Figure 1. The adult Asian citrus psyllid feeding at the characteristic 45 angle.

Worldwide, Huanglongbing (HLB), more commonly known as Citrus Greening, is the single most destructive and economically devastating disease of citrus in the history of agriculture. 1 The specific variety of HLB that is currently wreaking havoc in the state of Florida is caused by the phloem-inhabiting, gram-negative bacterium, Candidatus Liberibacter asiaticus (Las). 2 The bacteria requires a vector to be transmitted from one tree to another. The vector responsible for spreading Las is the Asian citrus psyllid (ACP), Diaphorina citri. It is about 4 mm in length, with a brown mottled body and feeds at a characteristic 45 angle(figure 1). Figure 1. The adult Asian citrus psyllid feeding at the characteristic 45 angle. The psyllid has five nymph stages and the adult stage can jump or fly short distances. The adult female can lay up to 800 eggs during her lifetime. Once a psyllid has acquired the bacteria, it can be transmitted to healthy trees over the course of the psyllids life. The first symptoms usually don t appear for up to two years from infection making early detection difficult and leaving infected trees in the orchard available for continued transmission by psyllids. Typical early symptoms are yellowing of isolated shoots, asymmetrical blotchy yellowing of the leaves, vein corking, yellow veins, and defoliation. Fruit may be small, misshapen, lop-sided, taste medicinal, bitter, or sour, and contain aborted seeds. Once symptoms appear the tree will continue to decline over a period of 3-8 years and may not produce any useable fruit during this period. Death for the tree is certain and unavoidable. Page 1 of 5

Figure 2. A citrus leaf displaying the vein corking that is typical of an HLBinfected tree. Figure 3. The orange on the right is typical of those produced by an HLBinfected tree. Notice the blackened, aborted seeds in the smaller than average sized fruit. Believed to have first been discovered in southern China in 1919, HLB has since spread to virtually all of the Asian countries, Africa, the middle east, Brazil, Mexico, Cuba, Costa Rica, Guatemala, the Honduras, Jamaica, the Dominican Republic, and the United States. It was first discovered in Florida in Miami- Dade County in 2005 and is now in all 31 citrus producing counties (Figure 4). More recently it has been discovered in Texas in December of 2011 and finally in California in March of 2012. (Figure 4.) Page 2 of 5

Florida is the largest producer of citrus in the United States and the second largest producer of orange juice in the world. The estimated economic impact of the citrus industry to the state of Florida during the 2007/08 season was approximately 9 billion dollars and generated over 75,000 jobs. Since the discovery of HLB Florida s growers have suffered 1.3 billion dollars in lost revenue, the state s economy has suffered 3.63 billion dollars in lost revenue and 6,611 jobs have been lost due to a reduction in orange juice production. 3 The potential losses are so great that California s response to the single tree discovered to be HLB-positive in a home-owners backyard in Los Angeles County was to destroy the tree, treat all trees within 800 meters and institute a 93 square mile quarantine. It is currently estimated that 43% of Florida citrus trees have been infected and extrapolation from current data has resulted in predictions that 100% of current citrus plantings in Florida will be affected by HLB within the next decade. 3 Hope, though too late for many, seems to be right around the corner. Erik Mirkov, a scientist at Texas A&M s Texas AgriLife Research and Extension Center has created a genetically modified citrus tree using bacteria-fighting proteins naturally found in spinach. Second, third, and fourth generation trees have now been produced and have proven successful in high psyllid population controlled environments. The trees have been planted in orchards near Lake Okeechobee for field tests in March of 2012. Growers are hopeful, but there is no doubt that, even if successful, by the time orchards can replace declining trees with HLB-resistant trees, many more growers will have been put out of business by an invasive bacteria, too small to be seen with the naked eye, that entered our state less than ten years ago. Introduction Prior to this lesson, students should have lab experience with DNA extracton, electrophoresis and PCR (thermocyclers). This lesson could probably be modified to incorporate beginner activities for those students with no exposure, but was not written that way. In this lesson, students learn about some of the job responsibilities of an agricultural technician. These include being able to identify plant diseases, insects, as well as run laboratory equipment and properly prepare samples for Page 3 of 5

