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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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Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
1967
With the use of psyllid to transmit the HLB pathogen, it was confirmed that trees with "citrus dieback" symptoms were positive for HLB.
Capoor et al
Indian Journal of Agricultural Sciences 37(6):572-576
1968
Citrus dieback in India is reported to have many similarities with greening disease of South Africa. The similarities with huanglongbing in China could not have been mentioned because Lin’s work on HLB was still not known out of China.
Fraser and Singh
Proceedings of 4th Conference, IOCV:141-144, University of California, Division of Agricultural Sciences, Riverside, CA.
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.
Schneider
Phytopathology 58:1155-1160
1969
HLB in Africa was found to be heat-sensitive and occurs only in areas below 30-32°C. Trioza erytreae, the African psyllid vector, was also found to thrive only in cool environments.
Catling
Journal of Entomology Society South Africa 32:209-223;
1970
"Mycoplasma-like organism" observed in citrus phloem tissue infected with HLB through electron microscopy.
Laflèche and Bové
Comptes Rendus de L'Academie des Sciences, Paris, 270:1915-1917
1970
These are the first two reports on bacterial structures associated with HLB, a disease until then considered to be caused by a virus. The 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).
Laflèche and Bové
Fruits 25: 455-465 C.R. Acad. Sci. Paris 270:1915-17
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) or Author(s)
Publication/Journal
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.
Saglio et al.
Physiologie Végétale 9: 569-82.
1971
As greening was found to be associated with bacteria, antibiotics, such as tetracyclines, were injected into affected trees in an effort to control the disease.
Schwarz and van Vuuren
Plant Disease Reporter 55: 747-750.
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
1974
First demonstration under phytotron conditions that African HLB was heat-sensitive (no symptom development at 32°C), while Asian HLB (from India, the Philippines) was heat tolerant (good symptom development at 32°C).
Bové et al.
Proceedings of 6th Conference, IOCV:12-15, University of California, Division of Agricultural Sciences, Riverside, CA.
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Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
1974
The cell envelope of the organism associated with greening was found to resemble that of a Gram-negative bacterium. However, the presence of peptidoglycan, a characteristic component of the bacterial cell wall, could not be demonstrated.
Moll and Martin
Proceedings of Conference Les Mycoplasmes/Mycoplasmas, INSERM 33: 89-96
1976
The African citrus psyllid vector, Trioza erytreae, is not only vector of the African HLB bacterium, but can also transmit the Asian HLB bacterium.
Massonié, Garnier, and Bové
Proceedings of 7th Conference (1976) IOCV: 18-20, University of California, Division of Agricultural Sciences, Riverside, CA.
1977
Through electron microscopy, the HLB causal organism was shown to possess a cytoplasmic membrane and a bacterial cell wall.
Garnier and Bové
Fruits 32:749-752
1980
Greenhouse-grown HLB-affected citrus plants having absorbed penicillin through the roots grew better, produced more roots and larger symptomless shoots and leaves than untreated controls. In contrast, Penicillin had no effect on Stubborn-affected citrus plants. 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.
Aubert and Bové; Bové et al.
Proceedings of 8th Conference, (1980) IOCV: 103-108; 91-102 University of California, Division of Agricultural Sciences, Riverside, CA.
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Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
1980
First successful biological control of Trioza erytreae and Diaphorina citri, the two psyllid vectors of HLB, in Reunion Island.
Etienne and Aubert
Proceedings of 8th Conference, (1980) IOCV: 118-121 University of California, Division of Agricultural Sciences, Riverside, CA.
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 and Bové
Phytopathology 73: 1358-1363
1984
The HLB-associated bacterium was shown to possess a cell wall of the Gram-negative type with an outer cell wall membrane and a peptidoglycan layer, thus establishing the Gram negative nature of the HLB-associated bacterium.
Garnier, Danel and Bové
Annales de l'Institut Pasteur. Microbiologie 135(1): 169-179; Proceedings of 9th Conference, IOCV: 115-124 University of California, Division of Agricultural Sciences, Riverside, CA.
1986
The ACP vector, Diaphorina citri, is not only vector of the Asian 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.
Lallemand, Fos, and Bové
Fruits 41: 341-343
1991
First extensive review on citrus greening disease was published.
Da Graça
Annual Review of Phytopathology 29:109-36
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Year(s)
Milestone
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1992
By random cloning of total DNA from periwinkle plants affected by Indian HLB, the first genome fragments of the HLB-associated 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.
Villechanoux et al.
