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.     

Multispecies Phytophthora disease patterns in declining beech stands

Decline diseases are typically caused by complex abiotic and biotic interactions and characterized by a suite of symptoms indicative of low plant vigour. Diseased trees are frequently infected by Phytophthora, but the complex interactions between pathogen, host and the heterogeneous forest environment mask a comprehensive understanding of the aetiology. In the present study, we surveyed European beech (Fagus sylvatica) stands in Swiss forests with recent increases in bleeding lesions for the presence of Phytophthora. We used a combined approach of analysing soil and bark samples from trees displaying bleeding lesions and trees free from bleeding lesions. Soil baiting revealed a higher prevalence of Phytophthora spp. around trees with bleeding lesions than around trees without bleeding lesions. For the bark samples from bleeding lesions, we used several detection methods. Phytophthora spp. were detected in 74% of the trees by an immunological on‐site diagnostic kit, in 64% by a specific PCR assay, and 38% by isolation on selective media. All samples tested were negative for P. ramorum using qPCR. Overall, nine Phytophthora species were identified by ITS sequencing, the most common of which were P. plurivora, P. gonapodyides, P. × cambivora and P. syringae. We identified distinct species in bleeding lesions and the rhizosphere of the same host tree which suggests a multispecies Phytophthora disease patterns in these declining beech. Among the recovered species, P. × cambivora and P. × serendipita were identified as hybrid genotypes with the former abundant in bleeding lesions.     

Phytophthora root and collar rot of mature Fraxinus excelsior in forest stands in Poland and Denmark

In recent years, Common ash (Fraxinus excelsior) throughout Europe has been severely impacted by a leaf and twig dieback caused by the hyphomycete Chalara fraxinea. The reasons for its current devastating outbreak, however, still remain unclear. Here, we report the presence of four Phytophthora taxa in declining ash stands in Poland and Denmark. Phytophthora cactorum, Phytophthora plurivora, Phytophthora taxon salixsoil and Phytophthora gonapodyides were isolated from rhizosphere soil samples and necrotic bark lesions on stems and roots of mature declining ash trees in four stands. The first three species proved to be aggressive to abscised roots, twigs and leaves of F. excelsior in inoculation experiments. Soil infestation tests also confirmed their pathogenicity towards fine and feeder roots of ash seedlings. Our results provide first evidence for an involvement of Phytophthora species as a contributing factor in current decline phenomena of F. excelsior across Europe. Specifically, they may act as a predisposing factor for trees subsequently infected by C. fraxinea. Phytophthora species from ash stands also proved to be aggressive towards a wide range of tree and shrub species commonly associated with F. excelsior in mixed stands. Although damage varied considerably depending on the Phytophthora species/isolate–host plant combination, these results show that many woody species may be a potential source for survival and inoculum build‐up of soilborne Phytophthora spp. in ash stands and forest ecosystems in general.     

Detection of Chalara fraxinea in common ash (Fraxinus excelsior) using real time PCR

A real time PCR assay was developed for the detection of Chalara fraxinea in common ash. PCR primers and Taqman probes, based on the internal transcribed spacer region of the multi‐copy gene rDNA, were tested for specificity and sensitivity. The primers amplified an 81 bp fragment for C. fraxinea but did not amplify DNA from other Chalara species or from other fungi isolated from ash, whether pathogenic or saprophytic. The limit of detection was 5 pg of genomic DNA per PCR. Moreover, naturally‐infected samples were correctly diagnosed. A procedure for DNA extraction from woody tissues using an electric drill yielded DNA of an appropriate quality for real time PCR. This molecular method could be useful for routine analysis of this emergent pathogen and for epidemiological studies.     

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.     

Strategies of attack and defence in woody plant–Phytophthora interactions

This review comprises both well‐known and recently described Phytophthora species and concentrates on Phytophthora–woody plant interactions. First, comprehensive data on infection strategies are presented which were the basis for three models that explain invasion and spread of Phytophthora pathogens in different woody host plants. The first model describes infection of roots, the second concentrates on invasion of the trunk, and the last one summarizes infection and invasion of host plants via leaves. On the basis of morphological, physiological, biochemical and molecular data, scenarios are suggested which explain the sequences of reactions that occur in susceptible and tolerant plants following infections of roots or of stem bark. Particular emphasis is paid to the significance of Phytophthora elicitins for such host–pathogen interactions. The overall goal is to shed light on the sequences of pathogenesis to better understand how Phytophthora pathogens harm their host plants.     

