The authors acknowledge the Cahuilla people as the Traditional Custodians of the Land on which the experimental work was completed. We are grateful to Professor Norman Ellstrand at the University of California, Riverside, for providing lab space to carry out research activities for this project under the UCR California Agriculture and Food Enterprise (CAF&#201;) Initiative. This research was supported by the CDFA - Specialty Crop Block Grant Program (grant no. 18-0001-055-SC). Additional support was also provided by the CRB project 6100; USDA National Institute of Food and Agriculture, Hatch project 1020106; and the National Clean Plant Network-USDA Animal and Plant Health Inspection Service (AP17PPQS&#38;T00C118, AP18PPQS&#38;T00C107, AP19PPQS&#38;T00C148, &#38; AP20PPQS&#38;T00C049) awarded to Georgios Vidalakis.

<ol>
	<li>Verni&#232;re, C., 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=Interactions+between+citrus+viroids+affect+symptom+expression+and+field+performance+of+clementine+trees+grafted+on+trifoliate+orange.">Interactions between citrus viroids affect symptom expression and field performance of clementine trees grafted on trifoliate orange.</a> Phytopathology. 96 (4), 356-368 (2006).</li><li>Verni&#232;re, C., 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=Citrus+viroids:+Symptom+expression+and+effect+on+vegetative+growth+and+yield+of+clementine+trees+grafted+on+trifoliate+orange.">Citrus viroids: Symptom expression and effect on vegetative growth and yield of clementine trees grafted on trifoliate orange.</a> Plant Disease. 88 (11), 1189-1197 (2004).</li><li>Zhou, C., Talon, M., Caruso, M., Gmitter, F. G., 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=Chapter+19+-+Citrus+viruses+and+viroids.">Chapter 19 - Citrus viruses and viroids.</a> The Genus Citrus. , 391-410 (2020).</li><li>Trends and issues facing the U.S. citrus industry. Choices Magazine Online Available from: <a target="_blank" href="https://www.choicesmagazine.org/choices-magazine/theme-articles/trends-and-challenges-in-fruit-and-tree-nut-sectors/trends-and-issues-facing-the-us-citrus-industry">https://www.choicesmagazine.org/choices-magazine/theme-articles/trends-and-challenges-in-fruit-and-tree-nut-sectors/trends-and-issues-facing-the-us-citrus-industry</a> (2021)</li><li>Fruit and Tree Nuts Outlook. United States Department of Agriculture-Economic Research Service Available from: <a target="_blank" href="https://www.ers.usda.gov/webdocs/outlooks/98171/fts-370.pdf?v=5697">https://www.ers.usda.gov/webdocs/outlooks/98171/fts-370.pdf?v=5697</a> (2020)</li><li>Forsyth, J., Fruits Damiani, J. C. i. t. r. u. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrus+Fruits.+Types+on+the+market.">Citrus Fruits. Types on the market.</a> Encyclopedia of Food Sciences and Nutrition. , 1329-1335 (2003).</li><li>Bostock, R. M., Thomas, C. S., Hoenisch, R. W., Golino, D. A., Vidalakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+health:+How+diagnostic+networks+and+interagency+partnerships+protect+plant+systems+from+pests+and+pathogens.">Plant health: How diagnostic networks and interagency partnerships protect plant systems from pests and pathogens.</a> California Agriculture. 68 (4), 117-124 (2014).</li><li>Osman, F., Dang, T., Bodaghi, S., Vidalakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+multiplex+RT-qPCR+detects+three+citrus+viroids+from+different+genera+in+a+wide+range+of+hosts.">One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.</a> Journal of Virological Methods. 245, 40-52 (2017).</li><li>Wang, J., 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=Past+and+future+of+a+century+old+Citrus+tristeza+virus+collection:+A+California+citrus+germplasm+tale.">Past and future of a century old Citrus tristeza virus collection: A California citrus germplasm tale.</a> Frontiers in Microbiology. 4, 366 (2013).</li><li>Gergerich, R. C., 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=Safeguarding+fruit+crops+in+the+age+of+agricultural+globalization.">Safeguarding fruit crops in the age of agricultural globalization.</a> Plant Disease. 99 (2), 176-187 (2015).