Specific and Sensitive Detection of Phytophthora nicotianae By Simple and Nested-PCR

By D. Grote, A. Olmos, A. Kofoet, J. J. Tuset, E. Bertolini, M. Cambra. European journal of plant pathology, 2002

132 citations recorded by the source corpus.

Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method.

Subfields: Cell Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/d16a3d375bc56d7372577de2cc3a8bbe342ea087

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In Cell Biology

1. L. Schena
2. A. Ippolito
3. Franco Nigro
4. B. Schulz

In Plant Science

1. L. Schena
2. A. Ippolito
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4. Vincenza Soleti ligorio
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6. B. Schulz
7. L. Korsten

Citations in the corpus

• Real-time detection of Phytophthora nicotianae and P. citrophthorain citrus roots and soil (2004, background): “…have been proposed to identify and detect P. nicotianae and P. citrophthora (Érsek et al., 1994; Lacourt and Duncan, 1997; Ristaino et al., 1998; Grote et al., 2002; Ippolito et al., 2002; Kong et al., 2003); however, to our knowledge, this is the first report on the real-time PCR…”
• Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance (2012, methodology): “This method has been widely used for detection and/or further characterisation of numerous fungi (Aroca & Raposo, 2007; Grote et al., 2002; Hong et al., 2010; Ippolito et al., 2002; Langrell et al., 2008; Meng and Wang 2010; Mercado-Blanco et al., 2001; Qin et al., 2011; Wu et al., 2011).”
• Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR (2002, background): “Regarding the two species that attack citrus, P. nicotianae and P. citrophthora, specific primers for their identification have been developed (Èrsek et al., 1994; Lacourt and Duncan, 1997; Grote et al., 2002).”