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Appendix K
Huanglongbing Research Milestones (1956–2009)

Year(s)

Milestone

Principal Investigator(s) or Author(s)

Publication/Journal

1956

Infectious nature of huanglongbing disease (HLB) was demonstrated.

Lin Kung Hsiang

Acta Phytopathologica Sinica 2:1-42

1957

This is the only known report on Lin Kung Hsiang’s work to have appeared in the western world soon after the publication of Lin’s work in 1956 (See: Lin, K.H., 1956). Unfortunately, Ciccarone’s report remained essentially unknown too.

Ciccarone

Revista di Agrumicoltura 2: 45-50.

1965

Graft and insect vector (Trioza erytreae) transmissibility of greening was demonstrated.

McClean and Oberholzer

South Africa Journal of Agricultural Science 8:253-276; 297-298

1967

Transmission of HLB by the Asian citrus psyllid (ACP) was demonstrated.

Martinez and Wallace

Plant Disease Reporter 51:692-695



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Appendix K Huanglongbing Research Milestones (1956–2009) 271 Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) 1956 Infectious nature of huanglongbing disease (HLB) was Lin Kung Hsiang Acta Phytopathologica Sinica demonstrated. 2:1-42 Ciccarone Revista di Agrumicoltura 2: 45- 1957 This is the only known report on Lin Kung Hsiang’s work to have 50. appeared in the western world soon after the publication of Lin’s work in 1956 (See: Lin, K.H., 1956). Unfortunately, Ciccarone’s report remained essentially unknown too. 1965 Graft and insect vector (Trioza erytreae) transmissibility of McClean and Oberholzer South Africa Journal of greening was demonstrated. Agricultural Science 8:253-276; 297-298 1967 Transmission of HLB by the Asian citrus psyllid (ACP) was Martinez and Wallace Plant Disease Reporter 51:692- demonstrated. 695

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Year(s) Milestone Principal Investigator(s) Publication/Journal 272 or Author(s) Capoor et al Indian Journal of Agricultural 1967 With the use of psyllid to transmit the HLB pathogen, it was Sciences 37(6):572-576 confirmed that trees with "citrus dieback" symptoms were positive for HLB. Fraser and Singh Proceedings of 4th Conference, 1968 Citrus dieback in India is reported to have many similarities with IOCV:141-144, University of greening disease of South Africa. The similarities with California, Division of huanglongbing in China could not have been mentioned because Agricultural Sciences, Lin’s work on HLB was still not known out of China. Riverside, CA. Schneider Phytopathology 58:1155-1160 1968 Localized pockets of necrotic phloem were found scattered throughout vascular system of mature leaves in greening-affected sweet orange shoots in South Africa. Leaf mottle associated with HLB are thought to be caused by the reaction to the blockage of the translocation stream. Catling Journal of Entomology Society 1969 HLB in Africa was found to be heat-sensitive and occurs only in South Africa 32:209-223; areas below 30-32°C.Trioza erytreae, the African psyllid vector, was also found to thrive only in cool environments. 1970 "Mycoplasma-like organism" observed in citrus phloem tissue Laflèche and Bové Comptes Rendus de infected with HLB through electron microscopy. L'Academie des Sciences, Paris, 270:1915-1917 Fruits 25: 455-465 1970 These are the first two reports on bacterial structures associated with Laflèche and Bové C.R. Acad. Sci. Paris HLB, a disease until then considered to be caused by a virus. The 270:1915-17 structures were restricted to the phloem sieve tubes and were thought to be mycoplasmas, i.e bacteria lacking a cell wall. They were observed not only with South Africa greening, but also with Reunion and Indian greening. They were shown only a few months later to be not mycoplasma-like (see Saglio et. al., 1971). 1971 HLB bacterium seen in citrus with "likubin" disease. Chen et al Phytopathology 61:598

