Huanglongbing (Citrus Greening): What ARS Is Doing

First detected in Florida in 2005; by 2008, it had been identified in most of the citrus growing counties in the state. Despite everyone's best efforts, HLB now literally threatens the survival of Florida citrus and is a potential threat to the entire U.S. citrus industry.

ARS scientists are actively engaged in research with university and industry partners on the critical components of this disease such as etiology and epidemiology, the insect vector, and the plant host's response to HLB infection.

In February 2008, members of the citrus industry requested additional input from ARS scientists, especially from researchers outside of the citrus field, to draw on the most diverse expertise possible to elicit new ideas about research needs, gaps, priorities, and opportunities.

In response, ARS convened a workshop in April 2008 at the U.S. Horticultural Research Laboratory in Fort Pierce, Florida, especially inviting ARS scientists who are leaders in their scientific fields but who are not ordinarily engaged in citrus research. These ARS scientists brought their collective expertise in animal and plant entomology, animal and plant pathology, genetics, horticulture, and microbiology to the workshop. The workshop was coordinated with the National Academy of Sciences (NAS), which had been asked by the Florida Department of Citrus to develop a Request for Proposals to manage grant funding for research leading to HLB management.

That meeting was followed in May 2008 by a workshop in Beltsville, Maryland, of key ARS citrus researchers to discuss potential new collaborations and research directions to address the needs identified at the April 2008 workshop.

On September 17-18, 2008, a group of ARS scientists, industry and university representatives from citrus producing states met with the federal regulatory agencies USDA-APHIS, FDA, and EPA to prepare for the advent of transgenic citrus to be used to combat HLB and canker to seek guidance on bringing these products to market.. This meeting was an excellent opportunity for each party to learn about what will be needed as progress is made toward this goal. Other potential new chemicals for insect control and management of the pathogen in citrus were also discussed. A team of scientists are preparing documents that summarize published data on citrus biology, pollination, gene flow and juice composition parameters; information that will be needed by the regulatory agencies as transgenic citrus products are developed.

Current ARS Research

ARS has initiated a significant research program, even before the 2008 workshops.

ARS scientists have established partnerships with industry and university colleagues, and these collaborations continue to grow and diversity as new knowledge is developed. The ultimate goal is to effectively manage major threats to the citrus industry in the U.S., in particular, citrus greening, or HLB, and canker. A broad approach is being taken to target the main components of the disease triangle; the host, the pathogen and associated organisms, and the insect vector. Insect management, repellants and control are critical to disease control. Manipulating citrus groves by varying nutrition and high density planting may reduce the impact of the diseases. As scientists learn more about the bacterium associated with HLB, methods for directly impacting its growth in the citrus host will be devised. Conventional breeding and genetically modified citrus varieties are being explored. All of these components of disease intervention are being investigated by ARS scientists and colleagues, and will be utilized for to benefit the national citrus industry.

Accomplishments

Because of the fastidious nature of HLB bacteria (Candidatus Liberibacter asiaticus), a high-throughput detection technology for HLB is critical for HLB research and disease management. Polyclonal antibodies against HLB pathogen have been developed by researchers at Fort Pierce, Florida, and a series of primers and probes were also developed for better detection of HLB using PCR or in situ hybridization. These technologies have been transferred to university and state regulatory research laboratories for HLB detection and other research purposes.

ARS scientists at Frederick, Maryland, determined the effect of imidacloprid insecticide on transmission of HLB. Following incorporation of the insecticide into sweet orange seedlings, infectious psyllids were aspirated on the seedlings and monitored for survival and disease transmission. Although most psyllids died within the first 24 hours, strong symptoms of HLB infection were evident by 8 weeks in most plants, indicating the insects could transmit the causal HLB bacterium before dying.

ARS scientists at Beltsville, Maryland developed and validated the first real-time and quantitative PCR assay for the greening pathogen. This assay was used by the USDA Animal and Plant Health Inspection Service (APHIS) and the Florida Department of Agriculture to confirm the presence of HLB in Florida. Producers will benefit from early detection and genetic improvement for resistance will be accelerated.

ARS scientists at Ft. Pierce and colleagues recently published the full genomic sequence of HLB bacterium, 'Candidatus Liberibacter asiaticus'. This is a major scientific advance, and will enable scientists to identify genes responsible for the disease, enhance capabilities to culture this organism and determine its true role as the causal agent.