Detection and quantification of Phytophthora species which are associated with root-rot diseases in European deciduous forests by species-specific polymerase chain reaction

Oligonucleotide primers were developed for the polymerase chain reaction (PCR)-based detection of selected Phytophthora species which are known to cause root-rot diseases in European forest trees. The primer pair CITR1/CITR2, complementing both internal transcribed spacer regions of the ribosomal RNA genes, gave a 711 bp amplicon with Phytophthora citricola. The Phytophthora cambivora specific primer pair CAMB3/CAMB4, producing a 1105bp amplicon, as well as the Phytophthora quercina specific primer pair QUERC1/QUERC2, producing a 842 bp amplicon, were derived from randomly amplified polymorphic DNA (RAPD)-fragments presented in this paper. All three primer pairs revealed no undesirable cross-reaction with a diverse test collection of isolates including other Phytophthora species, Pythium, Xerocomus, Hebeloma, Russula, and Armillaria. Under the PCR conditions described the detection of a well discernable amplicon was possible down to 100 pg (P. cambivora), 4pg (P. quercina), and 2pg (P. citricola) target DNA. This diagnostic PCR system was able to detect P. citricola, P. quercina, and P. cambivora in seedlings of pendunculate oak (Quercus robur) and European beech (Fagus sylvatica) which were artificially infected under controlled conditions.     

Incidence of Phytophthora species in oak forests in Austria and their possible involvement in oak decline

A survey on the occurrence of Phytophthora species in oak ecosystems in Austria was conducted from April to May 1999 and in June 2000. The investigations were carried out at 35 study sites distributed throughout the zone of oak forests in eastern Austria. Four oak species, including Quercus robur, Q. petraea, Q. cerris and Q. pubescens were considered in the survey. Rhizosphere soil samples were taken from sample trees, which consisted of healthy and declining trees as indicated by their crown transparency. Young oak leaflets were used as baits to recover Phytophthora species. The assemblage of Phytophthora spp. detected in Austrian oak forests consisted of five species, including Phytophthora quercina, P. citricola, P. gonapodyides, P. europaea and P. syringae. P. quercina and P. citricola were isolated from 11 and seven sites, respectively, and were thus the most common and most widely distributed species. The three other species were recovered only sporadically. P. citricola could be separated into two morphologically and genetically well‐characterized types (A and B). Phytophthora species, in particular the common P. quercina and P. citricola occurred on sites showing a wide variety of soil types, soil textures and moisture classes. There was mild evidence for connection between deteriorating crown status and the presence of Phytophthora spp. Furthermore, significant differences in contents of magnesium, as well as calcium, aluminium, nitrogen and carbon at different soil depths (0–10, 10–20 and 20–40 cm) were detected between Phytophthora‐infested and Phytophthora‐free sites. The results of the present study provide circumstantial evidence that Phytophthora species are involved in oak decline at certain sites in Austria.     

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.     

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.     

Occurrence and diversity of Phytophthora species in declining broadleaf forests in western Ukraine

In western Ukraine, forest decline and dieback of several broadleaved tree species have become increasingly evident during recent years, and surveys in some areas have shown symptoms indicative of Phytophthora infections. In this study, we aimed to determine the occurrence and diversity of Phytophthora species associated with several broadleaved tree species (Alnus glutinosa, Betula pendula, Castanea sativa, Fagus sylvatica and Quercus robur) from forest stands where dieback has been observed. Rhizosphere soil samples were collected from 14 forest stands during 2017 and 2018 and tested for the presence of Phytophthora species using morphological and molecular methods. Seven Phytophthora species (P. bilorbang, P. cactorum, P. gallica, P. gonapodyides, P. lacustris, P. plurivora and P. polonica), and two other clade six taxa were detected from the various forest types, several of which are probable agents responsible for decline. Four of the Phytophthora species (P. bilorbang, P. gallica, P. plurivora and P. polonica) have previously never been reported from broadleaf forests in Ukraine.     