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) Saglio et al. Physiologie Végétale 9: 569-82. 1971 Citrus stubborn disease is also associated with sieve tube-restricted bacterial structures. These structures could be cultured and shown to be mycoplasma-like, surrounded by a 70 A° thick cell envelope, which lacked a cell wall and was composed only of a unit membrane. Compared to the stubborn mycoplasma-like structures, those associated with greening had a 200A° thick envelope and therefore could not be mycoplasma-like. The 200A° envelope was characteristic not only of South African greening, but also of the disease in Reunion, the Philippines, and India. Schwarz and van Vuuren Plant Disease Reporter 55: 747- 1971 As greening was found to be associated with bacteria, antibiotics, 750. such as tetracyclines, were injected into affected trees in an effort to control the disease. 1973 HLB bacterium seen in citrus with "likubin" disease. Tanaka and Doi International Citrus Congress, Murcia-Valencia, pp. 352-353 1973 HLB bacterium seen in T. erytreae. Moll and Martin Phytophylactica 5:41-44 1973 HLB bacterium seen in D. citri (seen in salivary glands). Chen et al. Phytopathology 63(1):194-195 1973 HLB bacterium seen in citrus with "mottle leaf" disease. Tanaka and Doi International Citrus Congress, Murcia-Valencia, pp. 352-353 Bové et al. Proceedings of 6th Conference, 1974 First demonstration under phytotron conditions that African HLB IOCV:12-15, University of was heat-sensitive (no symptom development at 32°C), while Asian California, Division of HLB (from India, the Philippines) was heat tolerant (good symptom Agricultural Sciences, development at 32°C). Riverside, CA. 273

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Year(s) Milestone Principal Investigator(s) Publication/Journal 274 or Author(s) Moll and Martin Proceedings of Conference Les 1974 The cell envelope of the organism associated with greening was Mycoplasmes/Mycoplasmas, found to resemble that of a Gram-negative bacterium. However, the INSERM 33: 89-96 presence of peptidoglycan, a characteristic component of the bacterial cell wall, could not be demonstrated. 1976 The African citrus psyllid vector, Trioza erytreae, is not only vector Massonié, Garnier, and Proceedings of 7th Conference of the African HLB bacterium, but can also transmit the Asian HLB Bové (1976) IOCV: 18-20, bacterium. University of California, Division of Agricultural Sciences, Riverside, CA. 1977 Through electron microscopy, the HLB causal organism was shown Garnier and Bové Fruits 32:749-752 to possess a cytoplasmic membrane and a bacterial cell wall. Aubert and Bové; Bové et Proceedings of 8th Conference, 1980 Greenhouse-grown HLB-affected citrus plants having absorbed al. (1980) IOCV: 103-108; 91-102 penicillin through the roots grew better, produced more roots and University of California, larger symptomless shoots and leaves than untreated controls. In Division of Agricultural contrast, Penicillin had no effect on Stubborn-affected citrus plants. Sciences, Riverside, CA. A beneficial effect was also noted when penicillin was injected into the trunk of field-grown HLB-affected sweet orange trees in Reunion Island. Tetracycline had a beneficial effect on HLB- affected citrus as well as on stubborn-affected citrus. In view of the mode of action of penicillin (inhibits late step in peptidoglycan biosynthesis) and tetracycline (inhibits protein biosynthesis), these results strongly suggested that the bacterium associated with greening contained peptidoglycan, the characteristic component of the bacterial cell wall, indicating that the greening organism is a walled bacterium.

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) 1980 First successful biological control of Trioza erytreae and Etienne and Aubert Proceedings of 8th Conference, Diaphorina citri, the two psyllid vectors of HLB, in Reunion Island. (1980) IOCV: 118-121 University of California, Division of Agricultural Sciences, Riverside, CA. Garnier and Bové Phytopathology 73: 1358-1363 1983 First transmission of HLB from citrus to periwinkle through dodder. In periwinkle, like in citrus (see: Bové et al., 1974), the Asian HLB bacterium was found to be heat tolerant, while the African HLB bacterium was heat sensitive, showing that the temperature effect was due to the bacterium and not to the host. These observations were the first indications that the two bacteria were biologically different. Garnier, Danel and Bové Annales de l'Institut Pasteur. 1984 The HLB-associated bacterium was shown to possess a cell wall of Microbiologie 135(1): 169-179; the Gram-negative type with an outer cell wall membrane and a Proceedings of 9th Conference, peptidoglycan layer, thus establishing the Gram negative nature of IOCV: 115-124 University of the HLB-associated bacterium. California, Division of Agricultural Sciences, Riverside, CA. 1986 The ACP vector, Diaphorina citri, is not only vector of the Asian Lallemand, Fos, and Bové Fruits 41: 341-343 HLB bacterium, but can also transmit the African HLB bacterium. Since Trioza erytreae can also transmit the two bacteria (see: Massonié et al., 1976), each psyllid can transmit each one of the two bacteria. 1991 First extensive review on citrus greening disease was published. Da Graça Annual Review of Phytopathology 29:109-36 275

