Direct tissue print blotting followed by RT-qPCR for the detection of viroids in citrus germplasm

Authors: RAWSTERN, AMANDA - University Of California; Rodriguez, Esteban; URIBE, GERALDO - University Of California; DANG, T - University Of California, Riverside; BODAGHI, S - University Of California, Riverside; VILLA, N - University Of California; NEWMAN, VICKI - Retired ARS Employee; Moreland, Brittany; Keremane, Manjunath; Hartung, John; VIDALAKIS, GEORGIOS - University Of California; POLEK, MARYLOU - Retired ARS Employee; Krueger, Robert

Submitted to: Journal of Citrus Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: Viroid pathogens are small RNA that are not encapsulated with protein. Testing for viroid plant pathogens is generally done by extracting RNA from ground plant tissue, followed by qPCR assays. This report documents that the use of tissue-print blotting for RNA, followed by qPCR assays is effective for detecting citrus viroids. Tissue-print blotting for RNA followed by qPCR is documented to successfully detect citrus viroids in the same samples as detected by traditional extract followed by qPCR. Use of tissue-print blotting for RNA extraction is considerably less expensive and time consuming as compared to traditional tissue extraction and grinding. This technique may be useful for initial screening and routine re-testing of citrus germplasm and certification materials.

Technical Abstract: Citrus viroids are widespread and can impact tree health by causing bark scaling, stunting, gumming, and browning of phloem tissues, resulting in tree decline and yield reduction. Citrus viroids can be readily transmitted via mechanical injuries, grafting, pruning/cutting tools, and from tree propagation using infected, symptomless budwood. Due to their pathogenic potential and ease of transmission, laboratory testing of therapied budwood source plants is required for producing and preserving viroid-free propagative materials in citrus germplasm programs. Tissue-print blotting for nucleic acid isolation followed by reverse transcription (RT)-qPCR has been shown to be cost efficient and enable higher throughput testing capacity than traditional tissue grinding and nucleic acid purification methods for the detection of citrus bacteria and viruses (e.g., ‘Candidatus Liberibacter sp.’ and citrus tristeza virus). However, this method has not been tested for citrus viroid detection. Elution of RNA from nitrocellulose membranes imprinted with viroid infected stems of various citrus hosts was compared to RNA extraction and purification from ground tissues for testing 65 trees infected with different viroid species in single and mixed infections. Our study demonstrated that tissue-print blotting RNA isolation followed by different RT-qPCR assays (i.e., universal detection or species specific singleplex) performed well within the guidelines for validation of qualitative PCR methods and successfully detected viroids in the same samples as the traditional assays using RNA extracted from ground tissues. Therefore, tissue-print blotting can be readily used for routine, economic, high throughput viroid testing of germplasm source trees.