Yasuhara-Bell, Jarred

Huanglongbing (aka: citrus greening) associated with ‘Candidatus Liberibacter asiaticus’ (CLas) is one of the most impactful citrus diseases throughout the world. The aim of this project was to support the sensitive and rapid detection of CLas to prevent the continued spread and destruction of US citrus crops by this psyllid vector-borne disease. Two multicopy targets specific to CLas were selected for assay development: the five-copy nrdB (ribonucleotide reductase-RNR) gene and a four-copy locus 4cp (4CP). Published RNR RPA primers were used to develop a one-pot RPA-CRISPR assay, and RPA primers for 4CP were designed. crRNAs for both RNR- and 4CP-RPA-CRISPR/Cas assays were designed, evaluated through preliminary testing, and the best candidate primers and crRNA combinations were selected. The 4CP assay was selected and subjected to a battery of validation tests and determined fit-for-purpose. A field extraction protocol was also developed using commercial reagents, syringes and filters to bring kit sensitivity to the field. When combined with the assay, the complete workflow was able to detect CLas in infected citrus, down to a single infected leaf per subsample; the field workflow showed similar sensitivity to the laboratory-based workflow. While the extraction and assay proved useful, particularly for symptomatic leaves, its performance with asymptomatic leaves for early detection was not evaluated. The complexity of the assay in its current form hinders widespread practical applicability. In the future, revalidation of lyophilized reagents in a convenient kit can be performed for practical deployment; preliminary testing has shown success.

‘ Candidatus Phytoplasma prunorum’ is associated with European stone fruit yellows disease, affecting wild and cultivated species of Prunus at different degrees of susceptibility, and so far is being mainly restricted to Europe. Here, we report draft genome sequences for ‘ Ca. Phytoplasma prunorum’ strains ESFY1 and LNS1, which represent the first available for this species. Strain ESFY1 is the causal agent of the European stone fruit yellows disease of P. persica in Germany, and LNS1 is the causal agent of leptonecrosis disease of P. salicina in Italy. Their draft genomes consist of 480,123 and 490,930 bases assembled into 28 and 16 contigs, with an average GC content of 21.1 and 20.7%, respectively. Analysis of average nucleotide identity (ANI) values between genomes further supported the delineation of ‘ Ca. P. prunorum’ as a species distinct from ‘ Ca. P. mali’, in agreement with previous phylogenetic data. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

‘Candidatus Phytoplasma prunorum’ has been associated with severe disease in Prunus spp., which are commodities of economic importance in the USA. The introduction and establishment of ‘Ca. P. prunorum’ in the USA could result in huge economic losses, thus creating a need for validated diagnostic tools, which are the cornerstone of successful surveillance, quarantine, and eradication measures. Whole-genome comparisons led to the identification of a diagnostic marker gene specific to ‘Ca. P. prunorum’ (PE639). The PE639 assay was duplexed with an 18S rDNA plant internal control and compared to modified 23S (phytoplasmas) and imp (‘Ca. P. mali’) assays. The PE639 assay produced congruent results to 23S and imp assays for all metrics, demonstrating high linearity, repeatability, intermediate precision, and reproducibility. The limit of detection was comparable for all assays tested, and all demonstrated 100% analytical specificity, selectivity, and diagnostic specificity for their respective target species. Assays metrics were consistent across two platforms, the ABI QuantStudio™ 5 and Bio-Rad CFX96™ OPUS. A synthetic gBlocks™ control was designed and validated to work with all assays, as well as conventional PCR assays targeting 16S rDNA and tuf genes. These validated assays and synthetic control represent beneficial tools that support efforts to protect USA agriculture and facilitate safe trade.