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Year(s) Milestone Principal Investigator(s) Publication/Journal 276 or Author(s) Villechanoux et al. Current Microbiology 24: 89- 1992 By random cloning of total DNA from periwinkle plants affected by 95; Current Microbiology 26: Indian HLB, the first genome fragments of the HLB-associated 161-166. bacterium were obtained: In-2.6, In-1.0, and In-0.6. Sequencing showed In-2.6 to be the nusG-rplKAJL-rpoBC gene cluster (beta operon), and In-1.0 coded for a bacteriophage DNA polymerase. In- 2.6, used as a hybridization probe, detected all Asian HLB strains but not the South African strain. 1993 Monoclonal antibodies specific for HLB bacteria were produced. Gao et al. Proceedings of 12th Conference, (1993) IOCV: 244- 249, University of California, Division of Agricultural Sciences, Riverside, CA Jagoueix, Bove, and International Journal of 1994 On the basis of 16SrDNA sequence analyses, the HLB-associated Garnier Systematic Bacteriology 44: bacterium was confirmed to be a Gram negative bacterium, 397-86 belonging to a new genus, Candidatus Liberobacter, in the alpha subdivision of the Proteobacteria. “Candidatus” indicated that the HLB-bacterium was not available in culture. Two liberobacter species were characterized by molecular, biological, and serological methods: Candidatus Liberobacter africanum (CLaf) in Africa and Candidatus Liberobacter asiaticum (CLas) in Asia. 1993 Transmission of HLB to tobacco by dodder was demonstrated. Garnier and Bové Proceedings of 8th Conference, (1993) IOCV: 212-219, University of California, Division of Agricultural Sciences, Riverside, CA Planet et al. Current Microbiology 30: 137- 1995 Part of the rplKAJL-rpoBC operon of the African liberobacter was 141 obtained as a 1.7 kb fragment (As-1.7). As-1.7, used as a probe, detected the African HLB strains, but not the Asian strains.

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) 1996 First PCR detection of the Asian and African liberibacters by Jagoueix, Bove, and Molecular and Cellular Probes amplification of 16S rDNA. Garnier 10: 43-50. Garnier et al. 1996 Already known to harbor both the African and the Asian psyllid Proceedings of 13th Conference, vectors of HLB, Reunion and Mauritius islands were also shown to IOCV: 392-394, University of carry both the African and the Asian liberibacters. California, Division of Agricultural Sciences, Riverside, CA 1997 Confirmation that CLas and CLaf are two different bacterial species Jagoueix et al. International Journal of based on the 16S/23S rRNA intergenic regions. Systematic Bacteriology 47(1):224-227 1997 Petroleum spray oil was tested against the ACP in Guangzhou, Rae et al. International Journal of Pest China; It was found that petroleum oil was as effective as an Management 43(1):71-75 organophosphate pesticide and an insect growth regulator in controlling psylla nymphs. Hocquellet, Bove, and Hocquellet, A., Bové, J.M., and 1999 Random Amplified Polymorphic DNA (RAPD) was used to Garnier Garnier, M. 1999. Isolation of identify additional genomic sequences of the HLB liberibacters. In DNA from the uncultured particular, the omp (outer membrane protein) gene was obtained. “Candidatus Liberobacter” species associated with citrus huanglongbing by RAPD. 1999 A PCR detection method based on the amplification of ribosomal Hocquellet et al. Molecular and Cellular Probes protein genes, which allows direct identification of the liberibacter 13(5):373-379 species by the size of the amplified DNA, was developed. 277

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Year(s) Milestone Principal Investigator(s) Publication/Journal 278 or Author(s) Proceedings of 14th 2000 The Asian psyllid vector of HLB, Diaphorina citri, was captured for Bové et al. Conference, IOCV: 207-212 the first time in southern Iran in 1997 during a mission on Witches' University of California, Broom Disease of Lime. Division of Agricultural Sciences, Riverside, CA Garnier et al. International Journal of 2000 Following a 1999 proposal in prokaryote nomenclature, Systematic and Evolutionary “Liberobacter”, “africanum”, and “asiaticum were respectively Microbiology 50: 2119-2125. renamed “Liberibacter”, “africanus”, and “asiaticus”. A new liberibacter was detected in an ornamental rutaceous tree, Calodendron capense, in South Africa. The liberibacter was identified as a subspecies of Ca. L. africanus and named “Ca. L. africanus subsp. Capensis”. Hung et al. Journal of Phytopathology- 2000 The HLB causal agent was shown to replicate in Chinese box Phytopathologische Zeitschrift orange (Severinia buxifolia) and wood apple (Limonia acidissima) 148(6):321-326 but not in common jasmine orange (Murraya paniculata var. paniculata) and curry leaf (Murraya euchrestifolia). Liu and Tsai Annals of Applied Biology 2000 This study was conducted to determine the effect of temperature on 137(3):201-206 the biology and life table parameters of the ACP. Findings include the following: 1) Average number of eggs produced per female significantly increased with increasing temperature; maximum number of eggs was produced at 28°C; 2) Population reared at 28°C had the highest intrinsic rate of increased and net reproductive rate and the shortest population doubling time and mean generation time compared with populations reared at 15-25°C; 3) The optimum range of temperature for D. citri population growth was found to be 25-28°C.

