Hui Wen Lee

Pectobacterium actinidiae is a significant kiwifruit canker pathogen reported from kiwifruit-growing areas in Asia and South America. To improve the diagnostic capability for this bacterium, a probe-based real-time PCR assay was developed for sensitive and fast detection of P. actinidiae. The highly specific assay targets the meta-pathway phenol degradation protein-like locus, unique to P. actinidiae. Validation showed that the assay is highly sensitive, detecting the pathogen down to 0.1 pg of pure genomic DNA or 10 to 50 colony-forming units in plant tissue matrix. To assess the competency of the tested DNA samples and avoid false-negative results, the assay was duplexed with plant internal control. This new method provides a valuable tool for specific and sensitive testing of imported and exported plant material to ensure freedom from P. actinidiae and prevent the spread of this pathogen into new areas. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

A highly pathogenic fungus characterized as Verticillium nonalfalfae multilocus sequence type 2 (MLST2) is an emerging fungal pathogen causing Verticillium wilt on kiwifruit. Although V. nonalfalfae MLST2 has not been reported outside Chile, there is a risk that this pathogen could spread through the global movement of germplasms to other countries. Current diagnostic methods for this fungus rely on a laborious and time-consuming plating assay for morphological identification and DNA sequence analysis. In this study, we describe the development and validation of a novel quantitative polymerase chain reaction (qPCR) assay for rapid and specific detection of V. nonalfalfae MLST2 in plant tissues. The assay targets the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and was shown to detect all tested isolates of V. nonalfalfae MLST2 with a detection limit of approximately 2 pg of pathogen genomic DNA. There was no cross-reaction with V. nonalfalfae MLST1, other Verticillium species, or non-target fungal species found on kiwifruit. This assay was duplexed with a plant internal control for simultaneous detection of the pathogen and cytochrome oxidase gene from the host plant. This new specific and sensitive qPCR assay is a valuable molecular diagnostic tool for rapid screening of imported plant material and would also be useful for testing samples collected from field surveillance activities to monitor the presence of V. nonalfalfae MLST2.

Citrus canker, a serious bacterial disease affecting the citrus industry worldwide, is caused by Xanthomonas citri subsp. citri (Xcc) pathotypes A, A* and Aw, and to a lesser extent by X. fuscans subsp. aurantifolii (Xfa). The recent citrus canker outbreak in Australia has emphasised the need to re-evaluate the efficiency of molecular assays used for detecting citrus canker bacteria. Two published probe-based qPCR assays targeting the pth and lrp genes were tested for Xcc, whereas a SYBR Greenbased qPCR assay was tested for Xfa. The Xcc pth gene and Xfa qPCR assays were shown to be specific towards all pathotypes of Xcc and Xfa, respectively. The detection limit for both assays were 1 pg of genomic DNA or 103 CFU in bacteria-spiked leaf sample. The Xcc lrp gene qPCR assay was able to discriminate Xcc pathotypes with a detection limit of 1 ng of genomic DNA or 106 CFU in bacteriaspiked leaf sample, but this assay showed cross-reaction with Xfa. To allow rapid high-throughput detection of all Xcc pathotypes, a duplex probe-based qPCR assay was developed by incorporating COX primers as an internal control for plant DNA into the pth gene qPCR assay.

Strigolactones (SLs) are multifunctional plant hormones regulating essential physiological processes affecting growth and development. In vascular plants, SLs are recognized by α/β hydrolase-fold proteins from the D14/DAD2 (Dwarf14/Decreased Apical Dominance 2) family in the initial step of the signaling pathway. We have previously discovered that N-phenylanthranilic acid derivatives (e.g. tolfenamic acid) are potent antagonists of SL receptors, prompting us to design quinazolinone and quinazolinedione derivatives (QADs and QADDs, respectively) as second-generation antagonists. Initial in silico docking studies suggested that these compounds would bind to DAD2, the petunia SL receptor, with higher affinity than the first-generation compounds. However, only one of the QADs/QADDs tested in in vitro assays acted as a competitive antagonist of SL receptors, with reduced affinity and potency compared with its N-phenylanthranilic acid ‘parent’. X-ray crystal structure analysis revealed that the binding mode of the active QADD inside DAD2's cavity was not that predicted in silico, highlighting a novel inhibition mechanism for SL receptors. Despite a ∼10-fold difference in potency in vitro, the QADD and tolfenamic acid had comparable activity in planta, suggesting that the QADD compensates for lower potency with increased bioavailability. Altogether, our results establish this QADD as a novel lead compound towards the development of potent and bioavailable antagonists of SL receptors.