Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.     

A TaqMan real‐time PCR assay for the detection of Phytophthora ‘taxon Agathis’ in soil,pathogen of Kauri in New Zealand

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.     

Specific hybridization real‐time PCR probes for Phytophthora ramorum detection and diagnosis

Sudden Oak Death, caused by Phytophthora ramorum, poses a serious threat to native American oaks, and is also present in Europe where it has been isolated from numerous European ornamental plant nurseries. Its proven aggressiveness against plants in the Fagaceae and Ericaceae and the damage it has caused in North America have lead to it being assigned quarantine status. The timely and accurate detection of P. ramorum is a critical aid in the study of the epidemiology and biology of this pathogen. As a regulated organism, the availability of a sensitive and reliable assay is essential when attempting to achieve early detection of the pathogen. In this work, new specific hybridization probes for a real‐time PCR amplification method were found to be rapid, robust and labour‐saving, and proved suitable for routine use in a molecular diagnostic laboratory.     

Rapid diagnosis of pathogenic Phytophthora species in soil by real‐time PCR

Real‐time PCR assays based on the TaqMan system and using ITS sequences were developed for the identification of Phytophthora species, including P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina, all of which are currently causing significant damage to roots of forest trees in both managed stands and natural ecosystems. Total genomic DNA was extracted from mycelia of aforementioned Phytophthora isolates. Species‐specific primers for P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina were designed based on ITS sequences of rDNA. The amplification efficiency of target DNA varied from 93.1% (P. pseudosyringae) to 106.8% (P. quercina). The limit of the detection was calculated as 100 – 1,000 fg DNA, depending on the Phytophthora species. In mixed soil samples, all Phytophthora species were detected for Ct values shifted by 0.7 – 2.1 cycles. Based on these real‐time PCR assays we were able to identify the five Phytophthora species. These techniques will be of value in the identification of these pathogens, which may cause up to 80 – 90% fine root loss in oak stands.     

Development and evaluation of a real‐time PCR seed lot screening method for Fusarium circinatum,causal agent of pitch canker disease

Fusarium circinatum is a serious pathogen of Pinus spp. worldwide, causing pitch canker disease. F. circinatum can contaminate seeds both internally and externally and is readily disseminated via contaminated seed. Many countries require screening of pine seeds for F. circinatum before they can be imported. The currently accepted screening method is based on culturing the pathogen on a semi‐selective medium and identifying it using morphological traits. This method is time‐consuming and does not allow for accurate identification of the pathogen to the species level. A bulk DNA extraction and real‐time PCR procedure to screen seeds for the presence of F. circinatum were developed in this study. The real‐time PCR method resulted in the detection of F. circinatum in 5 of 6 commercial seed lots tested and has a lower detection limit of 1 × 10^?5 ng of F. circinatum DNA per PCR. The culture‐based method detected Fusarium spp. in four of six of the same seed lots. The real‐time PCR method can be used to screen multiple seed lots in 2 days, whereas the culture‐based method requires a minimum of 1–2 weeks. This new real‐time PCR seed screening method allows for fast, sensitive and accurate screening and can be adapted to handle larger volumes of seeds.     

Rapid and accurate detection of Ceratocystis fagacearum from stained wood and soil by nested and real‐time PCR

A nested and real‐time PCR assay was developed for the rapid and accurate detection of Ceratocystis fagacearum, which is the causal agent of oak wilt in stained wood and soil. Based on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species‐specific primers, CF01/CF02, was designed. Whereas a 280‐bp product was amplified using the purified DNA from three isolates of C. fagacearum as the template, no PCR product was obtained from template of other 18 fungi. The detection sensitivity was 10 pg genomic DNA per 25‐μl PCR reaction volume. To increase detection sensitivity, a nested PCR was developed by using ITS1/ITS4 as the first‐round primers and CF01/CF02 in the second round, as it can detect 1 pg genomic DNA per 25‐μl PCR reaction volume. More importantly, CF01/CF02 primers were successfully adapted to real‐time PCR with a detection limit of 0.1 pg genomic DNA per 20‐μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil.     