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) Subandiyah et al. Zoological Science 17:983-989 2000 The endosymbiotic microbiota of the citrus psyllid was investigated using PCR and RFLP. The whole DNA of D. citri was found to contain sequences that are similar to that of mycetocyte symbionts of other psyllids, Oxalobacter and Herbaspirillum, Arsenophonous spp., Liberobacter spp. and Wolbachia spp. 2000 Taylor's power law and Iwao's patchiness regression models Tsai et al. Florida Entomologist indicated that D. citri populations were aggregated. 83(4):446-459 McFarland and Hoy Florida Entomologist 84(2): 2001 The ability of D. citri and its two parasitoids, Tamarixia radiata and 227-233 Diaphorencyrtus aligarhensis to survive at different relative humidities and temperatures was studied. This study showed that D. citri survived longer than the parasitoids at all conditions tested, indicating a lower net water loss rate. Tsai et al. Florida Entomologist 2002 Psyllid population levels on orange jasmine were found to be 85(3):446-451 positively related to the availability of new shoot flushes, which were in turn related to the weekly minimum temperature and rainfall. The study indicated that continuous flushes produced by orange jasmine could play an important role in maintaining high populations of psyllids when new shoot flushes are not available in citrus groves. 2004 First report of CLas associated with HLB in Brazil. Colleta-Filho et al. Plant Disease 88:1382. 2004 Using PCR, it was determined that Liberibacter asiaticus persists in Hung et al. Plant Pathology 53(1):96-102 the Asian citrus psyllid vector but it is not transovarially transmitted. 279

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Year(s) Milestone Principal Investigator(s) Publication/Journal 280 or Author(s) McKenzie and Puterka Journal of Economic 2004 Results from a laboratory and spray booth study indicated that Entomology 97(3):970-975 nymphal and adult D. citri and a mite complex can be controlled by high concentrations of sucrose octanoate, a synthetic analog of natural sugar esters found in leaf trichomes of wild tobacco (Nicotiana gossei Domin). 2004 Coccinellid beetles were found to be the most important biological Michaud Biological Control 29(2):260- control agents of high-density populations in central Florida and 269 that intraguild predation causes >95% mortality of immature stages of Tamarixia radiata. 2004 A rearing method was developed for ACP and its parasitoids, Skelley and Hoy Biological Control 29(1):14-23 Tamarixia radiata and Diaphorencyrtus aligerhensis. Bastianel et al. Applied Environmental 2005 Omp (outer membrane protein gene)-based PCR-RFLP analysis was Microbiology 71:6473-6478. shown to be a simple method for detecting and differentiating CLas isolates. The phylogeny tree based on the omp gene sequences of the African and Asian liberibacters was very similar to the tree based on the 16S rDNA sequences. 2005 Murraya paniculata, the preferred host of the Asian HLB psyllid Lopes, Martins, and Frare Summa Phytopathologica 31: vector, was also found to be a host of the two HLB bacteria present 48-49.; Fitopatologia Brasileira in São Paulo State, CLam and CLas. 31: 303. Texeira et al. Plant Disease 89: 107; 2005 Discovery and first report of a new liberibacter species associated International Journal of with HLB in São Paulo State, Brazil: Candidatus Liberibacter Systematic and Evolutionary americanus. The new liberibacter was also detected in Diaphorina Microbiology 55: 1857-1862; citri, suggesting that the Asian psyllid vector in Brazil was Proceedings of 16th transmitting not only CLas, but also CLam. Conference, IOCV: 325-340 University of California, Division of Agricultural Sciences, Riverside, CA

