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United States Department of Agriculture

Agricultural Research Service

Research Project: EMERGING DISEASES OF CITRUS, VEGETABLES, AND ORNAMENTALS

Location: Subtropical Plant Pathology Research

Title: Visualization of ‘Ca. Liberibacter asiaticus’ cells in the vascular bundle of citrus seed coats with fluorescence in situ hybridization and transmission electron microscopy

Authors
item Hilf, Mark
item Sims, Kenneth
item Folimonova, S -
item Achor, Diann -

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 28, 2012
Publication Date: May 29, 2013
Citation: Hilf, M.E., Sims, K.R., Folimonova, S.Y., Achor, D.S. 2013. Visualization of ‘Ca. Liberibacter asiaticus’ cells in the vascular bundle of citrus seed coats with fluorescence in situ hybridization and transmission electron microscopy. Journal of Phytopathology. 103:6:545-554.

Interpretive Summary: This paper describes continuing research on the question of whether ‘Ca. Liberibacter asiaticus’, which is the bacterium associated with the citrus disease huanglongbing, is transmitted through seed. Previous tests using the polymerase chain reaction (PCR) detected bacterial DNA in seed coats and in a few seedlings, but detecting the DNA does not mean that intact cells are present in the tissue. We used transmission electron microscopy and fluorescence in situ microscopy to see bacterial cells in citrus seeds from infected trees, demonstrating that intact bacterial cells are present in seeds and providing one of the first microscopy studies of ‘Ca. Liberibacter asiaticus’ based on light microscopy and not electron microscopy.

Technical Abstract: ‘Ca. Liberibacter asiaticus’ is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated, but previous studies using real-time PCR (qPCR) assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seeds. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for ‘Ca. Liberibacter asiaticus’ were seen in phloem sieve elements in the vascular bundle of ‘Conners’ grapefruit seed coats using TEM. FISH analyses utilizing probes complementary to the ‘Ca. Liberibacter asiaticus’ 16S rRNA revealed bacterial cells in the vascular tissue of intact seed coats of ‘Conners’ and ‘Inman’ grapefruit and in fragmented vascular bundles excised from seed coats of ‘Liane’ pummelo. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to ‘Ca. Liberibacter asiaticus’. No cells were observed in preparations of seeds from fruits from noninfected trees. A small library of clones of prokaryote 16S rRNA gene sequences amplified with degenerate primers from seed coats from a non-infected tree contained no ‘Ca. Liberibacter asiaticus’ sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as ‘Ca. Liberibacter asiaticus’, providing molecular genetic corroboration that the bacterial cells revealed by TEM and FISH in seed coats from infected trees were ‘Ca. Liberibacter asiaticus’.

Last Modified: 7/24/2014
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