Journal of Citrus Pathology

The Australian citrus budwood and seed scheme is one of the oldest in the world and has been instrumental in maintaining the health, productivity, and uniformity of Australian citrus plantings. The scheme is run as a non-profit company under the trading name of Auscitrus, with most facilities at Dareton in far southwest New South Wales (NSW). In this paper, we provide a brief history of the citrus industry in Australia, including key events that led to the creation of a seed and budwood scheme in NSW in 1928. The emergence of plant diseases such as Phytophthora gummosis, citrus tristeza and exocortis compelled the industry to adopt new propagation and cultural practices. Industry and government continue to support and strengthen Auscitrus and Australian citrus biosecurity in the areas of diagnostics and surveillance, germplasm management, public education, policy and strategy. Auscitrus is a key component for the Australian citrus industry to manage endemic and respond to exotic graft-transmissible diseases such as huánglóngbìng.

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.