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) 2005 Development of PCR techniques for the detection of CLam and Texeira et al. Molecular and Cellular Probes, CLas in citrus and psyllids. 19; 173-179; Proceedings of 16th Conference, IOCV: 432- 438 University of California, Division of Agricultural Sciences, Riverside, CA 2005; CLas was detected in Wampee (Clausena lansium Skeels) using Ding et al.; Deng et al. Journal of Plant Pathology 2007 nested PCR. 87(3):207-212; Plant Health Progress 2007 Okuda et al. Plant Disease 89(7):705-711 2005 Detection of HLB pathogen through loop-mediated isothermal amplification (LAMP) was demonstrated; The LAMP method, which does not use a thermocycler and electrophoresis apparatus, is deemed useful for under-equipped laboratories. 2005 Coccinellid species found to have potential important role as Pluke et al. Florida Entomologist predators of the psyllid in Puerto Rico. 88(2):123-128 2005 Third bacterial species was detected and identified as another causal Teixeira et al. Molecular and Cellular Probes organism of HLB in Brazil. The name Ca. Liberibacter americanus 19(3):173-179; International was proposed for this HLB pathogen. Detection of CLam in D. citri Journal of Systematic and indicated that this is also the vector for this liberibacter species. Evolutionary Microbiology 551857-1862 Part 5 2005 In greenhouse trials, a neem-based biopesticide containing 4.5% Weathersbee and McKenzie Florida Entomologist azadirachtin was found to reduce psyllid nymph populations; 88(4):401-407 however, no mortality was observed in psyllid adults that were exposed to 11-180 ppm azadirachtin. 2006 Guava intercropping was observed to reduce ACP/HLB incidence in Beattie et al. Unpublished Vietnam 281

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Year(s) Milestone Principal Investigator(s) Publication/Journal 294 or Author(s) Faghihi et al. Plant Pathology 58: 793. 2009 First report of HLB in the southern and southwestern parts of Iran. The Asian psyllid vector was detected in the area in 1997 (see Bové et al., 2000). Kim et al. Phytopathology 99(1):50-57 2009 Microarray analysis indicated that HLB infection significantly affected the expression of 624 genes whose encoded proteins fell into the following categories: sugar metabolism, plant defense, phytohormone, cell wall metabolism and 14 other categories. Anatomical examination indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. It was determined that HLB-associated blockage resulted from plugged sieve pores and not due to HLB bacterial aggregates since CLas does not form aggregates in citrus. Li et al. Phytopathology 99(2):139-144 2009 Systematic quantification of the distribution of CLas genomes in tissues of six citrus species using a quantitative polymerase chain reaction assay was performed. The study showed the ubiquitous presence of CLas in symptomatic citrus trees and the variation in distribution between individual trees and among samples of different tissues from the same trees.

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) Plant Disease 93: 208-214; 2009 While the new liberibacter described by Hansen et al.; see Hansen et Liefting et al.; Lin et al. International Journal of al., 2008) as Candidatus Liberibacter psyllaurus was characterized Systematic and Evolutionary from the tomato/potato psyllid, Bactericera cockerelli, the Microbiology 59: 2274-2276; liberibacter described by Liefting et al. (2009a, b) as Candidatus Journal of Plant Pathology 91: Liberibacter solanacearum was characterized from tomato and other 215-219. solanaceous plants. The two liberibacters represent one and the same organism: Ca. L. psyllaurus refers to the agent in the psyllid vector, Ca. L. solanacearum refers to the agent in plants. It is the agent associated with potato Zebra chip disease (PZCD) and the PZCD psyllid vector. It has been shown that the citrus liberibacter, CLas, can experimentally infect, and induce disease in at least two solanaceous plants: tobacco (Garnier and Bové, 1993) and tomato (Duan et al., 2008). It might be expected that, similarly, Ca. L. solanacearum will be shown to infect, and induce symptoms in citrus. Lopes et al. Phytopathology 99: 301-306. 2009 Percentages of transmission by graft inoculation were from 54.7 to 88.0% for Ca. L. asiaticus and 10.0 to 45.2% for Ca. L. americanus. Average bacterial titers in field trees were 6.67 log cells per gram of leaf midrib for CLas and 5.74 for CLam. The titer of CLas in field trees being higher than that of CLam, transmission of the former by the psyllid vector might be more efficient than that of the latter and explain why newly infected trees are more frequently infected with CLas than with CLam. Lopes et al. Plant Disease 93: 257-262. 2009 While CLas, not yet affected at 35°C, was confirmed to be heat tolerant, CLam, affected at 32°C, was found to be heat sensitive. Thus two of the citrus liberibacters are heat sensitive: CLaf and CLam. 295