Current Microbiology 24: 89-95; Current Microbiology 26: 161-166.
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
1994
On the basis of 16SrDNA sequence analyses, the HLB-associated bacterium was confirmed to be a Gram negative bacterium, 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.
Jagoueix, Bove, and Garnier
International Journal of Systematic Bacteriology 44: 397-86
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
1995
Part of the rplKAJL-rpoBC operon of the African liberobacter was 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.
Planet et al.
Current Microbiology 30: 137-141
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Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
1996
First PCR detection of the Asian and African liberibacters by amplification of 16S rDNA.
Jagoueix, Bove, and Garnier
Molecular and Cellular Probes 10: 43-50.
1996
Already known to harbor both the African and the Asian psyllid vectors of HLB, Reunion and Mauritius islands were also shown to carry both the African and the Asian liberibacters.
Garnier et al.
Proceedings of 13th Conference, IOCV: 392-394, University of California, Division of Agricultural Sciences, Riverside, CA
1997
Confirmation that CLas and CLaf are two different bacterial species based on the 16S/23S rRNA intergenic regions.
Jagoueix et al.
International Journal of Systematic Bacteriology 47(1):224-227
1997
Petroleum spray oil was tested against the ACP in Guangzhou, China; It was found that petroleum oil was as effective as an organophosphate pesticide and an insect growth regulator in controlling psylla nymphs.
Rae et al.
International Journal of Pest Management 43(1):71-75
1999
Random Amplified Polymorphic DNA (RAPD) was used to identify additional genomic sequences of the HLB liberibacters. In particular, the omp (outer membrane protein) gene was obtained.
Hocquellet, Bove, and Garnier
Hocquellet, A., Bové, J.M., and Garnier, M. 1999. Isolation of DNA from the uncultured “Candidatus Liberobacter” species associated with citrus huanglongbing by RAPD.
1999
A PCR detection method based on the amplification of ribosomal protein genes, which allows direct identification of the liberibacter species by the size of the amplified DNA, was developed.
Hocquellet et al.
Molecular and Cellular Probes 13(5):373-379
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Year(s)
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2000
The Asian psyllid vector of HLB, Diaphorina citri, was captured for the first time in southern Iran in 1997 during a mission on Witches' Broom Disease of Lime.
Bové et al.
Proceedings of 14th Conference, IOCV: 207-212 University of California, Division of Agricultural Sciences, Riverside, CA
2000
Following a 1999 proposal in prokaryote nomenclature, “Liberobacter”, “africanum”, and “asiaticum were respectively 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”.
Garnier et al.
International Journal of Systematic and Evolutionary Microbiology 50: 2119-2125.
2000
The HLB causal agent was shown to replicate in Chinese box orange (Severinia buxifolia) and wood apple (Limonia acidissima) but not in common jasmine orange (Murraya paniculata var. paniculata) and curry leaf (Murraya euchrestifolia).
Hung et al.
Journal of Phytopathology-Phytopathologische Zeitschrift 148(6):321-326
2000
This study was conducted to determine the effect of temperature on 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.
Liu and Tsai
Annals of Applied Biology 137(3):201-206
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Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
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.
Subandiyah et al.
Zoological Science 17:983-989
2000
Taylor's power law and Iwao's patchiness regression models indicated that D. citri populations were aggregated.
Tsai et al.
Florida Entomologist 83(4):446-459
2001
The ability of D. citri and its two parasitoids, Tamarixia radiata and 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.
McFarland and Hoy
Florida Entomologist 84(2): 227-233
2002
Psyllid population levels on orange jasmine were found to be 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.
Tsai et al.
Florida Entomologist 85(3):446-451
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 the Asian citrus psyllid vector but it is not transovarially transmitted.
Hung et al.
Plant Pathology 53(1):96-102
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2004
Results from a laboratory and spray booth study indicated that 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).
McKenzie and Puterka
Journal of Economic Entomology 97(3):970-975
2004
Coccinellid beetles were found to be the most important biological control agents of high-density populations in central Florida and that intraguild predation causes >95% mortality of immature stages of Tamarixia radiata.
Michaud
Biological Control 29(2):260-269
2004
A rearing method was developed for ACP and its parasitoids, Tamarixia radiata and Diaphorencyrtus aligerhensis.
Skelley and Hoy
Biological Control 29(1):14-23
2005
Omp (outer membrane protein gene)-based PCR-RFLP analysis was 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.
Bastianel et al.
Applied Environmental Microbiology 71:6473-6478.