</li><li>Moreno, P., Ambr&#243;s, S., Albiach-Mart&#237;, M. R., Guerri, J., Pe&#241;a, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrus+tristeza+virus:+A+pathogen+that+changed+the+course+of+the+citrus+industry.">Citrus tristeza virus: A pathogen that changed the course of the citrus industry.</a> Molecular Plant Pathology. 9 (2), 251-268 (2008).</li><li>Yokomi, R. K., 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=Identification+and+characterization+of+Citrus+tristeza+virus+isolates+breaking+resistance+in+trifoliate+orange+in+California.">Identification and characterization of Citrus tristeza virus isolates breaking resistance in trifoliate orange in California.</a> Phytopathology. 107 (7), 901-908 (2017).</li><li>Selvaraj, V., Maheshwari, Y., Hajeri, S., Yokomi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+detection+tool+for+VT+isolates+of+Citrus+tristeza+virus+by+immunocapture-reverse+transcriptase+loop-mediated+isothermal+amplification+assay.">A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture-reverse transcriptase loop-mediated isothermal amplification assay.</a> PLoS One. 14 (9), 0222170 (2019).</li><li>Babcock, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+impact+of+California's+citrus+industry+in+2020.">Economic impact of California's citrus industry in 2020.</a> Journal of Citrus Pathology. 9, (2022).</li><li>Gottwald, T. R., Polek, M., Riley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History,+present+incidence,+and+spatial+distribution+of+Citrus+tristeza+virus+in+the+California+central+valley.">History, present incidence, and spatial distribution of Citrus tristeza virus in the California central valley.</a> International Organization of Citrus Virologists Conference Proceedings (1957-2010). 15, (2002).</li><li>Yokomi, R., 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=Molecular+and+biological+characterization+of+a+novel+mild+strain+of+citrus+tristeza+virus+in+California.">Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California.</a> Archives of Virology. 163 (7), 1795-1804 (2018).</li><li>Fuchs, M., 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=Economic+studies+reinforce+efforts+to+safeguard+specialty+crops+in+the+United+States.">Economic studies reinforce efforts to safeguard specialty crops in the United States.</a> Plant Disease. 105 (1), 14-26 (2021).</li><li>The real cost of HLB in Florida. Citrus Industry Magazine Available from: <a target="_blank" href="https://citrusindustry.net/2019/07/30/the-real-cost-of-hib-in-florida/">https://citrusindustry.net/2019/07/30/the-real-cost-of-hib-in-florida/</a> (2019)</li><li>McRoberts, N., 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=Using+models+to+provide+rapid+programme+support+for+California's+efforts+to+suppress+Huanglongbing+disease+of+citrus.">Using models to provide rapid programme support for California's efforts to suppress Huanglongbing disease of citrus.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 374 (1776), 20180281 (2019).</li><li>Albrecht, C., 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=Action+plan+for+Asian+citrus+psyllid+and+huanglongbing+(citrus+greening)+in+California.">Action plan for Asian citrus psyllid and huanglongbing (citrus greening) in California.</a> Journal of Citrus Pathology. 7 (1), (2020).</li><li>Navarro, L., 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=The+Citrus+Variety+Improvement+Program+in+Spain+in+the+period+1975-2001.">The Citrus Variety Improvement Program in Spain in the period 1975-2001.</a> International Organization of Citrus Virologists Conference Proceedings (1957-2010). 15 (15), (2002).</li><li>Vidalakis, G., Gumpf, D. J., Polek, M. L., Bash, J. A., Ferguson, L., Grafton-Cardwell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+California+Citrus+Clonal+Protection+Program.">The California Citrus Clonal Protection Program.</a> Citrus Production Manual. , 117-130 (2014).</li><li>Dang, T., Rao, A. L. N., Lavagi-Craddock, I., Vidalakis, G., 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=High-throughput+RNA+extraction+from+citrus+tissues+for+the+detection+of+viroids.">High-throughput RNA extraction from citrus tissues for the detection of viroids.