Bodaghi, Sohrab

‘Candidatus Liberibacter asiaticus’ (CLas), the agent associated with the Huanglongbing (HLB) citrus disease, is commonly detected using quantitative polymerase chain reaction (qPCR) with hydrolysis probes. Internal standards are typically included in the qPCR assays to reduce the risk of false negatives caused by inhibitors. When the internal standard is detected but CLas is not, it is generally assumed that the pathogen is absent from the tested sample. However, our study shows that trace amounts of CLas may go undetected if the internal standard is either overly abundant or too dissimilar to CLas. To overcome these limitations, we developed a synthetic internal standard (IS) that uses the same primer as the CLas target sequence, along with three to four downstream nucleotides, but with a unique internal sequence derived from smooth hammerhead shark (Sphyrna zygaena; IS-SHK). This sequence matches the G/C content and melting temperature of the CLas target and was specifically selected to minimize potential interference from other nucleic acid materials in citrus samples. To minimize competition between the IS-SHK standard and the CLas target, an average of only 21 molecules of IS-SHK standard is added to each qPCR reaction. Therefore, when IS-SHK standard is detected at expected levels and CLas is not, the absence of CLas is confirmed rather than inhibition of the detection. Conversely, the absence of both IS-SHK standard and CLas suggests the presence of a qPCR inhibitor, warranting retesting of the sample.

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. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens , a culturable surrogate for the unculturable HLB-associated bacterium “ Candidatus Liberibacter asiaticus,” we tested the hypothesis that members of the citrus microbiome produce potential anti-“ Ca . Liberibacter asiaticus” natural products with potential anti-“ Ca . Liberibacter asiaticus” activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens .

Citrus production throughout the world is being severely threatened by Huanglongbing (HLB), which is a disease associated with the bacteria ‘Candidatus Liberibacter asiaticus’ (CLas), africanus, and americanus. This Resource Announcement provides amplicon-based next generation sequencing (NGS) datasets of the bacterial and fungal rRNA internal transcribed spacer (ITS) region from CLas-infected citrus budwood, leaves, and roots from five orchards located in different geographical regions in Florida (USA). To our knowledge, this is the first amplicon-based NGS study (i) that describes the fungal taxa associated with citrus and (ii) that provides comparative analyses of the bacterial and fungal taxa associated with budwood, leaves, and roots from the same citrus trees. This report also provides the sample metadata linked to these sequence datasets including HLB severity rating, tissue type, citrus rootstock, citrus scion, geographical region, and year trees were planted. When analyzed with other similar datasets, we anticipate that researchers will be able to obtain a greater understanding of the factors that shape the citrus microbiome as well as identify individual microorganisms or consortia of microorganisms that play a role in HLB suppression or exacerbation.