diagnostic testing. This is much more than the old-fashioned farmer image that most students grew up with. Students will learn how to identify the Asian citrus psyllid and fill out a taxonomic chart identifying its characteristics. Students will learn how to identify symptoms of and HLB-positive tree, leaves, and fruit. Next, students will be given samples to diagnose as HLB-positive or HLB-negative using only photographs, physical samples, an iodine-starch test, and PCR. Once all the diagnoses have been made the students will learn which samples are actually HLB-positive and which are HLB-negative and students will tally the results to statistically analyze which test was most effective for cost benefit purposes. Finally, students will gather all of their information and create a brochure to be handed out at the local farmer s market in our community to bring awareness of HLB to our citizens and assist them in diagnosing their own backyard citrus trees. Objectives At the end of this lesson, students should be able to explain what citrus greening is and why it is of such concern to growers and the citrus industry. Students should be able to list symptoms of an HLB-infected tree and to pick out HLBinfected leaves with a fair amount of accuracy. Students should have a good understanding of why it is important for growers to have a correct diagnosis and which type of test is most likely to provide that. Students should also appreciate another use of DNA extraction, electrophoresis, and PCR as used in the agricultural field. Materials and Supplies Cost (at time of publishing) Item Description Company (if applicable) Catalog Number Liquid Nitrogen Can be obtained from nearest college or university Usually free of cost Absolute EMD Ethanol (500 ml) Fisher Scientific NC9054029 $46.30 DNeasy Plant MniKit for 50 reactions Qiagen $193.00 Forward Primer Reverse Primer Integrated DNA Technologies Integrated DNA Technologies $8.03 (plus shipping & handling) $7.70 (plus shipping & handling) Master Mix for PCR (90 units) BioRad 166-5009EDU $92.50 Page 4 of 5

Tincture of Iodine Drugstore $2.99 Tweezers Razor blades Timers Stereoscopes are helpful, but not required (magnifying glasses will suffice) Thermocycler Electrophoresis Apparatus Mortar and Pestles Centrifuge Vortexer Hot water bath or dry bath HLB-positive and HLB-negative citrus leaves Camera 1 Halbert SE and Manjunath KL, Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: a literature review and assessment of risk in Florida Fla Entomol 87:330-353 (2004). 2 Pelz-Stelinski K, Stelinski LL and Tiwari S, Effect of Candidatus Liberibacter asaticus infection on susceptibility of Asian citrus psyllid, Daphorina citri, to selected insecticides Pest Manag Sci 67:94-99 (2011). 3 Hodges AW and Spreen TH, Economic impacts of citrus greening (HLB) in Florida, 2006/07-2010/11 1 EDIS document FE903, a publication of the Food and Resource Economics Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Fl 32761. Published January 2012. 4 Stover E, Castle WS, Spyke P, The citrus grove of the future and its implications for Huanglongbing management. Proc. Fla. State Hortic. Soc. 121: 155-159 (2008) Page 5 of 5