</a> In Viroids: Methods and Protocols. 2316, (2022).</li><li>Osman, F., Vidalakis, G., Rao, A. L. N., Lavagi-Craddock, I., Vidalakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+detection+of+viroids+using+singleplex+and+multiplex+quantitative+polymerase+chain+reaction.">Real-time detection of viroids using singleplex and multiplex quantitative polymerase chain reaction.</a> Viroids: Methods and Protocols. 2316, (2022).</li><li>Li, R., 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=A+reliable+and+inexpensive+method+of+nucleic+acid+extraction+for+the+PCR-based+detection+of+diverse+plant+pathogens.">A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens.</a> Journal of Virological Methods. 154 (1-2), 48-55 (2008).</li><li>Saponari, M., Manjunath, K., Yokomi, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+of+Citrus+tristeza+virus+in+citrus+and+aphids+by+real-time+reverse+transcription-PCR+(TaqMan).">Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan).</a> Journal of Virological Methods. 147 (1), 43-53 (2008).</li><li>Damaj, M. B., 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=Reproducible+RNA+preparation+from+sugarcane+and+citrus+for+functional+genomic+applications.">Reproducible RNA preparation from sugarcane and citrus for functional genomic applications.</a> International Journal of Plant Genomics. 2009, 765367 (2009).</li><li>Dang, T., 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=First+report+of+citrus+leaf+blotch+virus+infecting+Bearss+lime+tree+in+California.">First report of citrus leaf blotch virus infecting Bearss lime tree in California.</a> Plant Disease. 104 (11), 3088 (2020).</li><li>Manchester, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+UV+methods+for+measurement+of+protein+and+nucleic+acid+concentrations.">Use of UV methods for measurement of protein and nucleic acid concentrations.</a> BioTechniques. 20 (6), 968-970 (1996).</li><li>Teare, J. M., 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=Measurement+of+nucleic+acid+concentrations+using+the+DyNA+QuantTM+and+the+GeneQuantTM.">Measurement of nucleic acid concentrations using the DyNA QuantTM and the GeneQuantTM.</a> BioTechniques. 22 (6), 1170-1174 (1997).</li><li>Imbeaud, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+of+RNA+quality+assessment+using+user-independent+classifiers+of+microcapillary+electrophoresis+traces.">Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.</a> Nucleic Acids Research. 33 (6), 56-56 (2005).</li><li>Menzel, W., Jelkmann, W., Maiss, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+four+apple+viruses+by+multiplex+RT-PCR+assays+with+coamplification+of+plant+mRNA+as+internal+control.">Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control.</a> Journal of Virological Methods. 99 (1-2), 81-92 (2002).</li><li>Vidalakis, G., Rao, A. L. N., Lavagi-Craddock, I., Vidalakis, G., 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=SYBR+Green+RT-qPCR+for+the+universal+detection+of+citrus+viroids.">SYBR Green RT-qPCR for the universal detection of citrus viroids.</a> Viroids: Methods and Protocols. , 211-217 (2022).</li><li>Arredondo Vald&#233;s, R., 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=A+review+of+techniques+for+detecting+Huanglongbing+(greening)+in+citrus.">A review of techniques for detecting Huanglongbing (greening) in citrus.</a> Canadian Journal of Microbiology. 62 (10), 803-811 (2016).</li><li>Li, S., Wu, F., Duan, Y., Singerman, A., Guan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrus+greening:+Management+strategies+and+their+economic+impact.">Citrus greening: Management strategies and their economic impact.</a> HortScience. 55 (5), 604-612 (2020).</li><li>. CDFA California Citrus Pest and Disease Prevention Program Operations Subcomittee Meeting. Meeting Minutes Available from: <a target="_blank" href="https://www.cdfa.ca.gov/citrus/docs/minutes/2019/OpsSubcoMinutes-11062019.pdf">https://www.cdfa.ca.gov/citrus/docs/minutes/2019/OpsSubcoMinutes-11062019.pdf</a> (2019)</li></ol>

