European Journal of Plant Pathology (2002) 108, 855-868

Antonio Ippolito, Leonardo Schena and Franco Nigro (2002)
Detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils by nested PCR
European Journal of Plant Pathology 108 (9), 855-868
Abstract: The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B-Pn6 and Pc2B-Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2-ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1 pg µl^-1 DNA for all the primer pairs (Ph2-ITS4, Pn5B-Pn6, and Pc2B-Pc7). In nested PCR, with primers Ph2-ITS4 in the first round, the detection limit was of 1 fg µl^-1 for both the primer sets (Pn5B-Pn6 and Pc2B-Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2-3 h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2-ITS4 in the first round. In the second round the primer pairs Pn5B-Pn6 and Pc2B-Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.
(The abstract is excluded from the Creative Commons licence and has been copied with permission by the publisher.)
Link to article at publishers website
Database assignments for author(s): Antonio Ippolito, Franco Nigro, Leonardo Schena

Research topic(s) for pests/diseases/weeds:
identification/taxonomy
surveys/sampling/distribution

---

Pest and/or beneficial records:

Beneficial	Pest/Disease/Weed	Crop/Product	Country	Quarant.
	Phytophthora citrophthora	Citrus (genus)	Italy
	Phytophthora nicotianae	Citrus (genus)	Italy