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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #367770
Research Project: Exotic Pathogens of Citrus: Curation, Diagnostics, and Interactions

Location: Molecular Plant Pathology Laboratory

Title: Enhanced serologically based detection of all Liberibacters associated with citrus Huanglongbing

Author
item DING, FANG - Hubei University
item PENG, SHU-ANG - Huazhong Agricultural University
item Hartung, John

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2020
Publication Date: 5/1/2020
Citation: Ding, F., Peng, S., Hartung, J.S. 2020. Enhanced serologically based detection of all Liberibacters associated with citrus Huanglongbing. Plant Disease. https://doi.org/10.1094/PDIS-12-19-2679-SC.
DOI: https://doi.org/10.1094/PDIS-12-19-2679-SC

Interpretive Summary: Huanglongbing (HLB) is the most destructive disease of citrus worldwide and in Florida. It is caused by one of two species of bacteria that cannot be grown in culture, called Liberibacters. The largest group of Liberibacters is originally from Asia and the other from Africa. Assays to detect the pathogen exist, but are laborious, expensive and are most useful to confirm a preliminary diagnosis of the disease based on symptoms. We have developed a novel set of assays based on antibodies that recognize the pathogen when it is pressed on a paper-like surface. The assay produces colored spots, is sensitive and is easily scaled to large numbers of samples. We have improved the assay by reducing non-specific background color and we have shown that it detects all strains of the Asiatic Liberibacter from everywhere in the world where it is found. We have also shown that the assay can detect all strains of the African Liberibacter as well. We have shown that the broad specificity of the assay is due to the presence of the same target for the antibody in all Liberibacters. The optimized protocol is efficient for recognition of both Liberibacters from citrus in different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the current urgent need for accurate, sensitive and high throughput screening of HLB, and may play an important role especially for plant inspection and quarantine programs.

Technical Abstract: ‘Candidatus Liberibacter spp.’ are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, wenestablished an in situ tissue print method for the detection of ‘Ca. L. asiaticus’ (CLas) in sweet orange.We optimized the protocol by preincubation of the anti-Omp antibody with 5% (w/v) extract of healthy rough lemon. This simple process eliminated cross reactions between citrus and the antibody. The optimized protocol enhanced the application of the polyclonal antibody, and we demonstrate detection of CLas from all parts of the world, including isolates from Japan, Thailand, Vietnam, Pakistan, Saudi Arabia, Brazil, the United States, and a selection of strains from China representative of the diversity extant there. The assay also was used to detect four isolates of ‘Ca. L. africanus’ (CLaf) representative of the diversity present in South Africa. The corresponding outer membrane genes of representative isolates were cloned and sequenced. The coding sequences were highly conserved, and isolates of CLas and CLaf shared 53.8 to 55.9% identity between species at the amino acid level. The optimized protocol is efficient for recognition of both CLas and CLaf in phloem cells of different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the urgent need for accurate, sensitive, and high-throughput screening of HLB bacteria, and may play an important role especially for plant inspection and quarantine programs.