2005
Murraya paniculata, the preferred host of the Asian HLB psyllid vector, was also found to be a host of the two HLB bacteria present in São Paulo State, CLam and CLas.
Lopes, Martins, and Frare
Summa Phytopathologica 31: 48-49.; Fitopatologia Brasileira 31: 303.
2005
Discovery and first report of a new liberibacter species associated with HLB in São Paulo State, Brazil: Candidatus Liberibacter americanus. The new liberibacter was also detected in Diaphorina citri, suggesting that the Asian psyllid vector in Brazil was transmitting not only CLas, but also CLam.
Texeira et al.
Plant Disease 89: 107; International Journal of Systematic and Evolutionary Microbiology 55: 1857-1862; Proceedings of 16th Conference, IOCV: 325-340 University of California, Division of Agricultural Sciences, Riverside, CA
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2005
Development of PCR techniques for the detection of CLam and CLas in citrus and psyllids.
Texeira et al.
Molecular and Cellular Probes, 19; 173-179; Proceedings of 16th Conference, IOCV: 432-438 University of California, Division of Agricultural Sciences, Riverside, CA
2005; 2007
CLas was detected in Wampee (Clausena lansium Skeels) using nested PCR.
Ding et al.; Deng et al.
Journal of Plant Pathology 87(3):207-212; Plant Health Progress 2007
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.
Okuda et al.
Plant Disease 89(7):705-711
2005
Coccinellid species found to have potential important role as predators of the psyllid in Puerto Rico.
Pluke et al.
Florida Entomologist 88(2):123-128
2005
Third bacterial species was detected and identified as another causal organism of HLB in Brazil. The name Ca. Liberibacter americanus was proposed for this HLB pathogen. Detection of CLam in D. citri indicated that this is also the vector for this liberibacter species.
Teixeira et al.
Molecular and Cellular Probes 19(3):173-179; International Journal of Systematic and Evolutionary Microbiology 551857-1862 Part 5
2005
In greenhouse trials, a neem-based biopesticide containing 4.5% azadirachtin was found to reduce psyllid nymph populations; however, no mortality was observed in psyllid adults that were exposed to 11-180 ppm azadirachtin.
Weathersbee and McKenzie
Florida Entomologist 88(4):401-407
2006
Guava intercropping was observed to reduce ACP/HLB incidence in Vietnam
Beattie et al.
Unpublished
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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).
Faghihi et al.
Plant Pathology 58: 793.
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.
Kim et al.
Phytopathology 99(1):50-57
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.
Li et al.
Phytopathology 99(2):139-144
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2009
While the new liberibacter described by Hansen et al.; see Hansen et al., 2008) as Candidatus Liberibacter psyllaurus was characterized from the tomato/potato psyllid, Bactericera cockerelli, the liberibacter described by Liefting et al. (2009a, b) as Candidatus Liberibacter solanacearum was characterized from tomato and other 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.
Liefting et al.; Lin et al.
Plant Disease 93: 208-214; International Journal of Systematic and Evolutionary Microbiology 59: 2274-2276; Journal of Plant Pathology 91: 215-219.
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.
Phytopathology 99: 301-306.
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.
Lopes et al.
Plant Disease 93: 257-262.
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2009
Phylochip analysis indicated that 47 orders of bacteria in 15 phyla 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.
Sagaram et al.
Applied and Environmental Microbiology 75(6):1566-1574
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. Confirmation in several laboratories is attempted.
Sechler et al.
Phytopathology 99:480-486.
2009
This study provides evidence that D. citri uses olfactory and visual cues in orientation to host plants which indicates the possibility of using plant volatiles to monitor and manage ACP.
Wenninger et al.
FCPRAC Annual Report 2008; Environmental Entomology 38(1):225-234
2009
Vibrational communication between male and female psyllids was studied. Findings include: 1) both sexes produced simple, low 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.
Wenninger et al.
Annals of the Entomological Society of America 102(3):547-555
2009
The size of the CLam genome was determined by pulse field gel electrophoresis, using Lam-infected periwinkle plants for bacterial 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.
Wulff et al.
International Journal of Systematic and Evolutionary Microbiology 59: 1984-1991.
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2009
Two field trials have been established in Florida to evaluate the resistance of transgenic citrus to citrus canker and citrus greening. The transgenic citrus tested carry the following genes: 1) gene encoding for a plant-based anti-microbial peptide and 2) a 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.
