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 scissors to cut out a small piece of leaf tissue, approximately 1 square inch in size. Place the cut tissue in the mortar to instantly freeze it. Add more liquid nitrogen, if needed. 
	NOTE: Insulated gloves should always be worn while handling liquid nitrogen.</li>
	<li>Quickly grind the plant tissue into a fine powder using the mortar. Continue grinding until the liquid nitrogen completely evaporates, and a fine green powder remains.</li>
	<li>Briefly chill a metal spatula in liquid nitrogen for 10 -&#160;15 s, then scoop the ground tissue inside the mortar into a 1.5 mL microcentrifuge tube, using the spatula.</li>
	<li>Add 600 &#181;L of Nuclei Lysis Solution (see Table of Materials) using a 1000 &#181;L pipette and vortex the sample for 1 - 3 s, then incubate in a 65 &#176;C water bath for 15 min.</li>
	<li>Add 3 &#181;L of RNase Solution to the lysate using a 10&#181;L pipette, invert the tube 2 - 5 times to mix it, and incubate at 37 &#176;C in a cabinet incubator for 15 min. 
	NOTE: Allow the mixture to cool to room temperature before proceeding.</li>
	<li>Add 200 &#181;L of Protein Precipitation Solution, vortex at high speed for 20 s, then centrifuge for 3 min at 13,000 x g to form a firm pellet. While the sample is being centrifuged, add 600 &#181;L of room temperature isopropanol to a fresh 1.5 mL microcentrifuge tube.</li>
	<li>Using a 1000 &#181;L pipette, carefully remove the supernatant from the centrifuged sample, transferring it to the fresh 1.5 mL microcentrifuge tube containing isopropanol. The pellet may now be discarded. 
	NOTE: Avoid contaminating the supernatant with protein by leaving a miniscule amount of supernatant above the pellet.</li>
	<li>Invert the tube until the DNA becomes visible as a mass of thread-like strands, then centrifuge at 13,000 x g for 1 min at room temperature.</li>
	<li>Decant the supernatant, add 600 &#181;L of 70% ethanol to the pellet, invert the tube several times to wash the DNA, and centrifuge at 13,000 x g for 1 min.</li>
	<li>Carefully aspirate the ethanol, leaving the loose DNA pellet, then open and invert the tube, resting on absorbent paper. Air dry at room temperature for 15 min.</li>
	<li>Add 100 &#181;L of DNA Rehydration Solution to the dried DNA pellet, then incubate the DNA in a 65 &#176;C water bath for 1 h, while mixing periodically by tapping the tube.</li>
	<li>Place 1 &#181;L of purified water on a microcuvette, and insert it into a spectrophotometer to be used as a blank. Calculate the concentration of DNA present by placing 1 &#181;L of sample on the microcuvette, and inserting into the spectrophotometer. DNA concentration (ng/&#181;L) can be calculated as (A260- A320) &#215; dilution factor &#215; 50 ng/&#181;L. 
	NOTE: The protocol can be paused here. The sample can be stored at 4 &#176;C.</li>
</ol>
2. Perform PCR on Genomic DNA Samples
<ol>
	<li>Combine 10 &#181;L 2x PCR Master Mix (see Table of Materials), 1 &#181;L forward primer (10 pM/&#181;L), 1 &#181;L reverse primer (10 pM/&#181;L) (see Table 2 for primer sequences), 100 ng of genomic DNA extracted from the sample, and purified water (up to 20 &#181;L final volume) in a 50 &#181;L PCR tube, flick to mix, and briefly centrifuge.</li>
	<li>Insert the PCR tube into the thermocycler and run accordingly (See Table 1). 
	NOTE: The protocol can be paused here. If necessary, store sample at 4 &#176;C after the reaction is complete.</li>
</ol>
3. Electrophorese the Amplified DNA in an Agarose Gel
<ol>
	<li>Weigh 0.4 g agarose powder and add it along with 1x TAE buffer to a clean 200 mL Erlenmeyer flask, filling up to 50 mL.</li>
	<li>Microwave on high for 1.5 min, shaking every 30 s, until agarose is fully dissolved. 
	NOTE: Insulated gloves should be worn while handling liquid agarose to prevent burns.</li>
	<li>Add 2.5 &#181;L of 10 mg/mL ethidium bromide to the liquid agarose and shake to mix, then pour the liquid agarose gel into a level gel mold. Quickly insert standard 12-tooth gel combs after pouring.</li>
	<li>Allow the gel to cool for 20 min at room temperature, then remove gel combs, and place the gel into a level gel box filled with 1x TAE buffer.</li>
	<li>Using a micropipette, load the total amplified DNA samples into wells. Add 5 &#181;L of DNA ladder (see Table of Materials) in a separate well from your samples.</li>
	<li>Electrophorese the samples for 35 min at a constant 90 V, then remove the gel and place in a UV transilluminator.</li>
	<li>Photograph and analyze the gel banding pattern while using UV transillumination at 302 nm.</li>
</ol>