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Year(s) Milestone Principal Investigator(s) Publication/Journal 296 or Author(s) Sagaram et al. Applied and Environmental 2009 Phylochip analysis indicated that 47 orders of bacteria in 15 phyla Microbiology 75(6):1566-1574 were present in citus leaf midribs while cloning and sequencing indicated 20 orders of bacteria in 8 phyla. Phylochip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. 2009 CLam found to be less heat tolerant than CLas. Lopes et al. Plant Disease 93(3):257-262 2009 Cultivation of all three citrus liberibacter species has been reported. Sechler et al. Phytopathology 99:480-486. Confirmation in several laboratories is attempted. 2009 This study provides evidence that D. citri uses olfactory and visual Wenninger et al. FCPRAC Annual Report 2008; cues in orientation to host plants which indicates the possibility of Environmental Entomology using plant volatiles to monitor and manage ACP. 38(1):225-234 Wenninger et al. Annals of the Entomological 2009 Vibrational communication between male and female psyllids was Society of America 102(3):547- studied. Findings include: 1) both sexes produced simple, low 555 amplitude vibrational signals at multiples of 17-250 Hz, ranging in duration from 140 to 700 ms; 2) vibrational frequency in males were significantly negatively correlated with mass; 3) latent period for initialization of calling was significantly shorter for males exposed to clean air, suggesting that in the absence of olfactory cues, psyllids might be more inclined to use acoustic signals to communicate with conspecifics. Wulff et al. International Journal of 2009 The size of the CLam genome was determined by pulse field gel Systematic and Evolutionary electrophoresis, using Lam-infected periwinkle plants for bacterial Microbiology 59: 1984-1991. enrichment. The genome size was found to be ~1.31 Mbp, a value close to 1.23 Mbp as found by Duan et al., 2009. The data also suggest the genome to be circular and to contain three ribosomal operons.

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) M. Irey http://www.aphis.usda.gov/biot 2009 Two field trials have been established in Florida to evaluate the echnology/status.shtml resistance of transgenic citrus to citrus canker and citrus greening. The transgenic citrus tested carry the following genes: 1) gene http://www.isb.vt.edu/cfdocs/fi encoding for a plant-based anti-microbial peptide and 2) a eldtests1.cfm proprietary gene with the trademark “Disease Block”. No field resistance data have been generated as of December 2009 but preliminary laboratory data for the transgenic citrus in both trials indicate efficacy against citrus greening. FCPRAC 2009 Progress Reports Brlansky FCPRAC Progress Report 2009 Results from this study indicate that D. citri which complete their (January, 2009) development on CLas(+) plants are more likely to acquire the pathogen compared to those individuals that feed on Las(+) plants as adults only. Therefore, the presence of CLas(+) trees on which psyllids can complete development is an important factor in the overall spread of HLB within a grove. Results from preliminary tests suggest that there appears to be some seasonality to the abundance of Las positive psyllids with one period of increase occurring in the late fall/early winter and a second smaller peak occurring in the spring. Brlansky and Davis FCPRAC Progress Report 2009 Results from this study provided (further) evidence that distribution (January, 2009); Proceeding of of the greening-associated pathogen varies widely within the International Research symptomatic, PCR-positive citrus trees and thus illustrates the Conference on Huanglongbing. importance of obtaining multiple samples from trees where an Orlando, FL infection is suspected. Using real-time PCR, it was determined that 80% of stumps had one or more sprouts that were CLas positive. This work confirms the importance of controlling sprouts from citrus stumps in order to minimize the spread of HLB within and between citrus groves. 297

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Year(s) Milestone Principal Investigator(s) Publication/Journal 298 or Author(s) Dawson FCPRAC Progress Report 2009 The response of 32 different citrus varieties or relatives to HLB was (January, 2009) studied. The effect of HLB on the plants was differentiated most dramatically by the rate of continuing growth. Although some trees See also Folimonova, S.Y., developed distinctive symptoms on leaves, growth was inhibited Robertson, C.J., Garnsey, S.M., only marginally, while with other trees when leaves became Gowda, S., and Dawson, W.O. chlorotic, growth ceased. Sweet orange, grapefruit, tangelo, and 2009. Examination of the some mandarins were extremely sensitive. Preliminary results in responses of different terms of most severe symptoms and reduced growth are: Valencia > genotypes of citrus to Hamlin > Rhode Red Valencia > Pineapple for sweet orange; for huanglongbing (citrus greening) grapefruit Duncan = Marsh > Rio Red. under different conditions. Phytopathology 99(12):1346- 1354 Dawson FCPRAC Progress Report 2009 DNA from highly infected citrus was sequenced. To date, we have (January, 2009) 55 million bases from a 454 run with an average read length of 235 bases. We also have 255 million bases from a Solexa sequencer at Oregon State University. An additional 8 billion bases should be available within 2 weeks from a SOLiD sequencer at UF. All of these data will be assembled with expertise from the University of Florida and Oregon State University. Several assembly programs are being tested for this purpose. These data are expected to provide a significant amount of citrus genome data as well as the citrus greening genome. We are attempting to use these data to fill gaps in the sequencing results of other groups. Gmitter FCPRAC Progress Report 2009 Development and validation of a novel and efficient adaptor-based (January, 2009) genomic subtractive enrichment protocol, which enables the nearly complete removal of host DNA and maximizes the likelihood of cloning only bacterial sequences from the extremely low titer of the bacterium present in infected tissues.