The authors declare no competing financial interests.

With the advent of HLB citrus disease, to reduce losses, the citrus industry, regulatory agencies, and diagnostic laboratories have been urged to rely on high-throughput nucleic acid extraction methods combined with low-throughput manual sample processing and pathogen detection assays such as qPCR34 for the testing of individual trees, in combination with disease management practices35. California's HLB positivity rate has gone from 0.01% in 2012 to 1.2% in 2020. Even thoug...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65159">Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering</a>. The Authors section was updated from:
Deborah Pagliaccia1,6 
Douglas Hill2 
Emily Dang1 
Gerardo Uribe1 
Agustina De Francesco1 
Ryan Milton2 
Anthony De La Torre2 
Axel Mounkam2 
Tyler Dang1 
Sohrab Botaghi1 
Irene Lavagi-Craddock1 
Alexandra Syed1 
William Grover3 
Adriann Okamba4,5 
Georgios Vidalakis2 
1Department of Microbiology and Plant Pathology, University of California Riverside 
2Technology Evolving Solutions (TES) 
3Department of Bioengineering, University of California Riverside 
4Ecole Sup&#233;rieure d&#39;Ing&#233;nieurs L&#233;onard de Vinci ESILV 
5University of California Riverside 
6California Agriculture and Food Enterprise (CAF&#201;), University of California Riverside
to:
Deborah Pagliaccia1,6 
Douglas Hill2 
Emily Dang1 
Gerardo Uribe1 
Agustina De Francesco1 
Ryan Milton2 
Anthony De La Torre2 
Axel Mounkam2 
Tyler Dang1 
Sohrab Bodaghi1 
Irene Lavagi-Craddock1 
Alexandra Syed1 
William Grover3 
Adriann Okamba4,5 
Georgios Vidalakis1 
1Department of Microbiology and Plant Pathology, University of California Riverside 
2Technology Evolving Solutions (TES) 
3Department of Bioengineering, University of California Riverside 
4Ecole Sup&#233;rieure d&#39;Ing&#233;nieurs L&#233;onard de Vinci ESILV 
5University of California Riverside 
6California Agriculture and Food Enterprise (CAF&#201;), University of California Riverside

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65159">Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering</a>. The Authors section was updated.