M. Irey
http://www.aphis.usda.gov/biotechnology/status.shtml
http://www.isb.vt.edu/cfdocs/fieldtests1.cfm
FCPRAC 2009 Progress Reports
2009
Results from this study indicate that D. citri which complete their 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
FCPRAC Progress Report (January, 2009)
2009
Results from this study provided (further) evidence that distribution of the greening-associated pathogen varies widely within symptomatic, PCR-positive citrus trees and thus illustrates the importance of obtaining multiple samples from trees where an 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.
Brlansky and Davis
FCPRAC Progress Report (January, 2009); Proceeding of the International Research Conference on Huanglongbing. Orlando, FL
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2009
The response of 32 different citrus varieties or relatives to HLB was studied. The effect of HLB on the plants was differentiated most dramatically by the rate of continuing growth. Although some trees developed distinctive symptoms on leaves, growth was inhibited only marginally, while with other trees when leaves became chlorotic, growth ceased. Sweet orange, grapefruit, tangelo, and some mandarins were extremely sensitive. Preliminary results in terms of most severe symptoms and reduced growth are: Valencia > Hamlin > Rhode Red Valencia > Pineapple for sweet orange; for grapefruit Duncan = Marsh > Rio Red.
Dawson
FCPRAC Progress Report (January, 2009)
See also Folimonova, S.Y., Robertson, C.J., Garnsey, S.M., Gowda, S., and Dawson, W.O. 2009. Examination of the responses of different genotypes of citrus to huanglongbing (citrus greening) under different conditions. Phytopathology 99(12):1346-1354
2009
DNA from highly infected citrus was sequenced. To date, we have 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.
Dawson
FCPRAC Progress Report (January, 2009)
2009
Development and validation of a novel and efficient adaptor-based 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.
Gmitter
FCPRAC Progress Report (January, 2009)
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Principal Investigator(s) or Author(s)
Publication/Journal
2009
The first transgenic grapefruit plants containing the LIMA construct 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 (January, 2009)
2009
A sweet orange genome "re-sequencing project", using next-gen 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.
Gmitter
FCPRAC Progress Report (January, 2009)
2009
It is estimated that for every tree that shows symptoms of HLB, 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.
Gottwald
FCPRAC Progress Report (April, 2009)
OCR for page 300
Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease
Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
2009
A new strain of Diaphorencyrtus aligarhensis (parasitoid of ACP) 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.
Hall
FCPRAC Progress Report (January, 2009)
2009
In this study, it was noted that fall and winter appear to be time periods when percentages of infected psyllids may be consistently the highest.
Hall
FCPRAC Progress Report (January, 2009)
2009
cDNA psyllid libraries were produced, which resulted in 17,000 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.
Hunter
FCPRAC Progress Report (January, 2009)
OCR for page 301
Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease
Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
2009
D. citri embryos were used to establish a series of primary cell lines 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.
Keyhani
FCPRAC Progress Report (January, 2009)
2009
Completion of assembly and annotation of all the Rutaceae 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.
Moore
FCPRAC Progress Report (January, 2009)
2009
The baseline susceptibility information (LC50s) for commonly used 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).
Rogers
FCPRAC Progress Report (January, 2009)
OCR for page 302
Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease
Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
2009
Results from a laboratory study that tested the effect of spray 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.
Salyani and Stelinski
FCPRAC Progress Report (January, 2009)
2009
In this study, it was noted that certain micro- and macro-element levels are significantly changed in greening infected trees. Data from this study also suggest that greening-induced nutritional changes may be detectable before the disease is detectable by PCR analysis
Spann
FCPRAC Progress Report (January, 2009)
OCR for page 303
Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease
Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
2009
Gamma-butyrolactone was identified as one of the behaviorally 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.
Stelinski
FCPRAC Progress Report (January, 2009)
2009
CLas was found to be present in bark tissue, leaf midrib, roots and different floral and fruit parts but not in the endosperm and embryo of infected fruit. Quantification analysis showed that CLas is not evenly distributed in planta and a relatively high concentration was 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
Wang
FCPRAC Progress Report (January, 2009); see also Satyanarayana et al. (Phytopathology, 2008)
OCR for page 304
Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease
Year(s)
Milestone
Principal Investigator(s) or Author(s)
Publication/Journal
2009
Microscopy analysis was performed to compare the phloem of HLB 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 (January, 2009).
2009
Microarray analysis (based on 33,879 expressed sequence tag sequences from several citrus species and hybrids) indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The upregulation of three key starch biosynthetic genes including ADPglucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves.
Wang
FCPRAC Progress Report (January, 2009). Co-author of Kim in Phytopathology article (2009)
