Name: colletotrichum fioriniae development in water and chloroform-based blueberry and cranberry floral extracts
Uploaded: 2019-04-12T14:00:00
Duration: 12 min 32 s
Description: Watch this Scientific Journal Video about Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts at JoVE.com

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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Compendium of Blueberry and Cranberry Diseases. , (1995).</li><li>DeMarsay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anthracnose fruit rot of highbush blueberry: biology and epidemiology. , (2005).</li><li>Madden, L. V., Yang, X. S., Wilson, L. L. <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+rain+intensity+on+splash+dispersal+of+Colletotrichum+acutatum.">Effects of rain intensity on splash dispersal of Colletotrichum acutatum.</a> Phytopathology. 86, 864-874 (1996).</li><li>Yang, X., Madden, L. V., Wilson, L. L., Ellis, M. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V-8+juice+agar+as+a+general+purpose+medium+for+fungi+and+bacteria.">V-8 juice agar as a general purpose medium for fungi and bacteria.</a> Phytopathology. 45, 461-462 (1955).</li></ol>

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.

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		<tr height="42">
			<td height="42" style="height:42px;width:324px;">0.22 &#181;m pore size, acetate sterilizing filter</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">101102-280</td>
			<td style="width:203px;">Blueberry floral extract (FE) clarification&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">200-1000 &#181;l pipette with tips</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, any make within range will be adequate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">40-200 &#181;l pipette with tips</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, any make within range will be adequate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">5-40 &#181;l pipette with tips</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, any make within range will be adequate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Air spray gun disposable paint spray cup with connection adapter&#160;</td>
			<td style="width:203px;">Harbor Freight</td>
			<td style="width:203px;">97098</td>
			<td style="width:203px;">Blueberry rainwater (rw-)FE collection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Autoclave</td>
			<td style="width:203px;">Amsco</td>
			<td style="width:203px;">3011</td>
			<td style="width:203px;">Equipment, media preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Bar mesh matting (plastic mesh sheet)</td>
			<td style="width:203px;">Winco</td>
			<td style="width:203px;">BL-240</td>
			<td style="width:203px;">Passive (pass)-FE collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Benchtop timer</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">06-662-47</td>
			<td style="width:203px;">Equipment, FE preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Black pressure/vacuum hose</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">62994-795</td>
			<td style="width:203px;">Vacuum filter component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:324px;">Buchner funnel</td>
			<td style="width:203px;">Coors USA</td>
			<td style="width:203px;">60240</td>
			<td style="width:203px;">Vacuum filter component, accepts 55 mm filter paper disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Bunsen burner&#160;&#160;</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Calcium carbonate</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">C64-500</td>
			<td style="width:203px;">Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Centrifuge</td>
			<td style="width:203px;">Sorvall&#160;</td>
			<td style="width:203px;">RC 5B Plus</td>
			<td style="width:203px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Centrifuge tubes (15 ml)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">05-527-90</td>
			<td style="width:203px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Centrifuge tubes (50 ml)</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">10025-694</td>
			<td style="width:203px;">Equipment, rw-FE collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Cheesecloth (grade 50)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">AS240</td>
			<td style="width:203px;">Equipment, FE preparation</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:324px;">Chloroform</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">JT9175-3</td>
			<td style="width:203px;">Chemical, trichloromethane: assay grade, &#8805; 99% pure, for molecular biology, peroxide-free</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Corn Meal Agar (CMA)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">B11132</td>
			<td style="width:203px;">&#160;Pre-mix media, isolate storage on slants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Cotton-blue stain</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:203px;">61335</td>
			<td style="width:203px;">Lactophenol cotton-blue stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Curved forceps (45&#730;)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">10-270</td>
			<td style="width:203px;">Equipment, flower processing and coverslip inversion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Difco Agar</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">90004-032</td>
			<td style="width:203px;">Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Drill-press</td>
			<td style="width:203px;">Delta</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, rw-FE collection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">EASYpure LF Ultrapure water</td>
			<td style="width:203px;">Barnstead</td>
			<td style="width:203px;">D738</td>
			<td style="width:203px;">Equipment, deionized water source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Ethanol (95%)</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Filter flask (500 ml)</td>
			<td style="width:203px;">Pyrex</td>
			<td style="width:203px;">No. 5340</td>
			<td style="width:203px;">Vacuum filter component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Freezer (set to -20&#730; C)</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, storage of active-FE, pass-FE, rw-FE&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Fume hood</td>
			<td style="width:203px;">Hamilton</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, chloroform usage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Funnel (7 X 7 cm)&#160;</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">60820-110</td>
			<td style="width:203px;">Cranberry rw-FE collection, FE preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Generic glass slide&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">22-038-101</td>
			<td style="width:203px;">Bioassay conductance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Generic plastic pump spray bottle</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">16126-454</td>
			<td style="width:203px;">pass-FE collection, at least 250 ml capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Glass cell culture tubes</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Storage of ch-FE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Glass coverslips (22 x 22 mm)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">12-542B</td>