Erratum: Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

Graft-transmissible phloem-limited pathogens of citrus, such as viroids, viruses, and bacteria, have caused devastating epidemics and serious economic losses in every citrus-producing area of the world. Citrus viroids are limiting production factors because of the exocortis and cachexia diseases they cause in economically important citrus types, such as trifoliate, trifoliate hybrids, mandarins, clementines, and tangerines1,2,3. In California, these viroid-sensitive citrus types are the basis of the growing and profitable market of &#34;easy-peelers&#34;, following the shifting trend in consumers&#39; preference for fruits that are easy to peel, segmented, and seedless4,5,6. Thus, citrus viroids are regulated under the California Department of Food and Agriculture (CDFA) &#34;Citrus Nursery Stock Pest Cleanliness Program-Senate Bill 140&#34;, and the laboratories of CDFA&#39;s Plant Pest Diagnostics Branch perform thousands of citrus viroid tests annually7,8,9,10. Citrus tristeza virus (CTV) has been responsible for the death of over 100 million citrus trees since the beginning of the global epidemic in the 1930s3,9,10,11. In California, stem pitting and trifoliate breaking resistance isolates of the virus pose a serious threat to the $3.6 billion California citrus industry12,13,14. Consequently, CDFA classifies CTV as a regulated class-A plant pest, and the laboratory of the Central California Tristeza Eradication Agency (CCTEA) performs extensive field surveys and thousands of virus tests every year15,16. The bacterium &#34;Candidatus Liberibacter asiaticus&#34; (CLas) and the huanglongbing (HLB) disease are estimated to have caused close to $9 billion of economic damage to Florida as a result of a 40% reduction of citrus acreage, a 57% decrease in citrus operations, and a loss of almost 8,000 jobs17,18. In California, a hypothetical 20% reduction in citrus acreage due to HLB was predicted to result in more than 8,200 job losses and a reduction of over half a billion dollars in the state&#39;s gross domestic product. Therefore, the Citrus Pest and Disease Prevention Program spends over $40 million annually on surveys to test, detect, and eradicate CLas from California14,17,19,20.
A key element of the management of citrus viroids, viruses, and bacteria is the use of pathogen-tested propagative materials (i.e., budwood) for tree production. Pathogen-tested citrus budwood is produced and maintained within comprehensive quarantine programs that employ advanced pathogen elimination and detection techniques10,21. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, tests thousands of budwood samples every year from citrus varieties newly imported into the state and the USA, as well as citrus budwood source trees, to protect California&#39;s citrus and support the functions of the National Clean Plant Network for Citrus10,17,22. To handle the large volume of citrus testing, high-throughput, reliable, and cost-effective pathogen detection assays are a fundamental component for the success of programs such as the CCPP7,10,22.
While molecular-based pathogen detection assays such as polymerase chain reaction (PCR) have allowed for significant increases in throughput in plant diagnostic laboratories, in our experience, one of the most critical bottlenecks in the implementation of high-throughput protocols is the plant tissue sample processing step. This is particularly true for citrus because the currently available protocols for the processing of phloem-rich tissues such as leaf petioles and budwood bark are labor-intensive, time-consuming, and require expensive and specialized laboratory equipment. These protocols require hand-chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation8,23,24. For example, at the CCPP diagnostic laboratory, sample processing includes (i) hand-chopping (6-9 samples/h/operator), (ii) freeze-drying (16-24 h), (iii) pulverization (30-60 s), and (iv) centrifugation (1-2 h). The process also requires specialized supplies (e.g., heavy-duty safe-lock tubes, stainless steel grinding balls, adapters, blades, gloves) and multiple pieces of costly lab equipment (e.g., ultra-low freezer, freeze-dryer, tissue pulverizer, liquid nitrogen cryostation, refrigerated centrifuge).