Kathy Savage Oviedo High School 601 King Street, Oviedo FL 32765 Kathy_savage@scps.k12.fl.us (cell) 407-443-9688 Title: Citrus Greening is not Green! Abstract: This action plan is designed to allow students to use newly obtained biotechnology lab skills within the context of a real-world problem. This is designed to be a project that encompasses prior skills and knowledge allowing students to discover problems for themselves and apply what they learn to a community awareness project. The heart of this project will be Citrus Greening or Huanglongbing, a disease affecting citrus trees in the state of Florida. In this module students will study how the disease affects citrus trees; how to identify it morphologically and in the lab using an iodine test and PCR, study the cause and factors affecting its spread, track its spread within our state, and design a community awareness brochure. Tests on leaves and psyllids will be performed using kits available from Qiagen. Students will use statistics from government websites to determine how fast HLB is spreading and graphically display their results. Classes will take a field trip to a local citrus grove to observe, learn how to diagnose and identify affected trees, look for psyllids, and collect leaves for testing. Rationale: Students will have the opportunity to study Citrus Greening (Huanglongbing or HLB), a vector-borne bacterial disease that is devastating Florida s citrus industry at an alarming rate. Since it was first detected in Florida in 2005, HLB is now found in all of the states citrus producing counties. Once a tree has become infected, the fruit it produces becomes bitter and salty making it virtually inedible. Death for the tree is inevitable as there is no cure for this disease. The vector that carries and transmits HLB in Florida is a small Asian psyllid that can hop, fly, or be carried longer distances by winds. It is estimated that every citrus grove in the state is within one mile of an infected tree. According to the Florida Department of Citrus, the industry employs approximately 76,000 workers and has an annual economic impact of 9 billion dollars. (1) The United States leads the world in grapefruit production supplying the world with over 30% of its grapefruit, and is the third largest overall citrus producer in the world. The majority of the citrus grown in the United States comes with Florida as the major producer. In fact Florida produces three times as many tons of oranges and four times as many tons of grapefruit as its closest competitor, California. (2) With over ½ million acres of citrus groves and 74 million trees, Florida is second only to Brazil in orange juice production and supplies approximately 80% of the orange juice in the United States during any given growing season. (3) These facts clearly demonstrate that any threat to the Florida Citrus Industry is a threat to the world citrus supply. This module would allow students to reinforce some laboratory skills and techniques they are already familiar with (electrophoresis, PCR) and use some that would be new to them (BLAST). In the past, lab skills have been used in an isolated fashion with various labs designed to teach the skill without a real- Page 1 of 6

world frame of reference. This module would allow students to see how these seemingly isolated and unrelated skills work together to identify and diagnose HLB in citrus trees. References Cited: 1. "Florida Grapefruit." Florida Department of Citrus. 2010. 30 Aug. 2011. <http://www.gofloridagrapefruit.com/about-fdoc/>. 2. "Background Statistics: U.S. Citrus Market." United States Department of Agriculture. 22 Jan. 2007. 28 Aug. 2011. <http://www.ers.usda.gov/news/citruscoverage.htm>. 3. "Citrus Facts." Florida Citrus. 2008. 30 Aug. 2011. <http://www.floridajuice.com/juice.php>. Lesson Plans: See Below Day One: Students will do a webquest on Citrus Greening (Huanglongbing) Day Two and Three: Discussion and Lecture on the citrus greening disease, it s vector the Asian Citrus Psyllid, and the Economic Impact for Florida using the following PowerPoint Presentations. These are excellent presentations with great graphics and include comments for the presenter. 1. The Asian Citrus Psyllid and the Citrus Disease Huanglongbing by the California Citrus Research Board http://casap.org/2009/2009conference/asian_citrus_psyllid.ppt 2. The Asian Citrus Psyllid & Huanglongbing by Protect U.S. Community Invasive Species Network at the University of Florida http://entnemdept.ifas.ufl.edu/hodges/protectus/greening%205-12-2011.pdf 3. Three Exotic Plant Diseases Threatening Florida by the Florida State Agricultural Response Team http://www.flsart.org/ppt/three%20diseases.ppt Day Four: Identify samples of citrus trees with HLB and compare to samples without HLB from a morphological perspective. If possible, taste test citrus that has been harvested from HLB infected trees. Discern whether or not they can tell infected Asian Citrus Psyllid from noninfected Psyllid by visual inspection under a stereoscope. Students will record their observations for each sample of leaf and psyllid and make their predictions as to infected vs. non-infected in a table. Day Five: Students will use the Iodine Starch Test to further solidify or change their initial predictions from the previous day s inspections. This protocol is available from the University of Florida IFAS website http://edis.ifas.ufl.edu/hs375. Page 2 of 6

