Fitness characteristics of the European lineages of Phytophthora ramorum

As an introduced pathogen, Phytophthora ramorum exists as four near-clonal evolutionary lineages, of which only EU1 and EU2 are established in the UK. EU1 has become widespread since the first findings in 2002 whereas EU2, detected in 2011, has a more limited distribution. Both lineages are epidemic in plantation-grown larch, sporulating asexually on needles, but also causing heavy dieback and mortality. To understand whether EU1 and EU2 pose different threats to forest health, we compared their growth characteristics on agar, pathogenicity on several hosts, and sporulation on Japanese larch needles. When pathogenicity was evaluated by measuring colonization at 20 °C in mature bark (phloem) of Japanese and European larch (Larix kaempferi and L. decidua), English oak (Quercus robur), and beech (Fagus sylvatica), Japanese larch was the most susceptible and oak the least susceptible. On average, EU2 isolates produced significantly larger lesions than EU1 isolates in Japanese larch and oak although not in the other hosts. With tests using young saplings of Japanese and European larch, damaging bark lesions formed at both 10 °C and 20 °C, but EU2 was significantly more pathogenic at 20 °C on both hosts compared with EU1. In contrast, both lineages caused similar amounts of necrosis on inoculated leaves of rhododendron (Rhododendron ponticum). Moreover, EU2 isolates usually sporulated less abundantly on larch needles compared with EU1 isolates, suggesting a trade-off in pathogenicity and sporulation between lineages. As EU2 tends to have smaller sporangia than EU1, this could also reduce the inoculum potential of EU2.     

Potential susceptibility of Australian native plant species to branch dieback and bole canker diseases caused by Phytophthora ramorum

Susceptibility to branch dieback caused by Phytophthora ramorum was tested using a detached branch assay for 66 Australian native plant species sourced from established gardens and arboreta in California. Six of these species were further tested for their susceptibility to bole cankers caused by P. ramorum using a sealed log assay. Isopogon formosus and Eucalyptus denticulata were identified as potentially highly susceptible Australian branch dieback hosts. Thirteen potentially tolerant Australian host species included Banksia attenuata, B. marginata, E. haemastoma, E. regnans, Pittosporum undulatum and Billardiera heterophylla. Eucalyptus regnans was identified as a potentially highly susceptible bole canker host, while E. diversicolor and E. viminalis were considered potentially tolerant species to bole cankers caused by P. ramorum. Phytophthora ramorum was able to infect all 66 species, as confirmed by reisolation. These results extend the known potential host range for P. ramorum, confirm it as a possible threat to Australian plant industries and ecosystems and highlight additional associated hosts that are important in the global horticultural trade, native forests and plantation forestry.     

Characterizing the variation in aggressiveness and sporulation of the NA1 and EU1 lineages of Phytophthora ramorum in Oregon

Phytophthora ramorum, the cause of sudden oak death, is an invasive pathogen present in parts of coastal California and south-western Oregon forests. The majority of these forest infestations have been caused by the NA1 clonal lineage. In 2015, the EU1 lineage of P. ramorum was isolated from a tanoak (Notholithocarpus densiflorus) tree located in a mixed-conifer forest of Curry County, Oregon. In order to evaluate the threat to Oregon forests of the EU1 lineage relative to the established NA1 lineage, a series of experiments was conducted comparing aggressiveness and sporulation of NA1 and EU1 isolates on logs and seedlings in the growth chamber and forest. There was no significant difference in lesion size on logs inoculated with NA1 and EU1 isolates for any of the tree species tested. Across all seedling experiments differences among isolates within lineage, in terms of both aggressiveness and sporulation, were more commonly observed than differences among lineages. Site to site variation in tanoak sporulation, as measured by rain bucket baiting, appears to be correlated with the number of P. ramorum-positive seedlings detected at each site.     

Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P. ramorum and P. kernoviae at the point of inspection

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.     

