SUMMARY New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real-time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras-related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.-specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.

Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.

Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a costeffective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.Phytophthora ramorum is the causal agent of extensive oak mortality (commonly known as sudden oak death) in coastal forests in California (27) and southern Oregon (12,25). This pathogen also causes ramorum leaf blight and dieback on a range of other plant species (9) and can have a profound and devastating effect on forest ecosystems. A distinct population of the same pathogen (6, 35) is found in a number of European countries (10,20,23,37), mostly causing dieback and leaf blight on a range of ornamental plants in nurseries and landscaped areas (2). There have also recently been a number of incidences of lethal bark cankers caused by P. ramorum in native and nonnative trees in Europe (7). P. ramorum has a broad and expanding host range (3,4,10,11,19,20,28,31), and as a result of the threat posed to forest ecosystems, the movement of a variety of its host species is subject to restrictions in Europe and the United States. Emergency European Community phytosanitary measures for P. ramorum were introduced in 2002 (1), and in the United States quarantine restrictions at both the federal and state levels control the movement of a variety of plant species from infested areas in California and Oregon (31). The availability of rapid and accurate detection methods for P. ramorum is critical to allow its prevalence to be monitored and to expedite management or eradication steps to prevent its introduction and minimize its spread.The identification of P. ramorum is not possible based on host symptoms alone due to the considerable variation in their expression and because a range of other causes can produce similar symptoms. These include infections by several other Phyt...

New polymerase chain reaction (PCR) primers were developed for the identification of the EU quarantine pest Monilinia fructicola. These allowed nine M. ffucticola isolates to be distinguished from other fungi, including six isolates of M. laxa and six isolates of M. fructigena (which also cause brown rot of stone and pome fruit). Three M. fnrcticola isolates from Japan and one from Australia did not react with a primer set previously published for M. fructicola. M. fnrcticola isolates could be subdivided into three groups based on the size of their nuclear rDNA small subunit. The subunits from the four non-reactive isolates were either smaller or larger than the reactive group. Further primers were developed which were specific for either M. laxa or M. fructigena. Another new primer identified both M. laxa and M. fructigena, and yet another M. laxa and M. fructicola. When used in combination, these primers specific for two species correctly identified unknown isolates of all three Monilinia species. The new primers designed in this study have been used to identify, rapidly and correctly, pustules taken directly from infected plum fruits, thus demonstrating their diagnostic potential. and 2 regions of the nuclear rRNA gene repeat were therefore produced ( Table 1). The effectiveness of these primers and the occurrence of group-I introns in isolates of M. fructicola is discussed.

Container-Based Sanitation (CBS) has rapidly progressed from its inception less than a decade ago to its recent classification as a type of improved sanitation facility by the Joint Monitoring Programme. CBS in many ways represents a sustainable service, as it addresses the entire sanitation service chain; offers a variety of service-based business models; and is affordable to people living in marginalized and informal urban settlements. At the same time, CBS services which have been operating for a number of years have grown relatively slowly. Taking CBS to scale will require solving several diverse challenges, particularly the need for government mandates; regulation; and innovative financing. This paper presents the collective views of some of the world's leading CBS practitioners in an effort to summarize the potential, research gaps, and major challenges to scaling CBS.

Plant health regulations to prevent the introduction and spread of Phytophthora ramorum and P. kernoviae require rapid, cost effective diagnostic methods for screening large numbers of plant samples at the time of inspection. Current on-site techniques require expensive equipment, considerable expertise and are not suited for plant health inspectors. Therefore, an extensive evaluation of a commercially available lateral flow device (LFD) for Phytophthora species was performed involving four separate trials and 634 samples. The assay proved simple to use, provided results in a few minutes and on every occasion a control line reacted positively confirming the validity of the test. LFD results were compared with those from testing a parallel sample, using laboratory methods (isolation and real-time PCR). The diagnostic sensitivity of the LFD (87·6%) compared favourably with the standard laboratory methods although the diagnostic specificity was not as stringent (82·9%). There were a small number ( n = 28) of false negatives, but for statutory purposes where all positive samples must be identified to species level by laboratory testing, overall efficiency was 95·6% as compared with visual assessment of symptoms of between 20-30% for P. ramorum and P. kernoviae . This work demonstrates the value of the LFD for diagnosing Phytophthora species at the time of inspection and as a useful primary screen for selecting samples for laboratory testing to determine the species identification.

Kauri Agathis australis, an iconic tree of New Zealand, is under threat from an introduced disease-causing pathogen provisionally named Phytophthora 'taxon Agathis' (referred to as PTA). This soilborne, Pythiaceous species belongs to the Chromista and causes a collar rot resulting in yellowing of the foliage and thinning of the canopy, which eventually causes death of the infected tree. The management and containment of this pathogen requires rapid and reliable detection in the soil. The current method for soil detection utilizes a soil bioassay involving lupin baits and soil flooding in a process that takes between ten and twenty days. We describe a real-time PCR assay based on TaqMan chemistry for the specific detection of PTA, which targets the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA. This TaqMan real-time PCR assay could be used with DNA extracted directly from bulk soil samples to enable rapid quantification of PTA within soil. The detection limit was 2 fg of PTA DNA from pure culture, or 20 fg in the presence of DNA extracted from soil. The assay was validated using soil samples taken from a PTA-infested site and soil spiked with a known concentration of oospores. We conclude that the TaqMan real-time PCR assay offers a more time-efficient method for detection of PTA in soil than existing methods.

Representative European wheat cultivars were tested under quarantine containment for their susceptibility to Tilletia indica, the cause of Karnal bunt of wheat. Fifteen winter and 15 spring wheat ( Triticum aestivum ) and 11 durum wheat ( Triticum durum ) cultivars were inoculated by boot injection just prior to ear emergence to test their physiological susceptibility. Selected cultivars were then re-tested by spray inoculation after ear emergence to determine their morphological susceptibility, which is a better predictor of field susceptibility. At maturity, the ears and seeds were assessed for incidence and severity of disease. For the physiological susceptibility tests, 13/15 winter wheat cultivars were infected and the percentage of infected seeds ranged from 1 to 32%. For spring cultivars, 13/15 cultivars were infected and the percentage of infected seeds ranged from 1 to 48%. For the durum cultivars, 9/11 were infected and the percentage of infected seeds ranged from 2 to 95%. Across all cultivars, 35/41 were infected. Based on historical Karnal bunt susceptibility categories using coefficients of infection, one cultivar was classed as highly susceptible, three as susceptible, 11 as moderately susceptible, 20 as resistant and only six as highly resistant. The spray-inoculation morphological susceptibility tests broadly confirmed the physiological susceptibility results, although lower levels of infection were observed. Overall, the range of susceptibility was similar to that found in cultivars grown in Karnal bunt affected countries. The results demonstrate that European wheat cultivars are susceptible to T. indica and thus could potentially support the establishment of T. indica if introduced into Europe.