Biology subjects

Larrea-Sarmiento, A.

Publications and source records attributed to Larrea-Sarmiento, A..

3 recordsLinked to original sources

bioRxiv2025

The "Grove-First" Framework: Starting in the Grove to Find Therapies for Huanglongbing

Citrus greening disease, also known as huanglongbing (HLB), is the most serious vector-borne bacterial disease of citrus world-wide1,2. There is an immediate global need to provide the citrus industry with relief from HLB and a return to profitable citrus production. Standard screening methods for HLB therapeutic treatments typically involve various laboratory-based assays to select treatments with antimicrobial properties3, which then advance to greenhouse and eventually field testing in a workflow that takes multiple years. Unlike traditional lab-first screening, we present a design of experiments framework4,5, referred to as Grove-First, that rapidly screens treatments with regulatory-friendly profiles in commercial citrus groves using trunk injection to select treatments that improve tree health and fruit yield over the course of a single growing season. Using this framework, we identified candidate treatments with effects comparable to or better than the standard oxytetracycline (OTC) on visual tree-health and/or yield indices in an initial screen of HLB-positive 8-year-old Valencia sweet orange trees. Expanded trials in commercial citrus groves allowed us to validate the initial screening results at other locations and in other citrus varieties. Grove-First rapidly accelerated the identification and large-scale field testing of HLB therapies, some of which are available for growers to use immediately and others that require further field testing and/or regulatory actions.

plant biology↗

bioRxiv2020

Multiplex recombinase polymerase amplification (RPA) assay developed using unique genomic regions and coupled with a lateral flow device for rapid on-site detection of genus Clavibacter and C. nebraskensis

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Gosss wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg and 100 fg was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1,000 fg when 1 {micro}L of host sap was added into the RPA reaction containing 10-fold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA for any plant pathogen. IMPORTANCEClavibacter species are prevalent worldwide as have the potential to result in systemic infection. In the past, detection attempts have relied on both molecular- and immunological-based assays; however, current detection methods are time consuming and laborious. Field-deployable tests are desirable to identify potential samples infected with Clavibacter species. This study demonstrates that the field-deployable isothermal multi-target recombinase polymerase amplification can be performed for the simultaneous detection of the genus Clavibacter in general (all species), and C. nebraskensis, in particular, without specialized equipment. Additionally, the multiplex RPA coupled with a LFD may confer the benefits of faster detection and discrimination of Clavibacter species that affect critical regions susceptible to infection. This user-friendly format offers a flexible assay to complement both nucleic acid amplification and novel diagnosis methods without the need for DNA purification; this assay may serve as a point-of-reference for developing multiplex RPA assay for other plant pathogens.

microbiology↗

bioRxiv2019

Synergetic effect of non-complementary 5’ AT-rich sequences on the development of a multiplex TaqMan real-time PCR for specific and robust detection of Clavibacter michiganensis and C. michiganensis subsp. nebraskensis

Clavibacter is an agriculturally important genus comprising a single species, Clavibacter michiganensis, and multiple subspecies, including, C. michiganensis subsp. nebraskensis which causes Gosss wilt/blight of corn and accounts for high yield losses - listed among the five most significant diseases of corn in the United States of America. Our research objective was to develop a robust and rapid multiplex TaqMan real-time PCR (qPCR) to detect C. michiganensis in general and C. michiganensis subsp. nebraskensis with enhanced reliability and accuracy by adding non-complementary AT sequences to the 5 end of the forward and reverse primers. Comparative genomic analyses were performed to identify unique and conserved gene regions for primer and probe design. The unique genomic regions, ABC transporter ATP-binding protein CDS/ABC-transporter permease and MFS transporter were determined for specific detection of C. michiganensis and C. m. subsp. nebraskensis, respectively. The AT-rich sequences at the 5 position of the primers enhanced the reaction efficiency and sensitivity of rapid qPCR cycling; the reliability, accuracy and high efficiency of the developed assay was confirmed after testing with 59 strains from inclusivity and exclusivity panels - no false positives or false negatives were detected. The assays were also validated through naturally and artificially infected corn plant samples; all samples were detected for C. michiganensis and C. m. subsp. nebraskensis with 100% accuracy. The assay with 5 AT-rich sequences detected up to 10- and 100-fg of C. michiganensis and C. michiganensis subsp. nebraskensis genome targets, respectively. No adverse effect was observed when sensitivity assays were spiked with host genomic DNA. Addition of 5 AT rich sequences enhanced the qPCR reaction efficiency from 0.82 (M = -3.83) and 0.91 (M = -3.54) to 1.04 (with optimum slope value; M = -3.23) for both C. michiganensis and C. michiganensis subsp. nebraskensis, respectively; a increase of 10-fold sensitivity was also obtained with C. michiganensis primer set. The methodology proposed here can be used to optimize the reaction efficiency and to harmonize the diagnostic protocols which have prodigious applications in routine diagnostics, biosecurity and microbial forensics.

plant biology↗