Survival of Phytophthora ramorum in Rhododendron root balls and in rootless substrates

This study assesses the survival of Phytophthora ramorum in the root ball of Rhododendron container plants as well as in different rootless forest substrates and a horticultural potting medium. Following inoculation of the root balls, the aboveground plant parts stayed symptomless, whilst the pathogen could be recovered with a novel non‐destructive baiting assay from the root balls until at least 8 months post‐inoculation. Plating of surface‐sterilized roots and direct microscopic analysis confirmed the presence of P. ramorum in the roots. Phytophthora ramorum could also be baited from the root balls of symptomless Rhododendron plants from commercial nurseries, even 2 years after acquisition. Survival of P. ramorum in rootless media was assessed after burying disks of infected leaf material below the soil surface in columns filled with four different undisturbed forest substrates or a potting medium, and incubated at an outdoor quarantine facility. Phytophthora ramorum could be recovered at least 33 months after burial from all substrates, with a significant increase in recovery after the winter period. These data suggest the possibility for long‐term symptomless presence of P. ramorum in root balls of commercial Rhododendron plants as well as survival in potting medium and different forest substrates under western European climate conditions. Symptomless presence in root balls can contribute to latent spread of this pathogen between nurseries. The novel baiting test, being non‐destructive, simple and applicable to a relatively large number of plants, can offer a valuable tool to test plants for the presence of Phytophthora species in root balls.     

Diagnostic sensitivity and specificity of different methods used by two laboratories for the detection of Phytophthora ramorum on multiple natural hosts

Five detection methods were comparatively tested on putative Phytophthora ramorum field samples from 41 wild plant species. The tested methods included two culture‐based assays, a DAS‐ELISA‐based polyclonal assay, a nested PCR‐based assay, and a TaqMan real‐time PCR assay. Diagnostic values including sensitivity, specificity, positive predictive value and negative predictive value were calculated for each method. The effects of host species, seasonality and host location were analysed and compared between two laboratories. Significant effects of season, host species and laboratory were detected. It is concluded that a combination of either culturing and molecular diagnosis or of two molecular assays is the most promising approach to diagnose this pathogen. Based on the results of this and other studies, diagnosis should occur as much as possible during wet and warm periods favourable to the pathogen, and proficiency tests should be performed to compare results obtained with molecular approaches in different laboratories. Furthermore, length of time lapsed between sample collection and processing strongly affected the diagnostic sensitivity of culture‐based methods, and therefore needs to be taken into account when comparing results from different laboratories.     

Efficacy of phosphonic acid, metalaxyl-M and copper hydroxide against Phytophthora ramorum in vitro and in planta

The ability of metalaxyl-M, phosphonic acid in the form of phosphonate, and copper hydroxide to inhibit different stages in the life cycle of Phytophthora ramorum , the causal agent of sudden oak death (SOD), was tested in vitro using 12 isolates from the North American forest lineage. In addition, experiments were conducted in planta to study the ability of phosphonic acid injections and metalaxyl-M drenches to control pathogen growth on saplings of California coast live oak ( Quercus agrifolia ), and of copper hydroxide foliar sprays to control infection of California bay laurel ( Umbellularia californica ) leaves. Phytophthora ramorum was only moderately sensitive to phosphonic acid in vitro , but was highly sensitive to copper hydroxide. In planta experiments indicated the broad efficacy of phosphonic acid injections and of copper hydroxide sprays in preventing growth of P. ramorum in oaks and bay laurels, respectively. Finally, although metalaxyl-M was effective in vitro , drenches of potted oak trees using this active ingredient were largely ineffective in reducing the growth rate of the pathogen in planta .     

Genetic diversity,sensitivity to phenylamide fungicides and aggressiveness of Phytophthora ramorum on Camellia,Rhododendron and Viburnum plants in Spain

Phytophthora ramorum has been detected in official plant health surveys on Rhododendron, Viburnum and Camellia in ornamental nurseries in northern Spain since 2003. A collection of 94 isolates of P. ramorum was obtained from 2003 to 2008 from plants with symptoms at different geographical locations. Isolates were identified based on morphology and sequence of the rDNA ITS region. Mating type, genetic variation, sensitivity to phenylamide fungicides and aggressiveness of these isolates were determined. All isolates belonged to the A1 mating type, ruling out the possibility of genetic recombination. Seven microsatellite markers were used to study genetic diversity; three out of the seven microsatellite markers were polymorphic within the Spanish population of P. ramorum. This study confirms that all Spanish isolates of P. ramorum belonged to the EU1 lineage. Twelve intralineage genotypes were detected, five that are unique to Spain (EU1MG38, EU1MG41, EU1MG37, EU1MG39 and EU1MG40) and seven that are also present in at least one other European country (EU1MG1, EU1MG29, EU1MG22, EU1MG13, EU1MG2, EU1MG18 and EU1MG26). Genotypes EU1MG37, EU1MG39 and EU1MG40 were isolated from Rhododendron from one region; EU1MG38 and EU1MG41 were isolated from Camellia from two different regions. Isolates of genotype EU1MG38 were resistant to metalaxyl and mefenoxam. The level of genetic diversity within the Spanish population of P. ramorum is limited and indicates a relatively recent clonal expansion.     

