents have been used to make plants transparent. However, conventional clearing reagents diminish fluorescent signals; therefore, it has not been possible to observe the cellular and intracellular structures with fluorescent proteins. Reagents were developed that can clear plant tissues by removing chlorophyll while maintaining fluorescent protein stability. A detailed protocol is provided here for the optical clearing of plant tissues using clearing reagents, ClearSee (CS) or ClearSeeAlpha (CSA). The preparation of cleared plant tissues involves three steps: fixation, washing, and clearing. Fixation is a crucial step in maintaining the cellular structures and intracellular stability of fluorescent proteins. The incubation time for clearing depends on the tissue type and species. In Arabidopsis thaliana, the time required for clearing with CS was 4 days for leaves and roots, 7 days for seedlings, and 1 month for pistils. CS also required a relatively short time of 4 days to make the gametophytic leaves of Physcomitrium patens transparent. In contrast, pistils in tobacco and torenia produced brown pigment due to oxidation during CS treatment. CSA reduced the brown pigment by preventing oxidation and could make tobacco and torenia pistils transparent, although it took a relatively long time (1 or 2 months). CS and CSA were also compatible with staining using chemical dyes, such as DAPI (4',6-diamidino-2-phenylindole) and Hoechst 33342 for DNA and Calcofluor White, SR2200, and Direct Red 23 for the cell wall. This method can be useful for whole-plant imaging to reveal intact morphology, developmental processes, plant-microbe interactions, and nematode infections.

Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning.

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

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

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