As in any industry, equipment engineering and the automation of processes are key to lowering costs, increasing throughput, and providing high-quality, uniform product and services. The citrus industry needs low-cost tissue-processing instruments that require minimum skill to operate and, as such, are easy to transfer to diagnostic laboratories and field operations to allow high sample-processing capacity for rapid downstream pathogen detection. Technology Evolving Solutions (TES) and the CCPP developed (i.e., design and fabricate) and validated (i.e., tested with citrus samples and compared to standard laboratory procedures) a low-cost (i.e., eliminated the need for specialized laboratory equipment) instrument for the rapid processing of phloem-rich citrus tissues (i.e., budwood), named the budwood tissue extractor (BTE). As seen in Figure 1, the BTE includes a base component for power and controls, plus a removable chamber for the processing of citrus budwood. The BTE chamber is composed of a grinding wheel specifically designed to strip the phloem-rich bark tissues from the citrus budwood. The shredded bark tissue is ejected rapidly through a slide port into a syringe containing extraction buffer, filtered, and made ready for nucleic acid extraction and purification without any additional handling or preparation (Figure 1). The BTE system also includes a paperless sample tracking application and an integrated weighing application, which record the sample processing information in an online database in real time.
The BTE system has increased the CCPP&#39;s lab diagnostic capacity by over 100% and has consistently produced citrus tissue extracts suitable for the purification of high-quality nucleic acids and the downstream detection of graft-transmissible pathogens of citrus using PCR assays. More specifically, BTE has reduced the time for tissue processing from over 24 h to ~3 min per sample, replaced laboratory instruments costing over $60,000 (Figure 2, steps 2-4), and allowed for the processing of larger sample sizes.
This paper presents the BTE high-throughput citrus bark tissue processing, nucleic acid extraction, and pathogen detection validation data with citrus budwood samples from source trees, including all the appropriate positive and negative controls from the CCPP Rubidoux Quarantine Facility and Lindcove Foundation Facility, respectively. We also present the throughput and processing time changes compared to the current laboratory procedure (Figure 2). In addition, this work provides a detailed, step-by-step protocol for citrus pathogen testing laboratories and demonstrates how the BTE can support the functions of pathogen-clean nursery stock, survey, and eradication programs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65159/65159fig01.jpg" /> 
Figure 1: Budwood tissue extractor. The BTE includes a base component for power and controls, plus a removable chamber for the processing of citrus budwood. The BTE chamber is composed of a grinding wheel specifically designed to strip the phloem-rich bark tissues from citrus budwood. The shredded bark tissue is ejected rapidly through a slide port into a syringe, filtered, and made ready for nucleic acid extraction and purification without any additional handling or preparation. Abbreviation: BTE = budwood tissue extractor. <a href="https://www.jove.com/files/ftp_upload/65159/65159fig01large.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/65159/65159fig02.jpg" /> 
Figure 2: Step-by-step comparison between the conventional hand-chopping lab procedure and BTE processing. BTE processing involves high-throughput citrus bark tissue processing, nucleic acid extraction, and pathogen detection. The time for each step is indicated in parentheses. <a href="https://www.jove.com/files/ftp_upload/65159/65159fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.08&quot; Hex Trimmer line</td><td>PowerCare</td><td>FPRO07065</td><td>Needed to replace blades.