In vitro leaf inoculation studies as an indication of tree foliage susceptibility to Phytophthora ramorum in the UK

Leaves of 11 coniferous and 23 broad-leaved tree species important to UK forestry were tested for their susceptibility to the quarantine pathogen Phytophthora ramorum using a detached leaf assay. Two European and two USA isolates were used. Wounded and unwounded leaves were dipped in zoospore suspensions during summer; conifers were also tested in winter. Successful infection of tissue and amount of necrosis were assessed. Highly susceptible broad-leaved hosts included Aesculus hippocastanum , Fraxinus excelsior , Quercus ilex , Ulmus procera and, to a lesser extent, Castanea sativa , Q. cerris and Q. petraea , together with Umbellularia californica and rhododendrons. Acer pseudoplatanus , Alnus glutinosa , Carpinus betulus , Corylus avellana , Fagus sylvatica , Prunus avium , Q. robur , Q. rubra and Q. suber showed consistently low susceptibility. Conifer species including Abies procera , Picea abies , P. sitchensis , Pseudotsuga menziesii , Sequoia sempervirens and Tsuga heterophylla were also susceptible. Pseudotsuga menziesii and A. procera were severely affected. Pinus contorta , P. nigra var. maritima and P. sylvestris were virtually resistant, while Taxus baccata was only slightly affected. Increased necrosis was apparent on leaves that were wounded prior to inoculation. These results extend the known range of trees that P. ramorum is able to attack and confirm its relative host-nonspecificity.     

Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis

Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification of DNA by PCR has revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real-time PCR detection assays targeting DNA of all four Phytophthora ramorum lineages, and a closely related species, P. lateralis. Nine assays were generated: three targeting DNA of all P. ramorum lineages, one for each lineage of P. ramorum, one for P. lateralis and one targeting DNA of P. ramorum and P. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection of DNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non-target species.     

Phytophthora species in oak ecosystems in Turkey and their association with declining oak trees

From 1999 to 2001, a survey on the occurrence of Phytophthora spp. in the rhizosphere soil of healthy and declining oak trees was conducted in 51 oak stands in Turkey. Seven Phytophthora spp. were recovered from six out of the nine oak species sampled: P .  cinnamomi , P .  citricola , P .  cryptogea , P .  gonapodyides , P .  quercina , Phytophthora sp. 1 and Phytophthora sp. 2. The most frequently isolated species, P .  quercina , was very common on slopes susceptible to drought. It occurred in four different climatic zones and on six Quercus spp., suggesting that it is native to oaks. The second most common species, P .  citricola , was separated into three subgroups: type C was recovered only in Anatolia, whereas A and B occurred only in the European part of Turkey. Phytophthora cinnamomi was recovered at one site only, and may not be involved in oak decline in Turkey. The other four species were recovered sporadically. On affected sites there was a significant association between deteriorating crown status and the presence of Phytophthora spp., particularly P .  quercina . The occurrence of Phytophthora species was significantly influenced by soil pH. Stem inoculation tests on oak seedlings revealed that Q .  petraea was the most susceptible species.     

Early survival of Quercus ilex subspecies from different populations after infections and co‐infections by multiple Phytophthora species

Forests in Europe are threatened by increased diversity of Phytophthora species, but effects on trees of simultaneous infections by Phytophthora and ecological consequences of their coexistence are unknown. This study explored variation in early survival of Quercus ilex to Phytophthora infections and assessed interactions between Phytophthora species when trees were co‐infected. Three Phytophthora species (P. cinnamomi, P. gonapodyides and P. quercina), seeds from 16 populations of Q. ilex (ballota and ilex subspecies) and two infection times were used as sources of variation in two experiments. The influence of Phytophthora species, Q. ilex subspecies and populations on plant germination and survival were analysed using generalized linear mixed models and survival analysis techniques. Germination rates were not influenced by Phytophthora spp. (P = 0.194) but by the subspecies and populations of Q. ilex (P < 0.001). In Phytophthora‐infested soils, Q. ilex subsp. ilex germinated at higher rates than Q. ilex subsp. ballota. Plant survival was strongly influenced by Phytophthora species (P < 0.001), not by the subspecies and populations of Q. ilex. Seedling mortality was reduced and delayed if a less virulent Phytophthora species infected plants prior to infection by a more virulent Phytophthora species. The results help to explain oak decline syndrome and the lack of natural and artificial regeneration of Q. ilex forests. Lack of interspecific variability of early survival to Phytophthora spp. discourages direct sowing for artificial reforestation programmes. Large, thick seeds, giving plants rapid growth, are advantageous traits when soils are infested with Phytophthora spp.     

