Source: AGRICULTURAL RESEARCH SERVICE submitted to
DEVELOPMENT AND IMPLEMENTATION OF NEW CONTROL STRATEGIES FOR CITRUS HUANGLONGBING (GREENING)
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
0418630
Grant No.
(N/A)
Project No.
6618-22000-034-33R
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Dec 11, 2009
Project End Date
Mar 31, 2012
Grant Year
(N/A)
Project Director
DUAN Y
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
219 SOUTH ROCK ROAD
FT PIERCE,FL 34945
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2120910104030%
2120920106070%
Goals / Objectives
1) Develop standardized protocols for screening and cleaning up the entire germplasm of the Florida citrus industry using our highly sensitive detection technology, and effective chemical compounds; 2) implement chemical control of citrus HLB by developing cost-effective application technology using the two effective chemical compounds; and 3) verify if Murraya paniculata is, or is not, a preferred reservoir of HLB pathogens, whether it can be used as trap plants for the control of citrus HLB.
Project Methods
1) Using the newly-generated genome information of HLB pathogen, 'Candidatus Liberibacter asiaticus', we will develop highly sensitive quantitive PCR and/or nested PCR to detect Las bacteria in the germplasms of citrus, and use the two effective chemicals to treat the budwoods and seeds to obtain real Las-free gerplasms; 2) using the chemicals, we will apply and modify the injection system for effective delivering the chemicals into citrus trees; and 3) we will determine the type(s) of Las bacteria that can survive in Murraya plants and the dynamics of the bacteria in the plants, as well as their possibility to be transmitted to citrus by the asian citrus psyllids.

Progress 10/01/10 to 09/30/11

Outputs
Progress Report Objectives (from AD-416) 1) Develop standardized protocols for screening and cleaning up the entire germplasm of the Florida citrus industry using our highly sensitive detection technology, and effective chemical compounds; 2) implement chemical control of citrus HLB by developing cost-effective application technology using the two effective chemical compounds; and 3) verify if Murraya paniculata is, or is not, a preferred reservoir of HLB pathogens, whether it can be used as trap plants for the control of citrus HLB. Approach (from AD-416) 1) Using the newly-generated genome information of HLB pathogen, 'Candidatus Liberibacter asiaticus', we will develop highly sensitive quantitive PCR and/or nested PCR to detect Las bacteria in the germplasms of citrus, and use the two effective chemicals to treat the budwoods and seeds to obtain real Las-free gerplasms; 2) using the chemicals, we will apply and modify the injection system for effective delivering the chemicals into citrus trees; and 3) we will determine the type(s) of Las bacteria that can survive in Murraya plants and the dynamics of the bacteria in the plants, as well as their possibility to be transmitted to citrus by the asian citrus psyllids. This project is related to objective 1: Characterize ecology, biology, epidemiology, genetics and host interactions of domestic, exotic, newly emergent and re-emerging pathogens. This is the second year of the two year research project. In addition to optimizing the APHIS recommended standardized protocol for detection of Huanglongbing (HLB) bacteria (Candidatus Liberibacter asiaticus), we have developed and standardized additional supersensitive detection method that targets the nearly identical tandem repeats of two prophage genes of Las bacteria. This method not only simplified the detection procedure, but also detected the HLB bacterium at very low titer (level), especially the seed-transmitted Las bacteria in seedlings. Since this detection method is 100-2000 fold more sensitive than the current standard quantitative polymerase chain reaction (qPCR) method, it eliminates the need for DNA isolation from plants and insects with a simple modified boil method for total DNA harvests. This new technology has been submitted for peer review publication, patent application and transferred to universities and regulatory institutions for extended applications in the detection and diagnostics of citrus HLB. We have evaluated some chemicals for the control of HLB bacteria in Las- infected periwinkle and citrus plants both in greenhouse and in the fields. A combination of two antibiotics (penicillin G and streptomycin) was effective for the control of the HLB bacteria using injections or soaking. Detection of Las bacterium from striped mealybug insects on infected periwinkle plants led us to investigate the hypothesis that the phloem- feeding mealybugs could (1) be infected with HLB and (2) transmit the disease to other plants in our greenhouse. Results confirmed that the mealybugs could carry Las bacterium and may be a potential vector of this bacterium. New application of thermotherapy was developed for the control of citrus HLB. Under constant heat treatment, most plants show undetectable levels of HLB bacterium at 270 days, no disease symptoms, and greatly improved plant growth and vigor. Preliminary testing at Pico farm using a prototype portable greenhouse also showed promising results. Our results suggest the extreme stress placed on the plants renders the bacteria harmless or unviable. We hypnotized that the pathogenic bacteria are altered to a non-pathogenic form and that a HLB prophage may be directly or indirectly responsible for this change.

Impacts
(N/A)

Publications


    Progress 10/01/09 to 09/30/10

    Outputs
    Progress Report Objectives (from AD-416) 1) Develop standardized protocols for screening and cleaning up the entire germplasm of the Florida citrus industry using our highly sensitive detection technology, and effective chemical compounds; 2) implement chemical control of citrus HLB by developing cost-effective application technology using the two effective chemical compounds; and 3) verify if Murraya paniculata is, or is not, a preferred reservoir of HLB pathogens, whether it can be used as trap plants for the control of citrus HLB. Approach (from AD-416) 1) Using the newly-generated genome information of HLB pathogen, 'Candidatus Liberibacter asiaticus', we will develop highly sensitive quantitive PCR and/or nested PCR to detect Las bacteria in the germplasms of citrus, and use the two effective chemicals to treat the budwoods and seeds to obtain real Las-free gerplasms; 2) using the chemicals, we will apply and modify the injection system for effective delivering the chemicals into citrus trees; and 3) we will determine the type(s) of Las bacteria that can survive in Murraya plants and the dynamics of the bacteria in the plants, as well as their possibility to be transmitted to citrus by the asian citrus psyllids. This research relates to inhouse objective 3: Develop or improve comprehensive integrated disease management strategies. We have developed a screening system for effective antibiotic/chemical treatment therapies using the Huanglongbing (HLB)-infected periwinkle cuttings. Some of these effective chemicals are being evaluated in citrus in greenhouse using HLB-infected citrus plants or seedlings. The effective compounds identified from periwinkle system are applied to HLB- infected citrus using various application methods. Preliminarily data indicated a couple of compounds are effective in suppressing the HLB bacteria though soaking treatment of citrus cuttings. We are trying different delivery system for better application of these chemical compounds to citrus for control of HLB disease. We have been comparing detection methods for HLB in Murraya plants, and we have selected DNA extraction methods and PCR primers that can be used for detection of HLB in Murraya. An ornamental nursery has provided for us access to a large number of potted Murraya plants for screening. A new psyllid colony was started on March 31 on plants that tested HLB positive. Once established, that colony can be used for experiments on psyllid acquisition and transmission of HLB between citrus and Murraya. Ten plantings of Murraya across St Lucie County and one planting in Palm Beach County were identified as urban research locations. Regular monitoring of Murraya and psyllids for HLB at six of these plantings was commenced.

    Impacts
    (N/A)

    Publications