Source: UNIVERSITY OF FLORIDA submitted to NRP
INFECTION PROCESSES ASSOCIATED WITH CITRUS CANKER AND CITRUS GREENING.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SPECIAL GRANT
Reporting Frequency
Annual
Accession No.
0215193
Grant No.
2008-34446-19462
Cumulative Award Amt.
(N/A)
Proposal No.
2008-04106
Multistate No.
(N/A)
Project Start Date
Sep 1, 2008
Project End Date
Aug 31, 2010
Grant Year
2008
Program Code
[RA]- Citrus Canker/Greening, FL
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
CITRUS RESEARCH & EDUCATION CENTER, LAKE ALFRED
Non Technical Summary
Florida annually accounts for more than 81% of the United States total citrus production. Florida grapefruit and orange production accounts for 32.3 and 18.50% respectively, of the world's annual production, and 81.1 and 68.7% of the USA's annual production. Annually, Florida produces more than 900 million gallons of orange juice. Citrus accounts for about 26% of Florida's agricultural sales. Nationally, Florida citrus sales account for $7.8 billion dollars annually. With the introduction of citrus greening into Florida in 2005, research challenges have arisen for which we are not fully prepared. Some immediate needs are related to the commitment to locate diagnostic indicators in the bacterial genes that can be used in a more sensitive diagnostic tool. Internal as well as external capabilities will be leveraged immediately with additional resources. In addition, immediate progress is being made by bringing together a team of bacteriologists to culture the greening bacterium, previously reported as not culturable. These two hurdles are major constraints to the development and delivery of both short and long-term solutions, but are goals that we plan to pursue aggressively. A consequence of implementing new management systems to cope with canker and HLB disease presence in the state are increased operational costs. To the degree that this increases the overall cost of production, grower economics will be affected. Thus, an ongoing economic analysis will need to be applied to all emerging tactics and impacts of these diseases. At present, a team of UF, IFAS, Florida Department of Citrus, and private economists are building a platform that will allow analysis of changes in production costs, expected returns with changes in supply, effects of acreage in citrus, and other major variables. These analyses will be used to determine the economics and profitability associated with the presence of these diseases in Florida as well as other relevant factors such as land conversion and hurricane impacts. In the short-term, better tools to detect these diseases and reduce their movement are vital. Over the long-term, development of citrus plants which can resist infection by citrus canker and greening is vital. It is doubtful that a chemical or other single approach will be available to stop field infection or prevent further spread. The need for continuing research exists to meet these ultimate goals of sustainable citrus production. Ultimately, the results of this research must be delivered to the affectged citrus growers and homeowners who are affected by these diseases. An aggressive delivery mechanism is in place through the Extension component of this project.
Animal Health Component
50%
Research Effort Categories
Basic
40%
Applied
50%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2120999104020%
2120999110050%
2120999116020%
2120999108010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1040 - Molecular biology; 1160 - Pathology; 1100 - Bacteriology; 1080 - Genetics;
Goals / Objectives
Development and deployment of management strategies requires in-depth understanding of the pathogens, their interactions with citrus plants and in the case of HLB, interactions with an insect vector. This project proposes to focus on elucidation of the basic aspects of HLB and canker as these diseases interact with the citrus plant. The objectives focus on better understanding how these pathogens invade and cause disease. These objectives include: 1); Understanding the biology and ecology of the canker or HLB pathogen; 2); Epidemiology of canker or HLB; 3);Genetic and physiological aspects of canker or HLB infection and disease manifestation; and 4); Development and delivery of educational programs through extension to provide the results of research on canker and HLB to diverse audiences
Project Methods
An aggressive research plan was elaborated at the 2nd International Citrus Canker and Greening Workshop held in Orlando in December, 2005. UF, IFAS has developed research plans against which resources can be applied, and a critical core set of objectives from that plan is proposed here. Overcoming several major obstacles to delivering solutions for greening are among the top priorities, and we have identified pathways to address these obstacles. There are a number of possible management strategies for HLB, and these include: Control spread of the disease by reducing the level of the psyllid Control the spread of the disease by removing sources of inoculum Use tolerant plants to reduce disease losses Engineer resistant plants Biological control of citrus greening There are several basic tools or achievements that are essential for all of the above management strategies to be advanced. Success of the research and outreach objectives proposed below for this program is necessary for advancement toward implementation of novel management strategies. As in previous years of this Special Grant, the UF, IFAS management team will seek competitive proposals from within and without UF, IFAS which address the objectives listed above. Resulting proposals will be subjected to peer review and the results will be provided to USDA, CSREES as a packet of sub-awards recommended for funding. It is anticipated that the funding from this continuation will supplement funding associated with currently funded work on the Special Grant, and new proposals will be encouraged as well. A condition for continued funding will be adequate demonstration of progress with existing funding. The management team of IFAS responsible for directing the call for proposals, the review and ultimately the funding decisions include the PDs and relevant disciplinary chairs. Due to the seriousness of the disease situation confronting U.S. citrus, collaborative relationships will be sought with scientists outside of the University of Florida, comprised primarily of existing programs at other Land Grant Institutions where capability exists. Other Universities and agencies may also be contacted directly for participation. Collaborators from outside of UF, IFAS will be funded through sub-contract or sub-awards arranged through the University of Florida, Division of Sponsored Research.

