Conceptualization, H.C. and Z. Q. F.; Investigation, H. C., J. C, and Z. Q. F; Resources, Z. Q. F.; Writing - Original Draft, I. A. P.; Writing &#8211; Review &#38; Editing, I. A. P., J. C., H. C., S. L. T., and Z. Q. F.; Visualization, H. C.; Supervision, Z. Q. F.; Funding Acquisition, Z. Q. F and F. Q. L.
Project supported by a South Carolina Improving Teacher Quality Higher Education State Grant titled, Enhancing Middle Grades Science Teachers&#39; Knowledge of Plant Processes, Structures, and Functions Through Engagement in Plant Sciences Research (Plant Science)
Funds awarded by the United Sates Department of Education (CFDA Number 84.367B)
The authors would like to thank Dr. Nian Wang of the University of Florida for providing genomic DNA samples from infected Citrus sinensis Valencia.

1. Isolate Genomic DNA from Plant Tissue Using a Genomic Extraction Kit
<ol>
	<li>Obtain citrus leaf tissue by cutting a whole fresh leaf from a citrus tree using clean scissors, and place the leaf in a clean plastic sandwich bag. 
	NOTE: The bag containing the leaf tissue should be placed in a 4 &#176;C refrigerator or on ice in an insulated container as soon as possible after collection to avoid spoilage.</li>
	<li>Add a small amount of liquid nitrogen to chill a mortar and pestle. While they are chilling, use s...

<ol>
	<li>Field Office, F. l. o. r. i. d. 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> Citrus October Forecast Maturity Test Results and Fruit Size. , (2016).</li><li>Singerman, A., Useche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+citrus+greening+on+citrus+operations+in+Florida.">Impact of citrus greening on citrus operations in Florida.</a> Food and Resource Economics Department, UF/IFAS Extension. , (2015).</li><li>Spreen, T. H., Hodges, 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> The economic impact of HLB on the florida citrus industry. , (2012).</li><li>UICEP. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology. , (2013).</li><li>Jagoueix, S., Bove, J. M., Garnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phloem-limited+bacterium+of+greening+disease+of+citrus+is+a+member+of+the+alpha+subdivision+of+the+Proteobacteria.">The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria.</a> Int J Syst Bacteriol. 44 (3), 379-386 (1994).</li><li>Fujikawa, T., Iwanami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+detection+of+citrus+greening+(huanglongbing)+bacterium+&amp;#34;Candidatus+Liberibacter+asiaticus&amp;#34;+by+DNA+amplification+with+new+16S+rDNA-specific+primers.">Sensitive and robust detection of citrus greening (huanglongbing) bacterium &amp;#34;Candidatus Liberibacter asiaticus&amp;#34; by DNA amplification with new 16S rDNA-specific primers.</a> Mol. Cell. Probes. 26 (5), 194-197 (2012).</li><li>Davis, M. J., Mondal, S. N., Chen, H., Rogers, M. E., Brlansky, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-cultivation+of+'Candidatus+Liberibacter+asiaticus'+with+actinobacteria+from+citrus+with+huanglongbing.">Co-cultivation of 'Candidatus Liberibacter asiaticus' with actinobacteria from citrus with huanglongbing.</a> Plant Dis. 92 (11), 1547-1550 (2008).</li><li>Department of Agriculture &amp; National Agriculture Statistics Service. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Citrus Fruits 2015 Summary (September 2015). , (2015).</li><li>Hocquellet, A., Toorawa, P., Bov&#233;, J. M., Garnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+the+two+Candidatus+Liberobacter+species+associated+with+citrus+huanglongbing+by+PCR+amplification+of+ribosomal+protein+genes+of+the+&#946;+operon.">Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the &#946; operon.</a> Mol. Cell. Probes. 13 (5), 373-379 (1999).</li><li>Mafra, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+genes+for+accurate+transcript+normalization+in+citrus+genotypes+under+different+experimental+conditions.">Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.</a> PLoS One. 7 (2), e31263 (2012).</li><li>Li, W., Hartung, J. S., Levy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+real-time+PCR+for+detection+and+identification+of+Candidatus+Liberibacter+species+associated+with+citrus+huanglongbing.">Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing.</a> J. Microbiol. Meth. 66 (1), 104-115 (2006).</li></ol>

It is critical that all steps are followed exactly to achieve optimum experimental conditions. Whole leaf tissue was used during this demonstration; however, the protocol can be modified to theoretically include any type of plant tissue. Previously, researchers have extracted genomic DNA from leaf vein tissue, rather than whole leaf tissue, and achieved similar results11. If the band corresponding to the citrus FBOX gene fails to appear, the result is invalid, and there may be one or more...

