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Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ' Candidatus Liberibacter asiaticus' ( C Las), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of C Las is real-time quantitative polymerase chain reaction (qPCR) using either the C Las 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of C Las-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect C Las in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the C Las RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to C Las, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting C Las in fresh citrus crude extracts from leaf midribs and roots from five California strains of C Las tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing C Las-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.

Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No significant trends of CTV infection on summed amounts of all sugar, phenolic, or terpenoid compounds were observed. However, individual compound levels were affected by CTV infections. Subsequent canonical discriminant analysis (CDA) that utilized profiles of individual amino acids, terpenoids, or phenolics successfully distinguished leaf samples to specific citrus varieties and identified infection status with good accuracy. Collectively, this study reveals biochemical patterns associated with severity of CTV infections that can potentially be utilized to help identify in-field CTV infections of economic relevance., Competing Interests: The authors have declared that no competing interests exist.

Spiroplasma citri is a bacterium that causes stubborn disease of citrus and infects other crops, ornamentals, and weeds. It is transmitted by leafhoppers in a circulative manner. Due to limited sequence data on S. citri, the bacterium was isolated from naturally infected Chinese cabbage grown on a farm in Fresno County, CA. DNA from S. citri CC-2 was extracted from a pure culture in LD8 and subjected to PacBio sequencing. Four contigs were obtained with a single circular chromosome of 1,709,192 bp and three plasmids of 40,210, 39,313, and 2,921 bp in size. The genome developed herein extends the sequence database of S. citri and is the first wholegenome sequence record of S. citri from California. [ABSTRACT FROM AUTHOR]