<ol><li>Field Office, F. lorida <a target="_blank" href="http://citrusindustry.net/wp-content/uploads/2016/10/cit1016.pdf">Citrus October Forecast Maturity Test Results and Fruit Size</a>. , USDA, NASS, Florida Field Office. (2016).</li>
<li>Singerman, A., Useche, P. <a target="_blank" href="https://pdfs.semanticscholar.org/2aa9/2db973c8f73893a0799ef82cb8883821127b.pdf">Impact of citrus greening on citrus operations in Florida.</a> Food and Resource Economics Department, UF/IFAS Extension. , EDIS (FE983) (2015).</li>
<li>Spreen, T. H., Hodges, A. <a target="_blank" href="https://conference.ifas.ufl.edu/citrus12/PDF%20Presentations/Fri-pdf/1045%20Spreen.pdf">The economic impact of HLB on the florida citrus industry</a>. , University of Florida. (2012).</li>
<li>UICEP. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Pathology%22">Pathology</a>. , (2013).</li>
<li>Jagoueix, S., Bove, J. M., Garnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+phloem-limited+bacterium+of+greening+disease+of+citrus+is+a+member+of+the+alpha+subdivision+of+the+Proteobacteria%5BTitle%5D)+AND+%22Int+J+Syst+Bacteriol%22%5BJournal%5D)">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/pubmed?term=((Sensitive+and+robust+detection+of+citrus+greening+%28huanglongbing%29+bacterium+%26%2334%3BCandidatus+Liberibacter+asiaticus%26%2334%3B+by+DNA+amplification+with+new+16S+rDNA-specific+primers%5BTitle%5D)+AND+%22Mol.+Cell.+Probes%22%5BJournal%5D)">Sensitive and robust detection of citrus greening (huanglongbing) bacterium &#34;Candidatus Liberibacter asiaticus&#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="https://apsjournals.apsnet.org/doi/abs/10.1094/PDIS-92-11-1547">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 & National Agriculture Statistics Service. <a target="_blank" href="https://www.cacitrusmutual.com/wp-content/uploads/2015/12/2015-USDA-Citrus-Fruits-Summary.pdf">Citrus Fruits 2015 Summary (September 2015)</a>. , (2015).</li>
<li>Hocquellet, A., Toorawa, P., Bové, J. M., Garnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+and+identification+of+the+two+Candidatus+Liberobacter+species+associated+with+citrus+huanglongbing+by+PCR+amplification+of+ribosomal+protein+genes+of+the+%CE%B2+operon%5BTitle%5D)+AND+%22Mol.+Cell.+Probes%22%5BJournal%5D)">Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the β 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/pubmed?term=((Reference+genes+for+accurate+transcript+normalization+in+citrus+genotypes+under+different+experimental+conditions%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Quantitative+real-time+PCR+for+detection+and+identification+of+Candidatus+Liberibacter+species+associated+with+citrus+huanglongbing%5BTitle%5D)+AND+%22J.+Microbiol.+Meth%22%5BJournal%5D)">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>

Huan Chen, Ian Arthur Palmer, Jian Chen, Ming Chang, Stephen L. Thompson, Fengquan Liu, and Zheng Qing Fu declare no conflicts of interest.

