Probe-based quantitative PCR and RPA-Cas12a molecular diagnostics for detection of the tomato pest Phthorimaea absoluta (Lepidoptera: Gelechiidae).

The tomato pest Phthorimaea absoluta Meyrick is highly invasive but has not yet invaded North America. However, several morphologically similar species are already present, making detection of P. absoluta presence and invasion challenging. We designed a quantitative PCR molecular diagnostic to differentiate P. absoluta, P. operculella (Zeller), or Keiferia lycopersicella (Walsingham) (Lepidoptera: Gelechiidae) DNA. Additionally, we developed an RPA-Cas12a molecular diagnostic that allows for the isothermal detection of P. absoluta DNA, eliminating the need for a thermocycler. The results of the RPA-Cas12a diagnostic can be visualized simply using a UV light source and cell phone camera. We expect these diagnostics to improve quarantine and prevention measures against this serious agricultural threat.

Genome Assembly and Population Sequencing Reveal Three Populations and Signatures of Insecticide Resistance of Tuta absoluta in Latin America.

Tuta absoluta is one of the largest threats to tomato agriculture worldwide. Native to South America, it has rapidly spread throughout Europe, Africa, and Asia over the past two decades. To understand how T. absoluta has been so successful and to improve containment strategies, high-quality genomic resources and an understanding of population history are critical. Here, we describe a highly contiguous annotated genome assembly, as well as a genome-wide population analysis of samples collected across Latin America. The new genome assembly has an L50 of 17 with only 132 contigs. Based on hundreds of thousands of single nucleotide polymorphisms, we detect three major population clusters in Latin America with some evidence of admixture along the Andes Mountain range. Based on coalescent simulations, we find these clusters diverged from each other tens of thousands of generations ago prior to domestication of tomatoes. We further identify several genomic loci with patterns consistent with positive selection and that are related to insecticide resistance, immunity, and metabolism. This data will further future research toward genetic control strategies and inform future containment policies.

Metabolite Signature and Differential Expression of Genes in Washington Navel Oranges (Citrus sinensis) Infected by Spiroplasma citri.

Spiroplasma citri is the pathogen that causes citrus stubborn disease (CSD). Infection of citrus with S. citri has been shown to cause leaf mottling, reduce fruit yield, and stunt tree growth. Fruit from trees exhibiting symptoms of CSD are misshapen and discolored. The symptoms of CSD are easily confused with nutrient deficiencies or symptoms of citrus greening disease. In this study, young Washington navel oranges (Citrus sinensis) were graft-inoculated with budwood originating from trees confirmed to be infected with S. citri. Leaf samples were collected monthly for 10 months for metabolomics and differential gene expression analyses. Significant differences in the concentration of metabolites and expressed genes were observed between control and S. citri-infected trees throughout the experiment. Metabolites and genes associated with important defense and stress pathways, including jasmonic acid signaling, cell wall modification, amino acid biosynthesis, and the production of antioxidant and antimicrobial secondary metabolites, were impacted by S. citri throughout the study, and even prior to symptom development. This work fills a current gap in knowledge surrounding the pathogenicity of S. citri and provides an updated mechanistic explanation for the development of CSD symptoms in S. citri-infected plants.

Multiplex detection of "Candidatus Liberibacter asiaticus" and Spiroplasma citri by qPCR and droplet digital PCR.

"Candidatus Liberibacter asiaticus" (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas-RNR (5 copies) and S. citri-SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of CLas and S. citri. ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.

A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing.

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrusaustraliasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.

Tuta absoluta (Lepidoptera: Gelechiidae) Success on Common Solanaceous Species from California Tomato Production Areas.

Tuta absoluta (Meyrick) is a devastating pest of tomato that has invaded many regions of the world. To date, it has not been detected in North America, but the pest reached Costa Rica in 2014 and seriously threatens the southern, southwestern, and western United States including California. Although the primary host of T. absoluta is tomato, several other species of Solanaceae may serve as alternative hosts. In our study, we aimed to assess the potential risk that other solanaceous crops and wild species that are often present in and around California tomato fields could serve as hosts. To accomplish this, we conducted greenhouse and laboratory studies to determine whether two common cultivars of fresh market tomato, two common cultivars of tomatillo, and the wild plants, Solanum nigrum L., Solanum sarrachoides (Sendtner), and Datura stramonium L., are suitable hosts for reproduction and development of the pest. According to our results, D. stramonium and tomatillo were unable to sustain T. absoluta larval development in either greenhouse studies or laboratory studies, and therefore, they are not likely to contribute to T. absoluta establishment during an invasion. On the contrary, the two other solanaceous weeds, S. nigrum and S. sarrachoides, share a similar potential as tomato to be reproductive and developmental hosts of T. absoluta, and might play an important role in the establishment of the pest in California.

Coupling a branch enclosure with differential mobility spectrometry to isolate and measure plant volatiles in contained greenhouse settings.

Volatile organic compounds (VOCs) are off-gassed from all living organisms and represent end products of metabolic pathways within the system. In agricultural systems, these VOCs can provide important information on plant health and can ordinarily be measured non-invasively without harvesting tissue from the plants. Previously we reported a portable gas chromatography/differential mobility spectrometry (GC/DMS) system that could distinguish VOC profiles of pathogen-infected citrus from healthy trees before visual symptoms of disease were present. These measurements were taken directly from canopies in the field, but the sampling and analysis protocol did not readily transfer to a controlled greenhouse study where the ambient background air was saturated with volatiles contained in the facility. In this study, we describe for the first time a branch enclosure uniquely coupled with GC/DMS to isolate and measure plant volatiles. To test our system, we sought to replicate our field experiment within a contained greenhouse and distinguish the VOC profiles of healthy versus citrus infected with Candidatus Liberibacter asiaticus. We indeed confirm the ability to track infection-related trace biogenic VOCs using our sampling system and method and we now show this difference in Lisbon lemons (Citrus×limon L. Burm. f.), a varietal not previously reported. Furthermore, the system differentiates the volatile profiles of Lisbon lemons from Washington navels [Citrus sinensis (L.) Osbeck] and also from Tango mandarins (Citrus reticulata Blanco). Based on this evidence, we believe this enclosure-GC/DMS system is adaptable to other volatile-based investigations of plant diseases in greenhouses or other contained settings, and this system may be helpful for basic science research studies of infection mechanisms.