Citrus greening, also known as huanglongbing, has caused a significant loss of citrus trees. One representative case is Florida, where the disease is forecasted to cause a 70% reduction in the production of Florida orange boxes from 244 million boxes in the 1997 - 1998 season to 70 million boxes as of the 2016 - 2017 season1. Over 90% of citrus trees in Florida are infected2, and it is estimated that the Florida citrus industry loses about one billion dollars each year due to citrus diseases, wherein citrus greening plays a major role3. Citrus greening is spread primarily by the invasive Asian citrus psyllid Diaphorina citri, but can also be spread by grafting of infected tissue. The disease causes yellowing of veins, asymmetrical yellow leaf mottling, premature defoliation, twig dieback, root decay, and death of the plant. Importantly, the disease causes the citrus plant to produce flowers out of season, which rarely produce fruit. The fruit that is produced by infected citrus plants are immature, green, and bitter tasting4.
The purpose of this method is to accurately and precisely identify the motile bacterium Candidatus liberibacter spp., the causative agent of citrus greening, living within the phloem of infected citrus trees5. Genomic DNA is extracted from whole leaf tissue, which contains the live bacteria. This extracted genomic DNA is used as a template for conventional PCR, where oligonucleotides complementary to the bacterium&#39;s 16S rDNA sequence are used to amplify this sequence. Oligonucleotides complementary to a citrus FBOX gene are used as an internal amplification control. We chose to use this method, because it has been proven to be successful in previous research6.
This method has the clear advantage of being simple, relatively inexpensive, and able to be performed in any normally equipped biochemistry lab. In addition, PCR remains the most accurate and precise method for detecting this pathogen, due to the difficulty of culturing this pathogen7, and the pathogen&#39;s ability to survive and reproduce for years within an asymptomatic host8. Many different specialty PCR assays have been used to successfully detect this pathogen; however, conventional PCR remains the simplest assay for accurate and specific detection, especially within asymptomatic hosts8.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Wizard Genomic DNA Purification Kit</td>
			<td style="width:175px;">Promega</td>
			<td style="width:111px;">A1120</td>
			<td style="width:111px;">Source of Nuclei Lysis, RNase, Protein Precipitation, and DNA Rehydration solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td>Air Products</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mastercycler pro with control panel</td>
			<td>Eppendorf</td>
			<td>6321000019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1250 W Microwave</td>
			<td>Panasonic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5424 table top centrifuge</td>
			<td>Eppendorf</td>
			<td>05-403-93</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isotemp 215 digital water bath</td>
			<td>Fisher scientific</td>
			<td>15-462-15Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metal spatula</td>
			<td>Sigma-Aldrich</td>
			<td>Z283274</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mortar and pestle</td>
			<td>Sigma-Aldrich</td>
			<td>Z247464</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSE Vortex Mixer</td>
			<td>Corning</td>
			<td>6775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 10 &#956;L tips</td>
			<td>TipOne</td>
			<td>1161-3730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 200 &#956;L tips</td>
			<td>TipOne</td>
			<td>1163-1730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 1250 &#956;L tips</td>
			<td>TipOne</td>
			<td>1161-1750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Variable Volume Pipettor Kit</td>
			<td>VWR</td>
			<td>75788-460</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethidium bromide</td>
			<td>Sigma-Aldrich</td>
			<td>E1510</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>IBI Scientific</td>
			<td>IB70041</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>17892</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Fisher Chemical</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris base</td>
			<td>Sigma-Aldrich</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#956;cuvette</td>
			<td>Eppendorf</td>
			<td>6138000018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stackable casting tray and combs</td>
			<td>Carolina</td>
			<td>213655</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini-Sub Cell GT System</td>
			<td>Bio-Rad</td>
			<td>1704487EDU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biospectrometer basic</td>
			<td>Eppendorf</td>
			<td>6135000009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 mL PCR tubes</td>
			<td>Fisher scientific</td>
			<td>AB-0620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL microcentrifuge tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>3439</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2X Taq Green PCR Master Mix</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forward Primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reverse Primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water, PCR grade</td>
			<td>Sigma-Aldrich</td>
			<td>3315953001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel Doc XR+</td>
			<td>Bio-Rad</td>
			<td>1708195</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 KB+ DNA ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>10787018</td>
			<td></td>
		</tr>
	</tbody>
</table>

specific and accurate detection of the citrus greening pathogen candidatus liberibacter spp. using conventional pcr on citrus leaf tissue samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

A positive result yields a distinct band corresponding to 500 bp for Candidatus Liberibacter asiaticus Las606/Lss6, and/or a distinct band corresponding to 700 bp for Laa2/Laj5, an insert in the 16S ribosomal &#946; operon9. The internal amplification control band must also appear at 400 bp. This band corresponds to amplification of the citrus FBOX gene10, and must appear f...

Watch this Scientific Journal Video about Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples at JoVE.com

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

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