			<td style="width:203px;">Bioassay conductance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Glass Van Tieghem cells (hand cut glass tubes)</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Chloroform (ch)-FE bioassay, (8 mm OD 6 mm ID)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Glass-pipette (1-100 &#181;l)</td>
			<td style="width:203px;">Hamilton Co. Inc.</td>
			<td style="width:203px;">#710</td>
			<td style="width:203px;">ch-FE bioassay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Glycerol</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:203px;">G5516</td>
			<td style="width:203px;">Lactophenol cotton-blue stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Hemocytometer</td>
			<td style="width:203px;">Bright-Line</td>
			<td style="width:203px;">5971R10</td>
			<td style="width:203px;">Equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Incubator (set to 25&#730; C, dark)</td>
			<td style="width:203px;">Percival&#160;</td>
			<td style="width:203px;">50036</td>
			<td style="width:203px;">Equipment, bioassay conductance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Lactic acid</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:203px;">W261106</td>
			<td style="width:203px;">Lactophenol cotton-blue stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Laminar flow hood</td>
			<td style="width:203px;">Labconco</td>
			<td style="width:203px;">3730400</td>
			<td style="width:203px;">Equipment, sterile work environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Metal probe (generic)&#160;</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Microcentrifuge tubes (2 ml)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">05-408-138</td>
			<td style="width:203px;">Aqueous treatment mixture storage and preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Microscope, Leica DMLB</td>
			<td style="width:203px;">Leica</td>
			<td style="width:203px;">020-519.010</td>
			<td style="width:203px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Mortar (ceramic)</td>
			<td style="width:203px;">Coors USA</td>
			<td style="width:203px;">60313</td>
			<td style="width:203px;">Vacuum filter component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Nitrile gloves&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">19-130-1597D</td>
			<td style="width:203px;">Flower collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Paper disks (cut paper towels)</td>
			<td style="width:203px;">Office Basics</td>
			<td style="width:203px;">KCC01510</td>
			<td style="width:203px;">humidity control in bioassay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Parafilm</td>
			<td style="width:203px;">Bemis</td>
			<td style="width:203px;">PM-996</td>
			<td style="width:203px;">Plastic paraffin film&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Pestle (ceramic)</td>
			<td style="width:203px;">Coors USA</td>
			<td style="width:203px;">60314</td>
			<td style="width:203px;">Vacuum filter component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Phenol crystals</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">A92-100</td>
			<td style="width:203px;">Lactophenol cotton-blue stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Plastic bags (~100 mm X 152 mm)</td>
			<td style="width:203px;">Uline</td>
			<td style="width:203px;">S1294</td>
			<td style="width:203px;">Equipment, flower refrigeration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Plastic cell culture dishes (9 cm diameter)&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">FB0875712</td>
			<td style="width:203px;">(Petri dish), bioassay conductance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Polytetrafluoroethylene (PTFE) lined caps&#160;</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">60927-228</td>
			<td style="width:203px;">Storage of ch-FE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Pyrex beakers (100 ml)</td>
			<td style="width:203px;">Pyrex</td>
			<td style="width:203px;">No. 1000</td>
			<td style="width:203px;">Preparation of ch-FE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Pyrex bread-pan</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">pass-FE collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Pyrex graduated cylinder</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, FE preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Refrigerator (set to 4&#730; C)</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, storage of ch-FE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Sealed plastic container (30 mm X 13 mm X 7 mm)&#160;</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Bioassay conductance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Sharp-pointed dissecting scissors</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">8940</td>
			<td style="width:203px;">Equipment, to cut cheese-cloth and paper disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Stainless steel mesh strainer</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">470149-756</td>
			<td style="width:203px;">Preparation of ch-FE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Step drill bit (step-bit)</td>
			<td style="width:203px;">Dewalt</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Equipment, rw-FE collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Sterile loop (combi-loop)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">22-363-602&#160;</td>
			<td style="width:203px;">Culture preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Telephone wire (internal wires)</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Blueberry rw-FE collection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:324px;">Test tube basket&#160;</td>
			<td style="width:203px;">VWR</td>
			<td style="width:203px;">470137-792</td>
			<td style="width:203px;">Readily available substitution for plastic mesh [strawberry] basket&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">V8 Juice</td>
			<td style="width:203px;">Campbell&#39;s Soup Company</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">Fungal media component</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:324px;">Vintage plastic mesh [strawberry] baskets</td>
			<td style="width:203px;">Donation</td>
			<td style="width:203px;">-</td>
			<td style="width:203px;">pass-FE collection, can substitute for test tube basket (470137-792)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:324px;">Vortex Genie (Vortex)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:203px;">12-812</td>
			<td style="width:203px;">Spore suspension preparation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:324px;">Whatman No. 1 Qualitative 55 mm circles</td>
			<td style="width:203px;">Whatman</td>
			<td style="width:203px;">1001-055</td>
			<td style="width:203px;">Vacuum filter component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">White plastic twist ties (100 mm)</td>
			<td style="width:203px;">Uline</td>
			<td style="width:203px;">S-566W</td>
			<td style="width:203px;">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 ...

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This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Construction of a Low-cost Mobile Incubator for Field and Laboratory Use

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water ...