Development of protocols using quantitative PCR (qPCR) for molecular diagnostic of the quarantine nematode A1 Pratylenchus crenatus

• Grant number: 12/19101-9
• Support Opportunities: Scholarships abroad - Research
• Start date: July 01, 2013
• End date: January 31, 2014
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Claudio Marcelo Gonçalves de Oliveira
• Grantee: Claudio Marcelo Gonçalves de Oliveira
• Host Investigator: Vivian Carol Blok
• Host Institution: Instituto Biológico (IB). Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). São Paulo , SP, Brazil
• Institution abroad: James Hutton Institute, Scotland

Abstract

Molecular detection of pest and pathogens relies on rapid and dependable methods for their identification and quantification. This project describes the development and evaluation of a novel qualitative and quantitative diagnostic method for Pratylenchus crenatus Loof, 1960, consider as A1 quarantine pest to Brasil, based on a fluorogenic 5' nuclease PCR assay (TaqMan). Primer/probe sets will be designed targeting the ITS-1 region of the ribosomal cistron (rDNA). The validation experiments will be carry out using extracted DNA from P. crenatus populations from United Kingdom, Holland and a population detected recently in Brazil. In order to assess the specificity, the primer/probe sets will be tested with samples of other Pratylenchus species. Experiments with dilutions of purified plasmid standards will be used to test the sensitivity of the TaqMan assay on detection of nematode DNA to the femtogram level. Quantification of the target DNA present in field potato samples will be performed. Also, the genetic diversity between P. crenatus populations will be studied based on molecular techniques and phylogenetic analysis. Complementary to the main objective, training on identification of Globodera pallida e G. rostochiensis will be implemented based on both traditional taxonomy and modern molecular techniques. These species are on the top list of main quarantine diseases, causing damages on potato tubers cultivated in Europe and should be treated as serious pests if introduced in Brazil. The use of these tools are expected to provide an accurate identification of P. crenatus and Globodera species, enabling researchers and extension services of quarantine service from Brazil to establish protocols for detecting plant-parasitic nematodes and, consequently, avoiding their introduction. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

OLIVEIRA, CLAUDIO M. G.; BLOK, VIVIAN; NEILSON, ROY; MROZ, TOMASZ; ROBERTS, DAVID. Hydrolysis probe-based PCR for detection of Pratylenchus crenatus, P. neglectus and P. penetrans. NEMATOLOGY, v. 19, n. 1, 1, p. 81-91, 2017. (12/19101-9)

OLIVEIRA, CLAUDIO M. G.; BLOK, VIVIAN; NEILSON, ROY; MROZ, TOMASZ; ROBERTS, DAVID. Hydrolysis probe-based PCR for detection of Pratylenchus crenatus, P. neglectus and P. penetrans. NEMATOLOGY, v. 19, n. 1, p. 11-pg., 2017-01-01. (12/19101-9)