Scientists at Ft. Pierce have evidence that suggests that guava, interplanted with citrus, prevents the spread of greening. Scientists in Japan and Viet Nam discovered that HLB did not occur in citrus groves when intercropped with guava, while control plots had over 30 percent infection. The mechanism may be volatile compounds produced by guava which disrupt the ability of the psyllid vector of HLB to locate its citrus host. Studies are underway in field plots to validate these preliminary findings and to identify the potential volatiles.

ARS Research Projects on Huanglongbing (Citrus Greening)

Exotic Pathogens of Citrus
LEAD SCIENTIST: John S. Hartung, Beltsville, Maryland

IPM Technologies for Subtropical Insect Pests
LEAD SCIENTIST: David Hall, Fort Pierce, Florida

Identification, Characterization, and Biology of Foreign and Emerging Insect-Transmitted Plant Pathogens
LEAD SCIENTIST: Vernon D. Damsteegt, Ft. Detrick, Maryland

Identification, Characterization, and Detection of Foreign and Newly Emerging Domestic Bacteria
LEAD SCIENTIST: Norm Schaad, Ft. Detrick, Maryland

Epidemiology and Management of Xylella Fastidiosa (XF) and other Exotic and Invasive Diseases and Insect Pests
LEAD SCIENTIST: Drake Stenger, Parlier, California

Management of Citrus and Date Genetic Resources and Information
LEAD SCIENTIST: Richard Lee, Riverside, California

Genetic Improvement of Citrus
LEAD SCIENTIST: Kim Bowman, Fort Pierce, Florida

Domestic, Exotic, and Emerging Diseases of Citrus, Vegetables, and Ornamentals (DEED)
LEAD SCIENTIST: Timothy Gottwald, Fort Pierce, Florida

ARS Research Publications on Citrus Greening/Huanglongbing (HLB)

Publication Abstract: Effect of Early Detection Greening on Juice Flavor and Chemistry

Publication Abstract: NPDN Citrus Greening Diagnostic and Detection Efforts

Asian Citrus Psyllid and Citrus Greening - A Closer Look at the Vector
Citrus and Vegetable Magazine

Huanglongbing, Citrus Greening: Preparing for the Future
Subtropics Newsletter

Twelve Polymorphic Microsatellite Loci from the Asian Citrus Psyllid, Diaphorina Citri (Kuwayama), the Vector for Citrus Greening Disease (Huanglongbing)
Molecular Ecology Notes

Sensitivity Of PCR-based Methods for Detection of Asiatic Strain of the Huanglongbing Bacterium in Symptomatic and Asymptomatic Citrus Tissues
Proceedings of the Huanglongbing-greening International Workshop, July 14-21, 2006, Ribeiro Preto, Brazil

Publication Abstract: Differences in Secondary Metabolites in Leaves from Trees Affected With the Greening (Hlb) Disease

Citrus Huanglongbing: The Pathogen and Its Impact
Plant Health Progress

Citrus Canker and Citrus Huanglongbing, Two Exotic Bacterial Diseases Threatening the Citrus Industries of the Western Hemisphere
Outlooks on Pest Management

Progress Report to the Citrus Industry: Florida Citrus Plant Protection Committee (FCPPC)
Citrus Industry

Detection And Identification of Candidatus Liberobacter Species Associated with Citrus Huanglongbing by Multiplex Real-time PCR
Journal of Microbiological Methods

Gene Expression in the Asian Citrus Psyllid: Vector of Citrus Greening (Hemiptera: Psyllidae)
Joint Proceedings of the 89th Annual meeting and 6th International Caribbean Conference of the Florida Entomologtical Society

Incidence Of the Asian Citrus Psyllid, Diaphorina Citri (Hemiptera: Psyllidae) and its Parasitoid Tamarixia Radiata (Hymenoptera: Eulophidae) in Florida Citrus
The 91st Annual Meeting of the Florida Entomological Society

Ribosomal Proteins of the Asian Citrus Psyllid, Diaphornia Citri
National Center for Biotechnology Information (NCBI)

Reovirus-like Sequences Isolated from Adult Asian Citrus Psyllid, (Hemiptera: Psyllidae: Diaphorina Citri)
Florida Entomologist

Comparison Of Visual Assessment to Polymerase Chain Reaction Assay Testing to Estimate the Incidence of the Huanglongbing Pathogen in Commercial Plantings in Florida
Proceedings of Florida State Horticultural Society

Gene Expression in Citrus Sinensis (L.) Osbeck after Infection with the Bacterial Pathogen Candidatus Liberibacter Asiaticus Causing Huanglongbing in Florida
Plant Science

Evaluation Of DNA Amplification Methods for Improved Detection of Candidatus Liberibacter Species Associated with Citrus Huanglongbing
Plant Disease

