Dataset Information

Real-Time PCR to Phenotype Resistance to the Citrus Nematode Tylenchulus semipenetrans Cobb.

ABSTRACT: As pest management options, such as nematicides, become more restrictive, developing rootstocks resistant to the citrus nematode is fundamental for citrus production. This study provides an updated methodology to screen for citrus nematode resistance in rootstock-breeding programs. We developed a novel method to extract female citrus nematodes from roots that is suitable for molecular work and a real-time-PCR-based nematode quantification method for Tylenchulus semipenetrans. These procedures allow scaling up screening to high-throughput workflows, increasing the chances of finding rootstock candidates that combine all the desired traits. Our results contribute to the growing literature supporting quantification of nematodes with molecular methods.

SUBMITTER: Ruiz M

PROVIDER: S-EPMC10346647 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Publications

Real-Time PCR to Phenotype Resistance to the Citrus Nematode <i>Tylenchulus semipenetrans</i> Cobb.

Ruiz Marta M Vo Annie Du AD Becker J Ole JO Roose Mikeal L ML

Plants (Basel, Switzerland) 20230704 13

As pest management options, such as nematicides, become more restrictive, developing rootstocks resistant to the citrus nematode is fundamental for citrus production. This study provides an updated methodology to screen for citrus nematode resistance in rootstock-breeding programs. We developed a novel method to extract female citrus nematodes from roots that is suitable for molecular work and a real-time-PCR-based nematode quantification method for <i>Tylenchulus semipenetrans</i>. These proced ...[more]

PMID: 37447106

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Project description:Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.

Dataset Information

Real-Time PCR to Phenotype Resistance to the Citrus Nematode Tylenchulus semipenetrans Cobb.

Publications

Real-Time PCR to Phenotype Resistance to the Citrus Nematode <i>Tylenchulus semipenetrans</i> Cobb.

Similar Datasets

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