Sporulation potential,symptom expression and detection of Phytophthora ramorum on larch needles and other foliar hosts

Phytophthora ramorum has caused extensive dieback and mortality of commercially grown Japanese larch (Larix kaempferi) in many parts of the UK, as infected foliage generates spores that then cause bark lesions and girdling cankers on trees. Following inoculation, individual needles of Japanese, European (L. decidua) and hybrid (L. × eurolepis) larch infected with P. ramorum can produce thousands of sporangia. Mean numbers of sporangia ranged from 806 to 1778 per cm² (hybrid larch and Japanese larch, respectively), surpassing mean sporulation levels on foliar hosts previously associated with P. ramorum outbreaks in Britain, namely Rhododendron ponticum, Castanea sativa and Vaccinium myrtillus. Sporulation on larch even exceeded that of California bay laurel (Umbellularia californica), which drives the sudden oak death epidemic in California. Inoculation of foliage selected at different times of year revealed that foliage age significantly affected sporulation levels, but this varied with host species. However, symptom development and sporulation were often not correlated. Symptoms on larch were frequently insignificant or even absent at certain times of year, with sometimes the only evidence of infection being the emergence of sporangia from needles, without any sign of discolouration or necrosis. Plating infected but symptomless needles onto Phytophthora selective medium also often failed to yield the pathogen. Symptomless infection of larch needles apparently occurs, but is only detectable with microscopy. More generally, it is suggested that diagnosis of Phytophthora infection in conifers is often underestimated due to isolation difficulties and delayed symptom expression.     

Histopathology of infection and colonization of Quercus ilex fine roots by Phytophthora cinnamomi

Quercus ilex is one of the European forest species most susceptible to root rot caused by the oomycete Phytophthora cinnamomi. This disease contributes to holm oak decline, a particularly serious problem in the ‘dehesas’ ecosystem of the southwestern Iberian Peninsula. This work describes the host–pathogen interaction of Q. ilex and P. cinnamomi, using new infection indices at the tissue level. Fine roots of 6‐month‐old saplings inoculated with P. cinnamomi were examined by light microscopy and a random pool of images was analysed in order to calculate different indices based on the measured area of pathogen structures. In the early stages of invasion, P. cinnamomi colonizes the apoplast and penetrates cortical cells with somatic structures. On reaching the parenchymatous tissues of the central cylinder, the pathogen develops different reproductive and survival structures inside the cells and then expands through the vascular system of the root. Some host responses were identified, such as cell wall thickening, accumulation of phenolic compounds in the middle lamella of sclerenchyma tissues, and mucilage secretion blocking vascular cells. New insights into the behaviour of P. cinnamomi inside fine roots are described. Host responses fail due to rapid expansion of the pathogen and a change in its behaviour from biotrophic to necrotrophic.     

Potential for eradication of the exotic plant pathogens Phytophthora kernoviae and Phytophthora ramorum during composting

Temperature and exposure time effects on Phytophthora kernoviae and Phytophthora ramorum viability were examined in flasks of compost and in a large‐scale composting system containing plant waste. Cellophane, rhododendron leaf and peat‐based inoculum of P. kernoviae and P. ramorum isolates were used in flasks; naturally infected leaves were inserted into a large‐scale system. Exposures of 5 and 10 days respectively at a mean temperature of 35°C in flask and large‐scale composts reduced P. kernoviae and P. ramorum inocula to below detection limits using semi‐selective culturing. Although P. ramorum was undetectable after a 1‐day exposure of inoculum to compost at 40°C in flasks, it survived on leaves exposed to a mean temperature of 40·9°C for 5 days in a large‐scale composting system. No survival of P. ramorum was detected after exposure of infected leaves for 5 days to a mean temperature of ≥41·9°C (32·8°C for P. kernoviae) or for 10 days at ≥31·8°C (25·9°C for Phytophthora pseudosyringae on infected bilberry stems) in large‐scale systems. Fitted survival probabilities of P. ramorum on infected leaves exposed in a large‐scale system for 5 days at 45°C or for 10 days at 35°C were <3%, for an average initial infection level of leaves of 59·2%. RNA quantification to measure viability was shown to be unreliable in environments that favour RNA preservation: high levels of ITS1 RNA were recovered from P. kernoviae‐ and P. ramorum‐infected leaves exposed to composting plant wastes at >53°C, when all culture results were negative.     