Publication Abstract Genome Sequence of Candidatus Liberibacter Asiaticus, the Casual Agent of Citrus Huanglongbing (Greening)

Effects Of a Particle Film on Biology and Behavior of Diaphorina Citri (Hemiptera: Psyllidae) and its Infestations in Citrus
Journal of Economic Entomology

Discovery of a Viral Pathogen in the Asian Citrus Pysllid
72nd Annual Meeting of the Florida Academy of Sciences in conjunction with the 85th Annual Meeting of the Georgia Academy of Sciences, March 14-15, Jacksonville, Florida

Daily Timing of Mating and Age at Reproductive Maturity in Diaphorina Citri (Hemiptera: Psyllidae)
Florida Entomologist

Optimized Quantification of Unculturable Candidatus Liberibacter Species Causing Citrus Huanglongbing in Host Plants by Real-Time PCR
Plant Disease

Publication Abstract: Genome Sequence of Candidatus Liberibacter Asiaticus, the Casual Agent of Citrus Huanglongbing (Greening)

Publication Proceedings: Concepts in Huanglongbing Epidemiology

Multiplex Taqman-based PCR for Sensitive and Accurate Quantification of Citrus HLB and CVC Pathogens; Candidatus Liberibacter and Xylella Fastidiosa
Proceedings of the Huanglongbing-greening International Workshop

Optimized Quantification of Unculturable Candidatus Liberibacter Species Causing Citrus Huanglongbing in Host Plants by Real-Time PCR
Plant Disease

First Report of Dodder Transmission of Huanglongbing from Naturally Infected Murraya paniculata to Citrus
Plant Disease

Incidence of Huanglongbing in Psyllids Under Natural Conditions in Florida
Phytopathology

Preliminary Spatial Pattern Analysis of Huanglongbing in Sao Paulo, Brazil
International Organization of Citrus Virologists Proceedings

Use of Psyllids in Early Identification of Huanglongbing Disease of Citrus
Phytopathology

Huanglongbing Epidemiology: an International Perspective
Proceedings of Huanglongbing-greening International Workshop, July 14-21, 2006, Ribeiro Preto, Brazil

Huanglongbing: The Dragon Arrives in the USA
Huanglongbing-greening International Workshop, July 14-21,2006, Ribeiro Preto, Brazil

Epidemiology of Huanglongbing in Sao Paulo
Huanglongbing-greening International Workshop, July 14-21, 2006, Ribeiro Preto, Brazil

Huanglongbing Epidemiology: Tracking the Dragon Through Time and Space
Second International Citrus Canker and Huanglongbing Workshop, Orlando, FL, November 7-11, 2005

Spatial Distribution of Huanglongbing Symptomatic Trees in Citrus Groves in Sao Paulo, Brazil
Second International Citrus Canker and Huanglongbing Workshop, Orlando, FL, November 7-11, 2005

Detection Of Candidatus Liberibacter Asiaticus from Citrus Huanglongbing Samples in China By Nested Conventional and Real-time PCR
Phytopathology

Biology, History and World Status of Diaphorina Citri
North American Plant Protection Organization Workshop, Taller Internacional Sobre Huanglongbing y el Psilido Asiatico de los Citricos, del 7 at 9 de Mayo de 2008, Hermosillo, Sonora

Progress Towards the Development of an Effective Risk Analysis Process for the Florida Citrus Nursery Industry to Mitigate the Impact of Citrus Canker and Huanglongbing
Proceedings from the International Citrus Canker and Huanglongbing Workshop, Orlando, FL, Nov. 2005

Intercropping Of Citrus and Guava Trees for Management of Huanglongbing
Florida Entomological Society Annual Meeting, July 15-18, 2007, Sarasota, Florida

Psyllids as a Tool in Evaluating the Efficiency of Different Management Practices for Control of Citrus Huanglongbing Disease
Phytopathology

Novel Molecular Technique for Rapid Cloning of Unknown Sequences From Unculturable Huanglongbing (HLB) Associated Bacterium, Candidatus Liberibacter
American Phytopathology Society Meeting

Murraya paniculata (Orange Jasmine), a Host and Possible Inoculum Reservoir for Candidatus Liberibacter Asiaticus, Causal Agent of Huanglongbing
Phytopathology

Large-scale Surveys for Multiple Pest Species; the Search for Citrus Canker and Huanglongbing in Florida
American Phytopathological Society Annual Meeting

Comparative Genomics and Phylogenetic Analyses of Newly Cloned Genomic Regions From the Citrus Huanglongbing (HLB)-associated Bacterium Candidatus Liberibacter
American Phytopathology Society