Day Six: Students will begin preparation for PCR testing of their samples using the protocol provided by Dr. Kirsten Stelinski from UF. Day Seven: Students will work on testing citrus samples and keep a record of results. Day Eight: Continued as above Day Nine: Students will graph results identified by location (city, county, etc.) and compare to actual data obtained from the State of Florida. Day Ten: Students will develop informational flyers/brochures for Seminole County Residents (to be given out at Great Day in the County and local craft fairs) that include morphological detection methods and phone numbers to call for plant inspection and testing. Day Fourteen: Field Trip to Lake Alfred Materials and Supplies: Thermocycler Hot Water Bath Mortar and Pestle Electrophoresis Apparatus 1.5 ml microfuge tubes with caps Microcentrifuge Autoclave Spatulas Stereoscopes Razor Blades Chemicals Required: Item Source for Purchase Catalog No. Cost Liquid Nitrogen University of Central Florida Free Chemistry Department Absolute EMD Ethanol Fisher Scientific NC9054029 $46.30 500 ml DNeasy Blood & Tissue Kit Qiagen $144.00 for 50 reactions DNeasy Plant MiniKit for Qiagen $193.00 50 reactions Primer IDT (Integrated DNA? Technologies) Loading Dye 1 ml 5x BioRad 166-401 EDU $50.00 Iodine Tincture or Tincture Drugstore? of Iodine or Lugol s solution Amazon Buses for Field Trip? Page 3 of 6

Total Cost $433.30 Bibliography: "Huanglongbing Florida Citrus Greening Information Video." World News, Inc.. 2011. 25 Aug. 2011. <http://wn.com/huanglongbing_florida_citrus_greening_information_video>. Protocol: Purification of Total DNA from Plant Tissue (Mini Protocol) Provided by Dr. Kirsten Stelinski Before starting: Buffer AP1 may develop a yellow color upon storage. This does not affect the procedure. All centrifugation steps are carried out at room temperature (15 25 C) in a microcentrifuge. Things to do before starting Buffer AP1 and Buffer AP3/E concentrate may form precipitates upon storage. If necessary, warm to 65 C to redissolve (before adding ethanol to Buffer AP3/E). Do not heat Buffer AP3/E after ethanol has been added. Buffer AW and Buffer AP3/E are supplied as concentrates. Before using for the first time, add the appropriate amount of ethanol (96 100%) as indicated on the bottle to obtain a working solution. Preheat a water bath or heating block to 65 C. Label tubes with sample numbers/codes. You will need: 2 sets of 1.5 ul microcentrifuge tubes, 1 set of QIAshredder spin columns (lilac), and ones set of DNeasy Mini spin columns (white). Autoclave mortars, pestles, and spatulas for use in grinding plant tissue. Procedure 1. Plant tissue (100 mg composite of petioles and midribs) can be ground to a fine powder under liquid nitrogen using a mortar and pestle. Allow the liquid nitrogen to evaporate and transfer the tissue powder to an appropriately sized tube. Do not allow the sample to thaw. 2. Add 400 μl Buffer AP1 and 4 μl RNase A stock solution (100 mg/ml) to a maximum of 100 mg (wet weight) disrupted plant or fungal tissue and vortex vigorously. Spin briefly to remove any sample that has collected on lid of tube. No tissue clumps should be visible. Vortex or pipet further to remove any clumps. Clumps of tissue will not lyse properly and will therefore result in a lower yield of DNA. Note: Do not mix Buffer AP1 and RNase A before use. 3. Incubate the mixture for 10 min- 30 min at 65 C. Mix 2 or 3 times during incubation by inverting tube. This step lyses the cells. 4. Add 130 μl Buffer AP2 to the lysate, mix, and incubate for 5 min on ice. This step precipitates detergent, proteins, and polysaccharides. 5. Centrifuge the lysate for 5 min at 20,000 x g (14,000 rpm). 6. Pour the lysate (avoid transferring perllet) into the QIAshredder Mini spin column (lilac) placed in a 2 ml collection tube, and centrifuge for 2 min at 20,000 x g (14,000 rpm). Page 4 of 6