Effect of electrical conductivity on survival of Phytophthora alni,P. kernoviae and P. ramorum in a simulated aquatic environment

This study investigated survival of the pathogens Phytophthora ramorum, P. alni and P. kernoviae as zoospores or sporangia in response to an important water quality parameter, electrical conductivity (EC), at its range in irrigation water reservoirs and irrigated cropping systems. Experiments with different strengths of Hoagland’s solution showed that all three pathogens survived at a broad range of EC levels for at least 3 days and were stimulated to grow and sporulate at ECs > 1·89 dS m^?1. Recovery of initial populations after a 14‐day exposure was over 20% for P. alni subsp. alni and P. kernoviae, and 61·3% and 130% for zoospores and sporangia of P. ramorum, respectively. Zoospore survival of these pathogens at ECs < 0·41 dS m^?1 was poor, barely beyond 3 days in pure water; only 0·3% (P. alni), 2·9% (P. kernoviae) and 15·1% (P. ramorum) of the initial population survived after 14 days at EC = 0·21 dS m^?1. The variation in rates of survival at different EC levels suggests that these pathogens survive better in cropping systems than in irrigation water. Containment of run‐off and reduction in EC levels may therefore be non‐chemical control options to reduce the risk of pathogen spread through natural waterways and irrigation systems.     

Phytophthora kernoviae and P. ramorum: host susceptibility and sporulation potential on foliage of susceptible trees1

Phytophthora kernoviae and P. ramorum are introduced, invasive pathogens in the UK. Both species are adapted for aerial dispersion and have a wide host range, many of which are common to both pathogens. The diseases they cause are foliar necrosis and shoot tip dieback on both tree and ornamental hosts, and bleeding cankers on tree hosts. Inoculum is produced on infected foliage but not on bleeding cankers in both cases. Proactive measures to prevent disease spread and to evaluate the risks posed by these pathogens are being undertaken. Amongst others, these include using the detached leaf assay to get an indication of tree foliage susceptibility, and inoculating wounded stems of saplings to get an idea of under‐canopy sapling and nursery tree susceptibility. The sporulation potential on selected susceptible hosts was assessed, and finally, surveys which are still ongoing were carried out in south‐west England. Results of this work are presented and discussed.     

Seasonal changes in susceptibility of Quercus suber to Botryosphaeria stevensii and Phytophthora cinnamomi

Monthly inoculations of both intact plants and excised shoots of Quercus suber with the pathogenic species Botryosphaeria stevensii and Phytophthora cinnamomi were performed to investigate seasonal changes in susceptibility of this forest tree species in relation to environmental parameters and plant water status. Infection symptoms were mainly detected on seedlings inoculated from spring to autumn (April through October) with either pathogen. Mean canker sizes also showed a seasonal pattern, the higher values being recorded in the same period as above. Lesion lengths were significantly ( P  < 0·001) related to environmental minimum temperature. Mean daily minimum temperatures within the range of 5–12°C clearly inhibited lesion development of P. cinnamomi , whereas B. stevensii showed a less pronounced decrease in canker expansion at the same temperature range. In excised shoots of Q. suber inoculated monthly with B. stevensii , a negative linear relationship was found between the studied range of plant relative water content (81–91%) and canker length. In contrast, the lesions caused by P. cinnamomi were not significantly ( P  = 0·32) related to any seasonal change in water content. Some control measures for the diseases caused by both pathogens are discussed on the basis of the seasonal changes in host susceptibility observed in this study.     

Sudden oak death: geographic risk estimates and predictions of origins

Ecological niche modelling techniques were applied to address the questions of the origins and potential geographic extent of sudden oak death, caused by the pathogen Phytophthora ramorum . Based on an ecological niche model derived from the phytopathogen's California distribution and distributions of potential host species, it was determined that the disease has high potential to colonize the southeastern United States, and that its likely source area is eastern Asia.