A new method to detect Lonsdalea quercina in infected plant tissues by real‐time PCR

Presymptomatic and accurate diagnoses of pathogens are essential for disease prediction and the timely application of bactericide. The bacterium Lonsdalea quercina (=Brenneria quercina) has been reported as the causal agent of drippy nut and bark canker disease on oak in California (US) and Europe. In recent years, it is also found on Populus × euramericana trees in Henan province of China. This bacterium causes longitudinal cankers of a few centimetres in size on the bark surface of the upper trunk. In this study, we developed two species‐specific PCR assays using primer pairs LqfF/LqfR and LqgF/LqgR for the rapid and accurate detection of the pathogenic bacteria in diseased plant tissues. The results show that the LqfF/LqfR primers amplified only a single PCR band of approximately 382 bp and the LqgF/LqgR primers yielded a PCR product of approximately 286 bp. The two primers were successfully adapted to real‐time PCR based on SYBR Green I used with the ABI 7500 system. The detection limit of the reaction was 0.1 pg genomic DNA per 20 μl PCR reaction volumes. The pathogen was mainly detected in the phloem of cankers as well as in the exudates of diseased trees, but was not found in the xylem or leaves. The size of pathogen in distribution was larger than the lesion. The results demonstrate that real‐time PCR assays can be used to detect the pathogen by extracting DNA directly from infected plant tissues. This method is a rapid, reliable method for the presymptomatic and accurate detection of L. quercina, providing a useful insight into epidemiological studies.     

Genetic diversity of European populations of the oak fine‐root pathogen Phytophthora quercina

The recently discovered oak‐specific fine root plant pathogen Phytophthora quercina is a significant factor in the current phase of European oak decline but its origins and ecology are poorly understood. A genome‐wide analysis of 260 amplified fragment length polymorphism (AFLP) markers was used to examine the genetic diversity of 72 isolates from five oak species at 28 sites in Germany (particularly Bavaria), Italy, France, Hungary and the UK. Within‐site diversity was examined at 16 sites. The limited genetic diversity (within and between sites) and lack of genetic substructuring according to geographic origin or host species suggest the rapid spread of a relatively recently introduced species. Two subgroups were distinguished and these may reflect an initial introduction of isolates of two different genetic backgrounds. The relatively low genetic diversity is probably because of the predominantly inbreeding (homothallic) nature of P. quercina. However, evidence of limited intra‐site diversity, temporal variation and the lack of clonality within the European population suggest that some diversity is being maintained by occasional outcrossing and turnover of a reservoir of long‐lived soil‐borne oospore (sexually derived) inoculum.     

The long‐term survival of Phytophthora cinnamomi in mature Banksia grandis killed by the pathogen

The ability of Phytophthora cinnamomi to survive long dry periods is the key to its persistence in the south‐west of Western Australia. It has been proposed that dead Banksia grandis are a significant long‐term reservoir for P. cinnamomi inoculum. To test this, 36 healthy B. grandis trees were inoculated in April 1999, and the presence of viable propagules in planta was determined between 2 and 34 months after tree death. By 10 months after inoculation, 75% of the trees had died, with the remaining seven trees dying by 22 months. The pathogen was more commonly recovered from bark than from wood, except from those trees that died at 22 months, and more commonly from above‐ground trunks than below‐ground trunks and roots until 8 months after plant death. In trees that died 12 months after inoculation, P. cinnamomi was recovered from 60% of trunk and root core samples at 3 months, declining to 33% at 10 months, 5.5% at 12 months and 0.1% at 34 months after tree death. In trees that died at 22 months, P. cinnamomi was recovered from 87% of trunk and root samples 2 months after tree death, decreasing to 0.5% by 33 months. This study suggests that the pathogen does not have a saprotrophic phase within dead B. grandis tissue, and B. grandis is unlikely to be a long‐term reservoir for P. cinnamomi. However, the manipulation of the density of B. grandis and the use of fire to facilitate the breakdown of dead Banksia trunks in the Eucalyptus marginata (jarrah) forest may reduce the spread and impact of P. cinnamomi.     

