An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘ Candidatus Liberibacter species’ (CLsp), ‘ Ca. Liberibacter asiaticus’ (CLas), ‘africanus’ (CLaf), and ‘americanus’ (CLam), associated with the huanglongbing disease of citrus. The multiplex assay was designed based on the qPCR assay published in 2006 by Li et al., considering all available CLsp 16S rRNA gene sequences in GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared with singleplex qPCR, no significant increase in quantitative cycle (Cq) values was detected. The specificity and sensitivity of the updated qPCR assay was optimal, and measuring the intra- and interassay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. [ ] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

This collaborative work by over 180 researchers from 40+ countries addresses the challenges posed by “phantom agents”—putative pathogenic agents named in literature without supporting data on their existence. Those agents remain on regulatory lists, creating barriers in trade and plant certification. Historically identified based solely on symptoms, these agents lack isolates or sequence data, making reliable detection or risk assessment impossible. After reviewing over 120 such agents across 10 key plant genera, we recommend their removal from regulatory lists and call for revised standards aligned with modern diagnostics. This effort seeks to streamline germplasm exchange, benefiting global agriculture by removing the constraints imposed by phantoms.

Citrus viroid V (CVd-V) and citrus viroid VI (CVd-VI) are members of the Apscaviroid genus found in citrus growing areas worldwide, but previously unreported from Australia. Other viroids, includin...

The effects of stubble management and crop rotation on suppression of soilborne pathogens was studied in wheat cropping soils taken from established trials at Moree, Warialda and Croppa Creek in northern New South Wales, Australia. Biological resilience was evaluated in two ways: by measuring severity of take-all when the pathogen (Gaeumannomyces graminis var. tritici) was introduced to soil, and by assessing displacement of Fusarium pseudograminearum, the cause of crown rot of wheat, from buried straw pieces. No effects of stubble management or rotation with sorghum were found on suppression of either the introduced or resident pathogens at the Moree site. At Warialda and Croppa Creek, the severity of take-all in pot bioassays was significantly lower in soils from zero-tillage plots with stubble retained than in cultivated plots where the stubble was burnt. This effect was seen in soils from both continuous wheat and wheat-chickpe a rotation treatments. There were no consistent differences between treatments in soil microbial biomass or respiration, or in soil carbon contents. Stubble retention also significantly increased the rate of displacement of F. pseudograminearum from infested straw at Croppa Creek, but not at Warialda. The results showed that, at two out of three sites, retention of crop residues increased the ability of the soil to resist the effects of introduction of a major plant disease.