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United States Department of Agriculture

Agricultural Research Service

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Research Project: DISSECTING THE DISEASE COMPLEX OF CITRUS HUANGLONGBING IN FLORIDA

Location: Subtropical Plant Pathology Research

2012 Annual Report


1a.Objectives (from AD-416):
To dissect the disease complex of citrus Huanglongbing (HLB) in Florida from the following five angles:.
1)to complete Las genome sequence and to conduct comparative genomics of the Liberibacter species;.
2)to validate Koch’s postulates by culturing the Las bacterium in vitro and development of artificial inoculation methods;.
3)to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development;.
4)to confirm if Las bacteria are seed-transmissible and their role in HLB development; and.
5)to monitor Las population dynamics in citrus and periwinkle plants after chemical and heat treatment.


1b.Approach (from AD-416):
Through molecular biology techniques, including state-of-the-art genomic sequencing, microarrays, and library construction, we will finish sequencing the Las genome, perform comparative genomics, seed transmission studies and microbial ecology analyses. Using a combination of long established and newly developed microbiology techniques, we will continue to pursue the culturing of Las. Greenhouse and lab experiments will be conducted to monitor Las population dynamics and when appropriate for the progress of other objectives.


3.Progress Report:

This research related to inhouse project objective: 1a. Characterize the etiology, molecular biology and genetics of ‘Candidatus Liberibacter asiaticus (Las),’ the bacterium associated with citrus huanglongbing (HLB); and 2a. Develop improved detection methods for GRSV, Las and Xanthomonads on citrus and strawberry.

A complete circular genome of Candidatus Liberibacter asiaticus (Las) was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration at USDA-ARS in Parlier, California, we have obtained and published a complete genome sequence of Ca. L. solanacearum and published in PLoS ONE 6(4):e1913, 2011. All bacterial artificial chromosome (BAC) clones of Las were sequenced, and sequence analyses revealed a potential mechanism of genome reduction. Based on the variations within the Las prophages, FP1 (CP001677.5) and FP2 (JF773396.1), twelve (A to H) different populations (genotypes) have been identified. Type A and B are located in FP1 and FP2, respectively. Typing revealed A, B and C as the three most abundant groups in libraries from psyllid, citrus and periwinkle, although psyllid contained much more type A sequence than the plant hosts. Typing also revealed periwinkle and dodder contained all population types, but psyllids did not. They had low to no titers of four types, including type D. In citrus a high titer of type D was associated with typical blotchy mottle symptoms. We are continuing to studies the different population types and their correlations with huanglongbing (HLB) phenotypes and disease severity.

We have characterized the adenosine triphosphate (ATP) translocase from Las and proved its function using a heterologous E. coli system (J. Bacteriol. 192:834-840, 2010). We are currently developing an antibody-based "drug" to target this protein, aimed at disrupting ATP import, which may be important for its survival. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange and trifoliate orange. A very low titer of Las was detected from the embryos and seedlings using nested polymerase chain reaction (PCR) and real-time PCR. Most, if not all the seedlings did not show typical huanglongbing (HLB) symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested "Detection of Las was not necessarily equal to the presence of "HLB disease" in plants." Psyllid transmission study on the Las-positive seedlings was performed. High percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is the first time that a one seed-transmitted HLB seedling was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed this seed-transmitted HLB.

Progress on culture of Las bacterium in vitro has been made. The Las bacterial growth reached Stationary Phase and Death Phase in 48-72 hours in the liquid cultures. We are looking into factors affecting further growth. Fifty-one BAC clones with overlapped Wolbachia endosymbiont of Diaphorina citri (wDia) genome sequences were screened using wDia specific primers from BamHI BAC library and were sequenced. The average size for the 51 clones was 85.4kb with 95-100% coverage and the average gas chromatography (GC) content is 34.3%. Assembly results indicated that due to large amount of repeat elements, such as transposase, only 13 BAC clones were able assembled into 1 scaffold. We are conducting the gap closing for each BAC clone and hope to get the full wDia genome soon.


Last Modified: 10/24/2014
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