</td></tr><tr><td>1 Hp, 8 gal air compressor</td><td>California Air Tools</td><td>8010</td><td>Quickly dry chambers after rinsed</td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>Globe Scientific</td><td>111558B</td><td>Store sample in after swishing with syinges</td></tr><tr><td>10 mL Syringe Set</td><td>Technology Evolving Solutions</td><td>TE006-F1-10A-G1000-E1</td><td>Syringe material is cut into. 1 L bottle with guanidine thiocyanate buffer. WARNING - contains guanidine thiocyanate, hazardous waste service required - do not mix with bleach</td></tr><tr><td>12&quot; Ruler</td><td>Westcott</td><td>&amp;lrm;16012</td><td>To measure trimmer line before cutting</td></tr><tr><td>12% Sodium Hypochlorite</td><td>Hasa</td><td>1041</td><td>Disinfects chambers after processing</td></tr><tr><td>-20 C Freezer</td><td>Insignia</td><td>NS-CZ70WH0</td><td>Store sample after processing</td></tr><tr><td>4&quot; x 12&quot; plastic bags</td><td>Plymor</td><td>FP20-4x12-10</td><td>Bags to hold branches during shipping. O-rings attach bag to BTE chamber to seal</td></tr><tr><td>6&quot; Cotton Swab</td><td>Puritan</td><td>806-PCL</td><td>Swab to remove clogs</td></tr><tr><td>7 Gallon Storage Tote</td><td>HDX</td><td>206152</td><td>Holds sodium hypochlorite solution to disinfect chambers and water to rinse chambers</td></tr><tr><td>Air blow gun</td><td>JASTIND</td><td>&amp;lrm;JTABG103A</td><td>Directs air into the chambers at high pressure</td></tr><tr><td>Black Sharpie</td><td>Sharpie</td><td>&amp;nbsp;S-19421</td><td>Mark 1.5 mL tubes so you can identify sample later</td></tr><tr><td>Bottle Top Dispensor</td><td>Brand</td><td>Z627569</td><td>Adjustable bottle top dispensor to dispense guandine into syringe</td></tr><tr><td>BTE Chamber</td><td>Technology Evolving Solutions</td><td>TE002BB-A05-E1</td><td>Used to process budwood. Includes O-rings, BTE Slide, slide plunger, drain valve, lid, blade set, and blade set removal tool</td></tr><tr><td>Dish Soap</td><td>Dawn</td><td>57445CT</td><td>Surfectant to improve sodium hypochlorite penetration into chamber</td></tr><tr><td>Fume hood with hepa filter</td><td>Air Science</td><td>P5-36XT-A</td><td>Fume hood with hepa filter (ASTS-030)&amp;nbsp; to limit possible contamination and protect against chemical spills</td></tr><tr><td>Insulated foam shipping container</td><td>PolarTech</td><td>261/J50C</td><td>Insulated shipping container to ship samples on ice after they are collected</td></tr><tr><td>Lab coat</td><td>Red Kap</td><td>KP14WH LN 46</td><td>Lab coat to limit possible contamination and protect against chemical spills</td></tr><tr><td>Laptop</td><td>Microsoft</td><td>Surface</td><td>Wifi capable laptop to run TES GUI. Needed for initial setup and provides more indepth information about the tissue processing base</td></tr><tr><td>NFC Capable Phone</td><td>Samsung</td><td>Galaxy S9</td><td>Phone to download and use TES phone app</td></tr><tr><td>NFC clip tag</td><td>Technology Evolving Solutions</td><td>TE005-Clip-E1</td><td>Sample tag that can be linked with trees. Made to function with TES phone app</td></tr><tr><td>NFC Collar Tag</td><td>Technology Evolving Solutions</td><td>TE005-Collar-E1</td><td>Tag that is attached to a tree. Made to function with TES phone app</td></tr><tr><td>Nitrile Gloves</td><td>Usa Scientific</td><td>3915-4400</td><td>Gloves to limit possible contamination and protect against chemical spills</td></tr><tr><td>Noise-Reducing Earmuff</td><td>3M</td><td>90565-4DC-PS</td><td>Protect ears while operating air compressor and tissue processing base</td></tr><tr><td>Polyurethane Recoil Air Hose</td><td>FYPower</td><td>&amp;lrm;510019</td><td>Attaches air gun to compressor</td></tr><tr><td>Saftey glasses</td><td>Solidwork</td><td>SW8329-US</td><td>Protect eyes for chemical and physical hazards</td></tr><tr><td>Spray bottle</td><td>JohnBee</td><td>B08QM81BJV</td><td>Spray bleach to deconatinate surfaces</td></tr><tr><td>Tissue Extractor Base</td><td>Technology Evolving Solutions</td><td>TE001-A-E1</td><td>System to process plant tissue. Needs BTE or LTE chambers to function. Includes power cable, blade adapter, and 8/32&quot; allen wrench</td></tr><tr><td>Tissue Processing Base Weight Scale</td><td>Technology Evolving Solutions</td><td>TE003-A05-200g-01-E1</td><td>200 g, 0.01 resolution weight scale that connects to tissue processing base to enforce weight ranges and/or link weights with sample. Includes scale, power cable, connection cable, 5ml syringe holder, tower air shield&amp;nbsp;</td></tr><tr><td>Vermiculite</td><td>EasyGoProducts</td><td>B07WQDZGRP</td><td>Needed to transport hazardous waste (guanidine thiocyanate) using a hazardous waste disposal service</td></tr><tr><td>Wire Cutter</td><td>Boenfu</td><td>&amp;lrm;BOWC-06002-US</td><td>Wire cutters to cut trimmer line</td></tr></tbody></table>

