Comparison of a baiting method and PCR for the detection of Phytophthora fragariae var. rubi in certified raspberry stocks*

In winter 2002/2003, a total of 136 root samples from 57 different raspberry stocks in Scotland were examined for the presence of raspberry root rot caused by the fungus‐like pathogen Phytophthora fragariae var. rubi. All stocks had been planted as propagation material entered at different grades in the Scottish certification scheme or had applied for plant passports. For detection, a modified ‘Duncan bait test’ was compared to a nested PCR method. The two tests identified the same infected stocks: PCR detected 10 positive samples from four different stocks, while the bait test picked up two additional positive samples coming from the same four stocks. The two tests had a similar level of reliability in this examination and a recommendation for one or the other depends mainly on the technical equipment and skills available in the laboratory.     

Phytophthora ramorum: the situation in Scotland1

In Scotland inspections for Phytophthora ramorum on plants in the horticulture nursery trade started in July 2001 and are currently carried out four times per year. In addition, approximately 130 established gardens have been inspected for the disease during the years 2003 and 2004. Phytophthora ramorum has been found on Rhododendron, Viburnum, and lilac (Syringa vulgaris); the most important host plant is Viburnum tinus. The pathogen is confined to nurseries and garden centres with the exception of one private garden. The first finding was in April 2002 with 17 more outbreaks the same year. Since then the number of outbreaks per year has declined dramatically to 6 in 2003, 5 in 2004 and 3 until November 2005. Altogether, there have been 21 different outbreaks sites since the first finding, some with repeated occurrences of the disease.     

Development of a quantitative real‐time PCR assay for the detection of Phytophthora austrocedrae,an emerging pathogen in Britain

A TaqMan real‐time PCR assay was developed for Phytophthora austrocedrae, an emerging pathogen causing severe damage to juniper in Britain. The primers amplified DNA of the target pathogen down to 1 pg of extracted DNA, in both the presence and absence of host DNA, but did not amplify any of the non‐target Phytophthora and fungal species tested. The assay provides a useful tool for screening juniper populations for the disease.     

A duplex PCR method for the simultaneous identification of Phytophthora ramorum and Phytophthora kernoviae

Phytophthora ramorum and Phytophthora kernoviae are two fungus‐like organisms affecting a wide range of hardy ornamental plants and trees. Emergency measures are implemented in the European Union for P. ramorum and aim to eradicate, or at least prevent the further spread of this harmful pathogen. Phytophthora kernoviae has so far been found only in New Zealand, the UK and Ireland, and is regulated on a UK level using the same measures as for P. ramorum. Both Phytophthora species have a similar host range and can be diagnosed using similar methods. Therefore a duplex PCR detection, based on the internal transcribed spacer (ITS) regions of the ribosomal DNA, was developed to enable simultaneous testing to reduce diagnostic times. The method was tested for its specificity and sensitivity, and on plant samples, and was shown to be reliable for identification of the two organisms.     

Euphresco Sendo: An international laboratory comparison study of molecular tests for <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis> detection and identification

An international test performance study (TPS) was organised to generate validation data for three molecular Synchytrium endobioticum tests: van den Boogert et al. (European Journal of Plant Pathology 113, 47–57, 2005), and van Gent-Pelzer et al. (European Journal of Plant Pathology, 126, 129-133, 2010) for the detection of S. endobioticum, and the pathotype 1(D1) identification test described by Bonants et al. (European Journal of Plant Pathology, 143, 495-506, 2015). Two TPS rounds were organised focussing on different test matrices, i.e. round 1: warted potato tissue, and round 2: resting spore suspensions. When using the tests for detection and identification of S. endobioticum in warted potato tissue, no significant differences were observed for diagnostic sensitivity, diagnostic specificity, overall accuracy, analytical sensitivity and robustness. When using the tests for detection and identification of S. endobioticum in resting spore suspensions, the van den Boogert and van Gent-Pelzer tests significantly outperform the Bonants test for diagnostic sensitivity and diagnostic specificity. For overall accuracy and analytical sensitivity, the van Gent-Pelzer significantly outperforms the van den Boogert and Bonants tests and is regarded as the test of choice when identifying S. endobioticum from resting spores. Tests regarded fit for purpose for routine testing of wart material and resting spore suspensions are proposed for the update of EPPO standard PM7/28(1) Synchytrium endobioticum.     

An improved method for qPCR detection of three Phytophthora spp. in forest and woodland soils in northern Britain

Using TaqMan qPCR assays, DNA of P. ramorum, P. kernoviae and P. austrocedri was detected in 500 g soil samples collected from twelve infected forest and woodland sites in northern Britain. Phytophthora DNA was also amplified in soil adhering to boots after walking transects along footpaths or animal trails. At two sites, Phytophthora DNA was detected in soil over a 4‐year period following removal of infected hosts. This new method enabling assessment of larger quantities of soil demonstrates the contamination risk of these pathogens in soil at infected sites and improves our understanding of the mechanisms of persistence and spread.     

Examination of latent infection in raspberry canes with Phytophthora rubi and P. idaei and transmission in micropropagation

Buds of raspberry canes from naturally and artificially infected root stocks were tested by nested PCR for the latent presence of Phytophthora rubi and P. idaei to establish the risk of introducing these pathogens into micropropagation. Fifteen out of 201 buds tested positive for P. rubi and only 1 out of 176 for P. idaei. These infections were not restricted to buds in the lower part of the cane and mostly do not appear to spread systemically up from the rootstock but seem to be caused by secondary infections of the canes. Buds from symptomless canes from infected root stocks were used for micropropagation and P. rubi could be detected in 5.8% of the obtained microplants after ten generations in one of the two trials. This indicates that micropropagation procedures are not absolutely infallible in eliminating infection and underlines the necessity of a zero tolerance towards diseases in the starting material.