Susceptibility of common tree species in Sweden to Phytophthora cactorum,P. cambivora and P. plurivora

In Sweden, invasive Phytophthora pathogens have been recognized as a growing threat to urban and production forests, calling for an urgent update of regeneration strategies for infested areas. Stem inoculation tests were performed to test the relative susceptibility of common conifer and broadleaved tree species Pinus sylvestris, Picea abies, Larix x eurolepis, Betula pendula, Quercus robur, Fagus sylvatica, Populus trichocarpa and Tilia cordata to the root pathogens Phytophthora cactorum, P. cambivora and P. plurivora commonly isolated from Swedish soils. Results indicate that all the species tested were susceptible and formed lesions following stem inoculation with all three Phytophthora species, but to varying degrees. Of particular interest are the high levels of susceptibility in P. trichocarpa to all three Phytophthora species compared to other tested tree species.     

Susceptibility of silver birch and black alder to several Phytophthora species isolated from soils in declining broadleaf forests in western Ukraine

In declining broadleaf forests in western Ukraine, several Phytophthora species including P. plurivora, P. bilorbang, P. polonica, P. gonapodyides and P. cactorum were recovered using soil baiting assays and identified using morphological and molecular methods. Pathogenicity tests of selected isolates were performed on black alder (Alnus glutinosa (L.) Gaerth.) and silver birch (Betula pendula Roth.) to assess susceptibility of these two tree species to the newly detected Phytophthora species. Phytophthora plurivora, P. bilorbang and P. polonica showed higher pathogenicity in both alder and birch compared to the other tested Phytophthora species.     

Relative susceptibility of oaks to seven species of Phytophthora isolated from oak forest soils

Isolates of Phytophthora cambivora, P. cinnamomi, P. citricola, P. europaea, P. quercetorum and two unidentified species were tested for their pathogenicity to eastern US oak species by root and stem inoculations. Experiments were conducted during two different periods and included 1‐, 2‐ and 20‐year‐old oaks grown under greenhouse and field conditions. Species of Phytophthora were pathogenic in varying degrees to the oak species tested. All species were pathogenic to fine and taproots of at least one oak species. The fine root damage caused by the species of Phytophthora ranged from 9 to 55% when compared to the controls. Roots were more susceptible during the fall inoculation period than the summer. With exception of Phytophthora sp1 and P. quercina‐like, all species of Phytophthora were pathogenic to oak stems with P. cinnamomi and P. citricola being the most aggressive. Quercus montana and Q. rubra were the most susceptible oak species to stem inoculation. Lesion sizes were considerably larger when 20‐year‐old trees were inoculated. Generally, no significant differences in lesion sizes were detected in greenhouse tests when the summer and fall inoculation periods were compared. However, on 2‐year‐old field‐grown seedlings, lesion sizes were considerably smaller or not significantly different from controls during the fall inoculation period, suggesting lower, late season temperatures may restrict lesion development.     

Identification of Phytophthora species baited and isolated from forest soil and streams in northwestern Yunnan province,China

Phytophthora species were surveyed by collecting soil samples and placing bait leaves in selected streams during June–October in the years 2005, 2006 and 2010 at three sites in oak forests in Diqing Tibetan Autonomous Prefecture of NW Yunnan province, China. Seventy‐three isolates of Phytophthora spp. were recovered from 135 baited leaf samples and 81 soil samples. Eight Phytophthora species were identified by observation of morphological features and ITS1‐5.8S‐ITS2 rDNA sequence analysis. The eight taxa included two well‐known species P. gonapodyides and P. cryptogea, two recently described species P. gregata and P. plurivora, two named but as yet undescribed taxa, P. taxon PgChlamydo and P. taxon Salixsoil, and two previously unrecognized species, Phytophthora sp.1 and P. sp.2. The most numerous species, P. taxon PgChlamydo, and the second most abundant species, P. taxon Salixsoil, were recovered at all three sites. Phytophthora cryptogea was detected only once at site Nixi. Phytophthora gregata and P. sp.2 were isolated from a stream only at site Bitahai, while the other three species were each found at two sites. Phylogenetic analysis revealed that the isolates belonged to three ITS clades, one species including six isolates in clade 2, six species including 66 isolates in clade 6 and one species in clade 8. There was a relatively rich species and genetic diversity of Phytophthora detected in the investigated regions where the forest biotic and abiotic factors affecting the growth and evolution of Phytophthora populations were diverse.     