It may be necessary to cut the end off the pipet tip to apply the lysate to the QIAshredder Mini spin column. The QIAshredder Mini spin column removes most precipitates and cell debris, but a small amount will pass through and form a pellet in the collection tube. Be careful not to disturb this pellet in step 7. 7. Add 675 μl Buffer AP3/Eto a new tube (not supplied). Transfer the flow-through fraction from step 6 to the cleared lysate without disturbing the cell-debris pellet, and mix immediately by pipetting or gently vortexing. Ensure that ethanol has been added to Buffer AP3/E. 8. Pipet 650 μl of the mixture from step 7, including any precipitate that may have formed, into the DNeasy Mini spin column placed in a 2 ml collection tube (supplied). Centrifuge for 1 min at _6000 x g (corresponds to _8000 rpm for most microcentrifuges), and discard the flow-through.* Reuse the collection tube in step 9. 9. Repeat step 14 with remaining sample. Discard flow-through* and collection tube. 10. Place the DNeasy Mini spin column into a new 2 ml collection tube (supplied), add 500 μl Buffer AW, and centrifuge for 1 min at 6000 x g (8000 rpm). Discard the flow-through and reuse the collection tube in step 11. Note: Ensure that ethanol is added to Buffer AW. 11. Repeat step 10 by adding 500 μl Buffer AW to the DNeasy Mini spin column, and centrifuge for 2 min at 20,000 x g (14,000 rpm) to dry the membrane. It is important to dry the membrane of the DNeasy Mini spin column since residual ethanol may interfere with subsequent reactions. Discard flow-through and collection tube. Note: Following the centrifugation, remove the DNeasy Mini spin column from the collection tube carefully so the column does not come into contact with the flow through, as this will result in carryover of ethanol. 12. Transfer the DNeasy Mini spin column to a 1.5 ml or 2 ml microcentrifuge tube (not supplied), and pipet 50 μl Buffer AE directly onto the DNeasy membrane. Incubate for 5 min at room temperature (15 25 C), and then centrifuge for 1 min at 6000 x g (8000 rpm) to elute. Store at -20 o C. Page 5 of 6

Conventional PCR to Examine the Presence of the HLB-associated Pathogen in Citrus Provided by Dr. Kirsten Stelinski 16S rdna, OI1/OI2 specific to the Ca. Liberibacter asiaticus sp. used for PCR amplification in a 25-μl reaction volume Forward primer: OI1 (5 -GCG CGT ATG CAA TAC GAG CGG CA-3 ) Reverse primer: OI2c (5 -GCC TCG CGA CTT CGC AAC CCA T- 3 ) PCR reaction 25-μl reaction volume consisting of: 1 μl of DNA template 200 nm (each) primer 200 μm (each) dntps 2.5 mm MgCl2 1 PCR buffer dh 2 O 1 unit Platinum Taq polymerase (Invitrogen, Carlsbad, CA) Amplification: 94 C for 2 min 35 cycles of 94 C for 30 s, 62 C for 30 s, and 72 C for 1 min Final extension cycle of 10 min at 72 C Analysis of PCR reactions: Expected product size: 1160 bp 1.0% agarose gel in 1 Tris-acetate-EDTA buffer (40 mm Tris, 20 mm acetic acid, 1 mm EDTA, ph 8.5) Any 1 kb DNA ladder should work e.g. Quick-Load 1 kb DNA Ladder (New England Biolabs) or 1 kb Plus DNA ladder (Invitrogen) Page 6 of 6