Next generation sequencing, and development of a pipeline as a tool for the detection and discovery of citrus pathogens to facilitate safer germplasm exchange

Authors: Keremane, Manjunath; SINGH, K - University Of California, Riverside; RAMADUGU, C - University Of California, Riverside; Krueger, Robert; Skaggs, Todd

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/26/2024
Publication Date: 1/30/2024
Citation: Keremane, M.L., Singh, K., Ramadugu, C., Krueger, R., Skaggs, T.H. 2024. Next generation sequencing, and development of a pipeline as a tool for the detection and discovery of citrus pathogens to facilitate safer germplasm exchange. Plants. 13(3). Article 411. https://doi.org/10.3390/plants13030411.
DOI: https://doi.org/10.3390/plants13030411

Interpretive Summary: Graft-transmissible pathogens of citrus have traditionally detected via biological indexing and, more recently, PCR-based assays. Next generations sequencing (NGS) for pathogen detection has been used in other crops but is not well established in citrus. This paper describes the development of an NGS-based pipeline for the detection of graft-transmissible citrus pathogens. NGS was able to detect the same pathogens in citrus as previously utilized assays. In addition, bacterial pathogens and pathogens of insect pests were detected. NGS also detected Beet western yellows virus (BWYV) in citrus, the first report of this. The pathogenicity of BWYV in citrus has not been established, however.

Technical Abstract: Citrus is affected by many diseases, and hence movement of citrus propagative materials is highly regulated in the USA. Currently used regulatory pathogen detection methods include biological and laboratory-based tech-nologies which are time-consuming, expensive, and have many limitations. There is an urgent need to develop alternate, rapid, economical, and reliable testing methods for safe germplasm exchange between industries. Citrus huanglongbing has devastated citrus industries leading to an increased need for germplasm exchanges between citrus growing regions for evaluating many potentially valuable hybrids for both HLB resistance and multilocational performance. In the present study, Next Generation sequencing (NGS) methods were used to sequence the transcriptomes of 21 test samples, including 15 well-characterized pathogen-positive plants. A workflow was designed in the ‘CLC Genomics workbench’ software for bioinformatics analysis of the se-quence data for the detection of pathogens. NGS was rapid and found to be a valuable technique for the detec-tion of viral and bacterial pathogens, and for the discovery of new citrus viruses, and complementary to the existing array of biological and laboratory assays. Using NGS methods, we report Beet western yellows virus, a new report from citrus, and a variant of the Citrus yellow vein-associated virus associated with “fatal yellows” disease.