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.

RNA extraction, purification, and quality using BTE-processed budwood citrus tissue and assessment of time for tissue processing 
We used budwood samples from 255 representative citrus trees for this test to compare the RNA quality from the BTE versus the standard procedure. Samples were processed by the budwood tissue extractor (BTE) (protocol steps 4.1-4.6 and Figure 2, right side, step 1, step 5, and step 6) or prepared following the regulatorily approved citrus budw...

Watch this Scientific Journal Video about Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering at JoVE.com

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

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 ...

automating-citrus-budwood-processing-for-downstream-pathogen

Research

JoVE Journal

Bioengineering

701 Views.  University of California Riverside. De gedemonstreerde techniek maakt gebruik van een goedkoop instrument om floëemrijk citrusweefsel snel te verwerken, waardoor arbeidsintensieve protocollen en gespecialiseerde laboratoriumapparatuur niet meer nodig zijn. Dit zorgt voor een snelle downstream detectie en beheer van citrus viroïden, virussen en bacteriën. Deze methode kan de doorvoer verhogen en de kwaliteit en uniformiteit van testdiensten verbeteren, wat de citrusindustrie ten goede komt en mogelijk helpt bij het diagnostic...

Video: Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

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 ...

Environment

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.

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.

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

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

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

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

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 ...

The Mushing, Sieving, and Floating Method for Detecting Fruit Fly Larvae

Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field

Fruit flies of the Tephritidae family are among the most destructive and invasive agricultural pests in the world. Many countries undertake expensive eradication programs to eliminate incipient populations. During eradication programs, a concerted effort is made to detect larvae, as this strongly indicates a breeding population and helps establish the spatial extent of the infestation. The detection of immature life stages triggers additional control and regulatory actions to contain and prevent any further spread of the pest. Traditionally, larval detection is accomplished by cutting individual host fruits and examining them visually. This method is labor intensive, as only a limited number of fruit can be processed, and the probability of missing a larva is high. An extraction technique that combines i) mushing host fruit in a plastic bag, ii) straining pulp through a series of sieves, iii) placing retained pulp in a brown sugar water solution, and iv) collecting larvae that float to the surface was tested. The method was evaluated in Florida with field-collected guava naturally infested by Anastrepha suspensa. To mimic low populations more representative of a fruit fly eradication program, mangos and papaya in Hawaii were infested with a known, low number of Bactrocera dorsalis larvae. The applicability of the method was tested in the field on guava naturally infested by B. dorsalis to evaluate the method under conditions experienced by workers during an emergency fruit fly program. In both field and laboratory trials, mushing and sieving the pulp was more efficient (required less time) and more sensitive (more larvae found) than cutting fruit. Floating the pulp in brown sugar water solution helped detect earlier instar larvae. Mushing and sieving fruit pulp of important tephritid hosts may increase the probability of detecting larvae during emergency programs.

Author Spotlight: Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field  

Increasing the detection of immature tephritid fruit flies in the field can trigger timely efforts to eliminate populations of these destructive pests. Detecting late instar larvae is faster and more accurate when mushing host fruit in a bag and passing the pulp through a series of sieves than hand cutting and visual inspection.

Fruit flies of the Tephritidae family are among the most destructive and invasive agricultural pests in the world. Many countries undertake expensive ...

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for trace detection of DNA viruses. Target DNA is amplified and recognized by RPA and CRISPR/Cas12a separately, which triggers the collateral cleavage activity of Cas12a that cleaves a fluorophore-quencher labeled DNA reporter and generalizes fluorescence. For POC detection, portable smartphone microscopy is built to take fluorescent images. Besides, deep learning models for binary classification of positive or negative samples, achieving high accuracy, are deployed within the system. Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae) was tested as an example for this DNA virus POC detection system, and the limits of detection (LoD) can achieve 10 aM within 40 min. Without skilled operators and bulky instruments, the portable and miniature RPA-CRISPR/Cas12a-SPM with artificial intelligence (AI) assisted classification shows great potential for POC DNA virus detection and can help prevent the spread of such viruses.

We present a protocol that combines recombinase polymerase amplification with a CRISPR/Cas12a system for trace detection of DNA viruses and builds portable smartphone microscopy with an artificial intelligence-assisted classification for point-of-care DNA virus detection.

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase ...

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

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