Fast and specific detection of the invasive forest pathogen Heterobasidion irregulare through a Loop‐mediated isothermal AMPlification (LAMP) assay

Heterobasidion irregulare is one of the most destructive fungal pathogens of pines in North America and was accidentally introduced into central Italy, where it has become invasive. The fungus is currently recommended for regulation by the European and Mediterranean Plant Protection Organisation (EPPO). In this work, an efficient diagnostic tool for the early detection of H. irregulare based on Loop‐mediated isothermal AMPlification (LAMP) coupled with two different DNA extraction methods was developed. The LAMP assay showed high specificity and good sensitivity, with a limit of detection of about 20 picograms of target DNA and time of detection of less than 40 min. The assay was successfully tested on a variety of different samples, including fungal fruiting bodies, infected plants and colonized wood. A survey on environmental samples collected in the field was also performed using the LAMP assay coupled with a rapid DNA extraction method. The possible applications of this molecular diagnostic tool encompass the monitoring of pine forests surrounding the current invasion area, laboratory or in‐field analyses of samples from suspected trees, and the surveillance in the ports of entry of wood imported from North America.     

Real‐time PCR assays for the detection of Heterobasidion irregulare,H. occidentale,H. annosum sensu stricto and the Heterobasidion annosum complex

A set of quantitative hierarchical real‐time PCR assays was developed for the detection of Heterobasidion irregulare, H. occidentale, H. annosum sensu stricto and of the entire Heterobasidion annosum complex. These assays enable specific and accurate detection and quantification of the target species from DNA extracted on airborne collected spores. Heterobasidion‐specific TaqMan? real‐time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96‐ or 384‐well plate format arrays for high‐throughput testing of large numbers of samples against multiple targets. Assays were validated for (i) specificity, (ii) sensitivity and (iii) repeatability. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and a hundred ITS gene region copies or one conidia count equivalent. Precision or repeatability of each assay revealed a mean coefficient of variation of 5.9%. These molecular tools are now available for rapid and reliable monitoring of one of the most significant pathogen species complex of temperate northern coniferous forest around the world.     

Land‐use changes influence the sporulation and survival of Phytophthora agathidicida,a lethal pathogen of New Zealand kauri (Agathis australis)

Phytophthora agathidicida is the accepted causal agent of dieback in remnant stands of long‐lived indigenous New Zealand kauri (Agathis australis) and poses a significant threat to the long‐term survival of this species. Little is known about the effect of key soil physicochemical characteristics on the growth of P. agathidicida. In this study, we investigated the growth of P. agathidicida in soils collected from adjacent areas under original kauri forest, short rotation pine (Pinus radiata) plantation forest and grazed pastures. A growth response assay was used to quantify asexual (sporangia) and sexual (oospore) spore counts over 8 days in soils sampled from each land‐use. Significantly higher numbers of sporangia (p < 0.001) and oospores (p < 0.01) were found in pasture and pine forest soil within 2 days of the growth assay trials, suggesting these soils may favour asexual/sexual reproduction in the early stages of P. agathidicida establishment compared to kauri forest soils. Additionally, oospore production significantly increased over 8 days in pine forest soil, suggesting that with an increase in inoculum loads, these soils potentially act as pathogen reservoirs. The soil physicochemical properties (e.g., pH, C and N, phosphorus content and electrical conductivity) investigated in this study did not significantly correspond to spore count data between land‐uses, suggesting that differences in growth response are driven by other edaphic factors not explored in the present study.     

Phytophthora ramorum is a generalist plant pathogen with differences in virulence between isolates from infectious and dead‐end hosts

Variation in virulence was examined among isolates of Phytophthora ramorum from epidemiologically important or infectious (non‐oak) and transmissive dead‐end (oak) hosts from North America. Twelve isolates representative of the genetic, geographic and host range of P. ramorum in the western United States were inoculated on leaves of Umbellularia californica (bay laurel or bay) and stems of Quercus agrifolia (coast live oak). In spite of extreme genetic similarity among the isolates employed, and even within the same genotype, significant differences in lesion size were measured, suggesting virulence in this pathogen is also controlled by epigenetic factors. A strong positive correlation between lesion size on bay laurel and coast live oak provides experimental evidence P. ramorum is a generalist pathogen that lacks host specificity. Isolates from non‐transmissive oaks were significantly less pathogenic both on oaks and bays than isolates from infectious hosts. These results are essential to further our understanding of the epidemiology and evolutionary potential of this pathogen. A quantitative differential in virulence of isolates from hosts with different epidemiological roles has been described for many animal diseases, but is a novel report for a plant disease.     