Progress 09/01/08 to 08/31/11

Outputs
OUTPUTS: Goals and objectives of the project were to understand the biology and disease development of citrus canker and greening, examine the epidemiology of canker or greening, and explore the genetic and physiological aspects of canker or infection and disease manifestation. We sought competitive proposals from within and outside of UF-IFAS to address the objectives and goals. Experiments were designed to develop a basic understanding of the bacteria causing canker and greening, and how the organisms induce disease. The greening bacterium is present in very low titer in citrus tissues, making detection difficult. Moreover, the greening bacterium has not yet been cultured, underscoring the difficulty in making rapid progress to successfully manage and cure the disease. Research focused on early detection methods, host range, susceptibility and tolerance determinants. Bacterial virulence mechanisms were explored to determine the genetic and physiological basis of infection and disease spread. Bacterial communication was explored, the presence and role of virulence genes, the possible presence of toxins, and the differential reaction of citrus cultivars and citrus relatives. In addition, outreach was a key element of the effort. Educational programs and products were developed and delivered to provide the results of research on canker and greening to diverse audiences. An existing team of extension professionals were added as collaborators to projects. Products and displays were created and disseminated throughout the life of the project. Venues included citrus trade shows, grower and producer meetings, and invited speeches to commodity and government officials. PARTICIPANTS: Nain Wang, UF-IFAS, Assistant Professor of Microbiology and Cell Science Kuang-Ren Chung, UF-IFAS, Associate Professor of Plant Pathology Pamela Roberts, UF-IFAS, Professor of Plant Pathology Svetlana Folimonova, UF-IFAS, Research Assistant in Virology TARGET AUDIENCES: A team of extension professional were added as collaborators. Products and displays were created and disseminated through the life of the project. Target audiences were growers, producers, scientists working in the area. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Host range surveys identified sensitive, intermediate, tolerant and extremely tolerant citrus types. In most cases titer in tolerant and sensitive varieties were similar. Phloem and cambial necrosis was verified. Assessment of Zanthoxylum fagara as a natural host for the greening pathogen indicated no bacteria present. Differential expression of 360 genes in Sweet orange plants (Valencia) and psyllids infected with Ca. L. asiaticus was evaluated using quantitative reverse transcription PCR. A large number of genes overexpressed in the plant were predicted to be localized to the cytoplasm. A number of genes involved in assembly of flagellar machinery were up-regulated. Several genes involved in protein export including secE, secA, ffh spase1 and spaseII were also up-regulated. ABC transporters including ssuB, proX, proW, pstS,C,A,B, AapM,P, ZnuB,C, SitD, LptG,B were up-regulated. Other overexpressed genes that may be involved in virulence of this bacterium were also identified. Some of the genes overexpressed including znuC, hemE and CLIBASIA_02135, a membrane protein involved in iron transport were selected, and expression was evaluated in citrus. Two hypothetical genes that were overexpressed were screened on Nicotiana benthamiana and showed symptoms. Transgenic citrus plants expressing these two genes were constructed. Polyclonal antibodies were generated. Studies confirmed HLB-affected phloem is clogged with amorphous proteins and calloses. HLB-affected leaves had lower chlorophyll content. Foliar application of low concentration of salicylic acid or NAA on HLB-positive trees every four months reduced callose deposition. Newly produced phloem was observed after treatments. Foliar application of GA3 or glucan synthesis inhibitors, or SDS had no detectable effect on phloem clogging or callose deposition in greenhouse trials. Compounds will be tested by trunk injection. Examination with the canker bacterium revealed many differentially expressed genes belonged to the HrpG and HrpX regulons. Totally, 123 genes were overlapped in the two regulons at the three selected time-points representing three growth stages of XCC. HrpG and HrpX regulated 24 type III secretion system genes, 23 type III secretion system effector genes, and 29 type II secretion system substrate genes. We found that 124 and 90 unknown genes were controlled by HrpG and HrpX. We investigated the role of Quorum sensing (QS). Comparison of the transcriptomes of QS mutants (rpfF, rpfC, and rpfG) with the wild type strain revealed that QS temporally regulates expression of a large set of genes, including genes that are involved in chemotaxis and flagellar biosynthesis, metabolism, and genes encoding virulence traits such as type II secretion system substrates, type III secretion system and its effectors. Cross-talk between the QS regulon and the HrpG regulon was identified, as 62 genes were shared by both regulons. The finding that QS is temporally regulated and that there is cross-talk between the QS regulon and the HrpG regulon have significantly advanced our understanding of QS in citrus canker infection, including how XCC attaches to, invades and grows in the host apoplast.

Publications

  • Folimonova, S.Yu., Garnsey, S.M., Dawson, W.O. (2008). Examination of host responses of different citrus varieties to HLB infection. Proceedings of the International Research Conference on Huanglongbing, Orlando, FL, number 13.7
  • Robertson, C. J., Folimonova, S. Yu., Dawson W. O. (2010). Interaction of Candidatus Liberibacter asiaticus with poncirus.The XVIII Conference of the International Organization of Citrus Virologists, Campinas, Sao Paulo, Brazil, November 07-12, 2010.
  • 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, pp. 1346-1354.
  • Rosales R, Burns JK (2011) Phytohormone changes and carbohydrate status in sweet orange fruit from Huanglongbing-infected trees. J Plant Growth Regul 30:312-321.