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

specific-accurate-detection-citrus-greening-pathogen-candidatus

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7.6K Views.  University of South Carolina. Citrus "vergroening" is een bijzonder destructieve ziekte die citrusvruchten wereldwijd. Hier gepresenteerd is een eenvoudige methode om met behulp van PCR en genomic DNA-extractie van citrus blad weefsel voor de nauwkeurige en precieze identificatie van het pathogeen citrus vergroening, Candidatus liberibacter spp.

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

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

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

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Immunology and Infection

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while &#34;Candidatus Liberibacter asiaticus&#34; has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California&#39;s citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step.
Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/&#181;L) that was comparable with the CCPP&#39;s hand-chopping protocol (77.84 ng/&#181;L). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

We engineered, fabricated, and validated an instrument that rapidly processes phloem-rich bark citrus budwood tissues. Compared to current methods, the budwood tissue extractor (BTE) has increased sample throughput and decreased the required labor and equipment costs.

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious ...

Bioengineering

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A simple method for rapid and portable Huanglongbing (HLB) diagnosis is presented here that combines recombinase polymerase amplification and a fluorescent reporter utilizing the nuclease activity of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a (CRISPR-Cas12a) system. The sensitivity of this technique is much higher than PCR. Furthermore, this method showed similar results to qPCR when leaf samples were used. Compared with conventional CLas detection methods, the detection method presented here can be completed in 90 min and works in an isothermal condition that does not require the use of PCR machines. In addition, the results can be visualized through a handheld fluorescent detection device in the field.

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

In this work, a rapid, sensitive, and portable detection method for Candidatus Liberibacter asiaticus based on recombinase polymerase amplification combined with CRISPR-Cas12a was developed.

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A ...

Biology

Molecule Delivery in Plants Using a Direct Infusion Device

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

Author Spotlight: Direct Infusion Device for Molecule Delivery in Plants  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

Rapid In Vivo Leaf Inoculation for Screening Poplar Stem Canker Resistance

In Vivo Leaf Inoculation: An Alternative Method to Assess the Disease Resistance of Hybrid Clones in Poplar Breeding of Stem Canker Disease

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. &#38; de Not. are the two major forest diseases in the poplar plantations in China, sometimes which can destroy all the poplar seedlings or severely damage mature poplar forests. Hybrid breeding is the most direct and efficient method of controlling and managing tree diseases. However, assessing disease resistance or selecting disease-resistance clones based on In vitro stem inoculation is inefficient, time-consuming, and expensive, limiting the development of hybrid breeding of poplar stem canker disease. In this study, we proposed an alternative method to assess disease resistance to stem canker pathogens through in vivo leaf inoculation. The test materials used in this method can be on 1-year-old poplar saplings or the annual branches of perennial poplars in the greenhouse or the field. The critical step of this alternative method is the selection of inoculating leaves: the 5-7th newly matured leaves might be the most suitable. The second critical step of the leaf inoculation method is to make wounds on plant leaves through needle pierces, providing sufficient lesions to measure disease severity. For the adequate number of leaves produced in the early stage of poplar breeding, this in vivo leaf inoculation contributes to the rapid, accurate, and large-scale screening of the disease-resistance poplar clones to stem canker pathogens. Moreover, this leaf inoculation method will also serve as an efficient method for screening pathotypes of stem canker disease pathogen C. chrysosperma, B.dothidea, or other poplar stem canker pathogens.

Author Spotlight: High-Throughput In Vivo Leaf Inoculation for Accelerating Disease Resistance Screening in Poplar Hybrid Breeding

We provide a step-by-step protocol for assessing poplar resistance to stem canker pathogens using an in vivo leaf inoculation method. This method is especially suitable for large-scale assessment of Cytospora chrysosperma and Botryosphaeria dothidea canker disease resistance in poplar breeding progeny in China.

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. &#38; de Not. are the two ...

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

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