Update on the 35‐year expansion of the invasive root pathogen,Phytophthora lateralis,across a landscape of Port Orford cedar (Chamaecyparis lawsoniana)

Port Orford cedar (Chamaecyparis lawsoniana) is endemic to northern California and southwestern Oregon and is considered a foundation species that plays critical roles in riparian areas and on nutrient‐poor soils. Since 1952, a non‐native, pathogenic oomycete (Phytophthora lateralis) has been spreading throughout the range of the cedar. Most spread occurs by vehicles carrying infested soil along gravel roads primarily used for timber harvest. In a previous study conducted in 1998 and 1999, Port Orford cedar and P. lateralis were censused in a 37‐km² study area and dendrochronology was used to reconstruct the history of pathogen invasion. That work, which represents the only detailed analysis of spread rates for P. lateralis, showed that the first successful invasion into the study area took place in 1977 and that 43% of the susceptible host sites (stream crossings) were infested by 1999. In the work presented here, all sites that were uninfested in 1999 were re‐censused in 2012, extending the historical reconstruction of P. lateralis spread to 35 years. Two new infestations were initiated between 1999 and 2012, suggesting that the rate of spread of P. lateralis has slowed greatly. Between 1980 and 1989, the average number of new site infestations was 1.8 infestations per year, while between 1990 and 1999 the average was 0.4 infestations per year and between 2000 and 2009 the average was 0.2 infestations per year. Several potential explanations for the reduced number of new infestations are discussed.     

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.     

PCR–DGGE method for in planta detection and identification of Phytophthora species

We have developed a method to detect multiple species of Phytophthora directly in infected plant tissues. The method is based on the polymerase chain reaction and uses Phytopththora‐specific primers and denaturing gradient gel electrophoresis (PCR–DGGE). The method distinguished most of the 16 Phytophthora species tested. Very closely related species might not, however, be identified using the method. The detection efficiency was high and successful in eight different plant tissues tested. The PCR–DGGE detection tool developed here will be a fast and inexpensive method suitable for pathogen surveys and research programmes.     

Development of species‐specific PCR primers on rDNA for the identification of European Armillaria species

Attempts to design species‐specific PCR primers from six European Armillaria species in the ribosomal RNA genes are reported. Primers were developed on the basis of the nucleotide sequence variability of the internal transcribed spacers (ITS) and the intergenic spacer (IGS1) of the ribosomal DNA. Four sets of primers gave specific PCR products for Armillaria tabescens, Armillaria mellea and Armillaria ostoyae. However, due to the high sequence similarities between Armillaria borealis and Armillaria ostoyae and between Armillaria cepistipes and Armillaria gallica no species specific amplification was obtained for these taxa.     

Development of two reverse transcription‐PCR methods to detect living pinewood nematode,Bursaphelenchus xylophilus,in wood

Pinewood nematode, Bursaphelenchus xylophilus, is an inhabitant of native pine species of North America, where its presence in trees is non‐pathogenic. By contrast, the introduction of this nematode to forests overseas has devastated some pine stands and is recognized as a pest of phytosanitary concern by some countries' National Plant Protection Organizations. The ability to detect B. xylophilus in internationally traded wood products is crucial to reduce the spread of this organism. Current molecular techniques for the detection of B. xylophilus rely on the presence of genomic DNA and thus will detect both living and dead nematodes without differentiation. The detection of dead nematodes could lead to unnecessary trade disruption. Therefore, accurate techniques for the detection of and differentiation between live and dead B. xylophilus are critical. We have developed an endpoint RT‐PCR assay and a SYBR Green 1 real‐time RT‐PCR assay, both of which selectively identify living pinewood nematode by detecting the presence of Hsp70 mRNA as a viability marker. Both of these assays may help overcome or resolve disputes involving the detection of pinewood nematode at the port of entry and can also be used to evaluate the efficiency of wood treatment procedures.