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) Gmitter FCPRAC Progress Report 2009 The first transgenic grapefruit plants containing the LIMA construct (January, 2009) were inoculated with HLB 18 months ago and remain RT-PCR negative with no symptoms, while the grafted HLB inoculum budsticks remain RT-PCR positive; this result suggests that LIMA may provide resistance or at least substantially delay disease. Citrus genes involved in systemic acquired resistance (SAR) have been identified and cloned, and several have already been engineered into transgenic plants, and are being tested against HLB and canker. Gmitter FCPRAC Progress Report 2009 A sweet orange genome "re-sequencing project", using next-gen (January, 2009) sequencing (454 of Roche Diagnostics) was initiated and is underway. This project is in concert with the ICGC sequencing initiative plans to use next-generation sequencing technologies on several diploid genomes, as an added resource to the Sanger haploid sequencing project conducted in parallel. Sequence reads already available have been aligned to 23 previously sequenced BAC clones, revealing excellent agreement between the previous Sanger sequence and the currently produced 454 sequence data. Gottwald FCPRAC Progress Report 2009 It is estimated that for every tree that shows symptoms of HLB, (April, 2009) there are on the average 13 trees (range is 2-52) that are infected but are asymptomatic. Latency of HLB is estimated to be from 6 to 36 months. 299

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Year(s) Milestone Principal Investigator(s) Publication/Journal 300 or Author(s) Hall FCPRAC Progress Report 2009 A new strain of Diaphorencyrtus aligarhensis (parasitoid of ACP) (January, 2009) was imported from China and is currently being reared and released to help reduce or eliminate ACP populations. Before release, temperature and relative humidity experiments, as well as parasitism rate experiments were conducted. Choice and no choice tests have shown that this parasitoid will parasitize 2nd thru 4th instar nymphs only and will host feed on 1st thru 4th instars of immature psyllids. Results from these experiments show that this strain is ideally suited for mass rearing and release into Florida as a biological control agent. 2009 In this study, it was noted that fall and winter appear to be time Hall FCPRAC Progress Report periods when percentages of infected psyllids may be consistently (January, 2009) the highest. Hunter FCPRAC Progress Report 2009 cDNA psyllid libraries were produced, which resulted in 17,000 (January, 2009) expressed sequence tags. The libraries were from adult, testes, and midgut tissues. These results aided efforts to identify the microbial fauna of the Asian citrus psyllid, enabling genetic promoters to be designed for further analyses. Eight sequences suggest there is an as yet unidentified phage within the psyllids. Sequences from a new Reovirus were identified and characterized. Sequences from a new Wolbachia species were produced. The internal anatomy of the psyllid was identified for tissue collection in anticipation of future cDNA library constructions. An inbred psyllid population was created F7 to produce the needed genomic material for a psyllid genome effort. Several classes of cathepsins were identified, as well as a new FK506-binding protein; these proteins have important functions in body formation, digestion, and in egg/embryo metabolism, plus cell maintenance. Approximately 60 proteins and all other sequences have been published in the public database, at NCBI for use by the research community.

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) Keyhani FCPRAC Progress Report 2009 D. citri embryos were used to establish a series of primary cell lines (January, 2009) and conditions for routine establishment of stable and transferable primary cell lines have been developed. To date, four cells lines Dce 10, Dce 17 and Dce 19(cell patch), and Dce 19(total) have undergone their first passage. All four cultures displayed cell growth after first passage of the cells into fresh media. Two cell lines, Dce 19 and Dce 17 have undergone their second passage, with clumps of cells appearing to grow. The current time of growth before passage is 4-5 weeks. We have successfully accomplished our second passage for two cells lines and anticipate continuing the cultivation of the cells. It is anticipated that efforts to begin cryopreservation of some cell aliquots will begin soon. Cell morphology of the various lines remains heterogeneous with some cells forming large clumps before spreading in the tissue culture flasks and whereas others remain in suspension. Moore FCPRAC Progress Report 2009 Completion of assembly and annotation of all the Rutaceae (January, 2009) sequences (ESTs and mRNAs) available in public databases (GenBank) and sequences from various previous studies (cold acclimation, canker response, salicylic acid treatment). With this information a new microarray based on Agilent technology was designed. Microarrays have been manufactured and a simple experiment (of known results) has been designed to test them before their release to the scientific community. Rogers FCPRAC Progress Report 2009 The baseline susceptibility information (LC50s) for commonly used (January, 2009) insecticides for psyllid control was determined. These insecticides include the following: fenpropathrin (Danitol 2.4EC), chlorpyrifos (Lorsban 4E), carbaryl (Sevin XLR), imidacloprid (Provado 1.6F), dimethoate (Dimethoate 4EC), zeta-cypermethrin (Mustang), phosmet (Imidan), thiamethoxam (Actara; expected to be labeled for use in FL citrus in 2009) and abamectin (Agri-mek 0.15EC). 301