Foliar susceptibility of eastern oak species to Phytophthora infection

Seven different Phytophthora species were used to test the foliar susceptibility of the common eastern US oak species and understory plants to Phytophthora infection. The Phytophthora species employed were Phytophthora cambivora, Phytophthora cinnamomi, Phytophthora citricola, Phytophthora europaea, Phytophthora quercetorum, Phytophthora quercina‐like and Phytophthora sp1. Inoculation of detached‐leaves with agar plugs containing mycelia of Phytophthora provided an estimate of their relative susceptibility. Lesions were always greater when foliage was wounded and young. On deciduous plants, lesion sizes were considerably reduced with the increasing foliar age, although with evergreen plants lesion sizes remained similar regardless of foliar age when more aggressive isolates were tested. Infections seldom resulted when foliage was not wounded. With young and mature foliage, P. citricola usually produced the largest lesions. Young foliage of Quercus rubra was the most susceptible to infection followed by Castanea dentata for both wounded and non‐wounded inoculations. Mature foliage of Hamamelis virginiana, Kalmia latifolia and Quercus alba were the most susceptible to wound and non‐wound inoculations.     

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.     

Analysis of the distribution of Phytophthora cinnamomi in soil at a disease site in Western Australia using nested PCR

The oomycete plant pathogen Phytophthora cinnamomi has infected a very large area of native vegetation in the south western corner of Australia. An important aspect of effective disease management depends on being able to accurately map areas of infestation. For this purpose, we have developed a nested polymerase chain reaction (PCR) protocol for the detection of P. cinnamomi in soil. The test uses two sets of primers developed from the rRNA ITS sequences of P. cinnamomi and can detect as little as 1 pg DNA. The degree of sensitivity was reduced with DNA extracted from soil although this depended on the type of soil. Soils with a high organic content, such as eucalypt forest soil and potting mix were more inhibitory than sandy soils. Inhibition by soil DNA could be reduced by the addition of bovine serum albumin and formamide to the reaction. Taq DNA polymerase was very sensitive to inhibitors compared with Tth⁺ or TaqF1*. In comparison with baiting (0–10% positive samples), nested PCR proved to be a very much more efficient (90–100% positive samples) method for the detection of P. cinnamomi in soil.     

Pathogenic fungi associated with pre‐ and post‐emergence seedling blight of pine and cypress in Fars Province,Iran

Seed and root rot of pine and cypress seedlings cause heavy annual losses to forest nurseries in Fars Province. Root and crown samples of various species of conifers, such as Tehran pine (Pinus eldarica), brutia pine (Pinus brutia), Arizona cypress (Cupressus arizonica), Shirazian cypress (Cupressus sempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) and oriental arborvitae (Thuja orientalis), showing chlorosis, necrosis, stunted growth, defoliation and root and crown rot symptoms were collected from pine and cypress nurseries across Fars province at various time intervals. Infected tissues were washed and cultured on acidified potato dextrose agar (PDA) and corn meal agar (CMA) amended with Delvocide and ampicillin, with and without surface sterilization, respectively, and incubated at 25°C for 3–5 days. Decaying seeds of all plant species were also collected from nursery seed stocks and cultured on PDA. Hyphal tip isolates were used for further studies. Species of Phytophthora, Pythium, Rhizoctonia and Fusarium were isolated from symptomatic seedlings of pine and cypress at different times during the growing season. Pathogenicity of isolates of Phytophthora, Pythium and Rhizoctonia was confirmed on seedlings of all plant species, whereas Fusarium sp. isolated from the seeds of Tehran pine was only pathogenic on seedlings of Tehran pine and Arizona cypress. Virulent isolates identified as Phytophthora nicotiana var. parasitica, Pythium ultimum, Pythium paroecandrum, Rhizoctonia solani and binucleate Rhizoctonia‐like fungus caused root and crown rot of pine and cypress seedlings. Pathogenic isolates of Fusarium identified as Fusarium proliferatum also caused pre‐emergence seed decay of pine and cypress. Isolates of Phytophthora and Pythium were the most virulent of these fungal isolates.