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Year(s) Milestone Principal Investigator(s) Publication/Journal 302 or Author(s) Salyani and Stelinski FCPRAC Progress Report 2009 Results from a laboratory study that tested the effect of spray (January, 2009) droplet size on ACP control indicate that smaller droplet sizes result in greater mortality of all ACP life stages than larger ones suggesting that ULV sprays may give more effective control of ACP in the field. Tests conducted in the field showed that for insecticides that are known to be effective against ACP, low volume applications are as effective as standard airblast sprays for psyllid control. Dibrom, Micromite (nymph activity only), Malathion, Portal *(nymph activity only), Dimethoate, Provado 1.6 F, Mustang, Danitol, and Delegate are effective against ACP when applied as low volume sprays. 2009 In this study, it was noted that certain micro- and macro-element Spann FCPRAC Progress Report levels are significantly changed in greening infected trees. Data (January, 2009) from this study also suggest that greening-induced nutritional changes may be detectable before the disease is detectable by PCR analysis s

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Year(s) Milestone Principal Investigator(s) Publication/Journal or Author(s) Stelinski FCPRAC Progress Report 2009 Gamma-butyrolactone was identified as one of the behaviorally (January, 2009) active compounds that likely comprise the pheromone of ACP. Testing in the laboratory has confirmed its behavioral activity to psyllids. This chemical is attractive to male psyllids and appears to repel females. Also, this chemical attracts the psyllid parasitoid, Tamarixia radiata. This information will be used to manipulate the parasitoids’ behavior to enhance mass rearing programs of this species, a prerequisite to its utilization as a biocontrol agent. Biologically active compound(s), which attract T. radiata, will be incorporated into an efficient attractant formulation. Such attractants may allow for monitoring T. radiata population densities, which will allow for tailoring spray programs such that peak parasitoid population densities can be avoided and conserved. Also, deployment of a potent attractant within groves. Alpha Scents has formulated gamma-butyrolactone into lures as well as an attract- and-kill formulation for direct psyllid control, which we will be evaluating this year as psyllid populations build up. 2009 CLas was found to be present in bark tissue, leaf midrib, roots and Wang FCPRAC Progress Report different floral and fruit parts but not in the endosperm and embryo (January, 2009); see also of infected fruit. Quantification analysis showed that CLas is not Satyanarayana et al. evenly distributed in planta and a relatively high concentration was (Phytopathology, 2008) found in fruit peduncles. A method for quantifying viable CLas cells was developed. This method (EMA-QPCR), which uses ethidium monoazide to differentiate between live and dead cells, could provide an accurate assessment of the amount of viable CLas cells in plant samples . 303

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Year(s) Milestone Principal Investigator(s) Publication/Journal 304 or Author(s) Wang FCPRAC Progress Report 2009 Microscopy analysis was performed to compare the phloem of HLB (January, 2009). infected citrus and healthy control. It was observed that 1) there was significantly greater accumulation of starch in phloem parenchyma cells of infected leaves, 2) callose deposition occurred more frequently in infected midribs. With the use of transmission electron microscopy, it was observed that sieve pores of the midribs of infected plant were plugged with an amorphous substance. Collapse of sieve tubes and companion cells were also observed in HLB- infected midribs but not in healthy midribs. TEM observations also indicated that the HLB bacterium can pass through the sieve plate pore, which suggests that it is unlikely that the HLB bacterium physically caused phloem blockage because multiple bacterial cells were not aggregating. It is likely that the host response results in sieve pore plugging. Wang FCPRAC Progress Report 2009 Microarray analysis (based on 33,879 expressed sequence tag (January, 2009). Co-author of sequences from several citrus species and hybrids) indicated that Kim in Phytopathology article HLB infection significantly affected expression of 624 genes whose (2009) encoded proteins were categorized according to function. The up- regulation of three key starch biosynthetic genes including ADP- glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves.