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.     

危险性林木新病原康沃尔疫霉对我国的风险分析

从传入和定殖可能性两方面分析了康沃尔疫霉(Phytophthora kernoviae)的风险性,并利用MAXENT生态位模型预测了其在我国的潜在适生区。结果表明:康沃尔疫霉(P.kernoviae)传入我国及其在我国南方沿海地区定殖具有高风险性,主要包括海南、广西、广东、福建、云南、台湾等省区,尤其花卉进出口基地云南省;上述地区应加强康沃尔疫霉(P.kernoviae)的检疫工作,实行严格的风险管理。     

Phytophthora diversity and the population structure of Phytophthora ramorum in Swiss ornamental nurseries

Invasive oomycete pathogens have been causing significant damage to native ecosystems worldwide for over a century. A recent well‐known example is Phytophthora ramorum, the causal agent of sudden oak death, which emerged in the 1990s in Europe and North America. In Europe, this pathogen is mainly restricted to woody ornamentals in nurseries and public greens, while severe outbreaks in the wild have only been reported in the UK. This study presents the results of the P. ramorum survey conducted in Swiss nurseries between 2003 and 2011. In all 120 nurseries subjected to the plant passport system, the main P. ramorum hosts were visually checked for above ground infections. Phytophthora species were isolated from tissue showing symptoms and identified on the basis of the morphological features of the cultures and sequencing of the ribosomal ITS region. Phytophthora was detected on 125 plants (66 Viburnum, 58 Rhododendron and one Pieris). Phytophthora ramorum was the most frequent species (59·2% of the plants), followed by P. plurivora, P. cactorum, P. citrophthora, P. cinnamomi, P. cactorum/P. hedraiandra, P. multivora and P. taxon PgChlamydo. The highest incidence of P. ramorum was observed on Viburnum × bodnantense. Microsatellite genotyping showed that the Swiss P. ramorum population is highly clonal and consists of seven genotypes (five previously reported in Europe, two new), all belonging to the European EU1 clonal lineage. It can therefore be assumed that P. ramorum entered Switzerland through nursery trade. Despite sanitation measures, repeated P. ramorum infections have been recorded in seven nurseries, suggesting either reintroduction or unsuccessful eradication efforts.     

Eradication of plant pathogens and nematodes during composting: a review

The effects of temperature–time combinations and other sanitizing factors during composting on 64 plant pathogenic fungi, plasmodiophoromycetes, oomycetes, bacteria, viruses and nematodes were reviewed. In most cases pathogen survival was determined by bioassays of unknown sensitivity and minimum detection limits of 5% infection or more. For 33 out of 38 fungal and oomycete pathogens, all seven bacterial pathogens and nine nematodes, and three out of nine plant viruses, a peak temperature of 64–70°C and duration of 21 days, were sufficient to reduce numbers to below the detection limits of the tests used. Shorter periods and/or lower temperatures than those quoted in these tests may be satisfactory for eradication, but they were not always examined in detail in composting systems. Plasmodiophora brassicae (clubroot of Brassica spp.), Fusarium oxysporum f.sp. lycopersici (tomato wilt) and Macrophomina phaseolina (dry root rot) were more temperature-tolerant, as they survived a peak compost temperature of at least 62°C (maximum 74°C) and a composting duration of 21 days. Synchytrium endobioticum (potato wart disease) survived in water at 60°C for 2 h, but was not examined in compost. For Tobacco mosaic virus (TMV), peak compost temperatures in excess of 68°C and composting for longer than 20 days were needed to reduce numbers below detection limits. However, TMV and Tomato mosaic virus (TomMV) were inactivated over time in compost, even at temperatures below 50°C. Temperatures in excess of 60°C were achieved in different composting systems, with a wide range of organic feedstocks. The potential survival of plant pathogens in cooler zones of compost, particularly in systems where the compost is not turned, has not been quantified. This may be an important risk factor in composting plant wastes.     

Susceptibility of Iberian trees to Phytophthora ramorum and P. cinnamomi

The capacity of Phytophthora ramorum to colonize the inner bark of 18 native and two exotic tree species from the Iberian Peninsula was tested. Living logs were wound-inoculated in a growth chamber with three isolates belonging to the EU1 and two to the NA1 clonal lineages of P. ramorum . Most of the Quercus species ranked as highly susceptible in experiments carried out in summer, with mean lesion areas over 100 cm² in Q. pubescens , Q. pyrenaica , Q. faginea and Q. suber and as large as 273 cm² in Q. canariensis , ca . 40 days after inoculation. Quercus ilex ranked as moderately susceptible to P. ramorum , forming lesions up to 133 cm² (average 17·2 cm²). Pinus halepensis and P. pinea were highly susceptible, exhibiting long, narrow lesions; but three other pine species, P. pinaster , P. nigra and P. sylvestris , were resistant to slightly susceptible. No significant difference in aggressiveness was found between the isolates of P. ramorum . In addition, there was evidence of genetic variation in susceptibility within host populations, and of significant seasonal variation in host susceptibility in some Quercus species. The results suggest a high risk of some Iberian oaks to P. ramorum , especially in forest ecosystems in southwestern Spain, where relict populations of Q. canariensis grow amongst susceptible understory species such as Rhododendron ponticum and Viburnum tinus . One isolate of P. cinnamomi used as positive control in all the inoculations was also highly aggressive to Iberian oaks and Eucalyptus dalrympleana .     

Induced antimicrobial activity in heat-treated woodchips inhibits the activity of the invasive plant pathogen Phytophthora ramorum

The antimicrobial activity of heat-treated woodchips of three woody host species against the invasive oomycete plant pathogen Phytophthora ramorum was investigated to assess the potential of heated woodchips to suppress disease. Results demonstrated that heat-treated woodchips of pine (Pinus sylvestris), Japanese larch (Larix kaempferi) and rhododendron (Rhododendron ponticum) inhibited recovery of P. ramorum spores and mycelium compared with similar material that had only been air-dried. Effects were most evident with pine and larch; inhibition was maintained even when larch woodchips were diluted with soil. In vitro assays using methanol crude extracts from woodchips of the three species showed they all had an inhibitory effect on P. ramorum zoospores and reduced chlamydospore germination compared with air-dried wood extracts. Chemical analysis of the extracts revealed several induced compounds were present but in different concentrations for each species. Coniferaldehyde was the most active inhibitor against spores and mycelium, while the dominant resin acids, dehydroabietic and abietic acid, decreased the minimum inhibitory concentration of phenolic compounds tested against P. ramorum but were ineffective when used alone. An array of compounds, including dehydroabietic acid, methyl abietate, α-pinene and 3-carene, occurred at elevated levels in the living tissue of Japanese larch bark attacked by P. ramorum. These compounds may be part of the induced resistance response of larch to P. ramorum. Results of a field trial using heat-treated and air-dried woodchips were consistent with the crude extract bioassay results, suggesting that heat-treated woody materials have potential to reduce the survival of P. ramorum under natural conditions.     

Infectivity and sporulation potential of Phytophthora kernoviae to select North American native plants

Phytophthora kernoviae exhibits comparable epidemiology to Phytophthora ramorum in invaded UK woodlands. Because both pathogens have an overlapping geographic range in the UK and often concurrently invade the same site, it is speculated that P. kernoviae may also invade North American (NA) forests threatened by P. ramorum, the cause of Sudden Oak Death. This paper addresses the susceptibility of select NA plants to P. kernoviae, including measures of disease incidence and severity on wounded and unwounded foliage. The potential for pathogen transmission and survival was investigated by assessing sporangia and oospore production in infected tissues. Detached leaves of Rhododendron macrophyllum, Rhododendron occidentale and Umbellularia californica, and excised roots of U. californica and R. occidentale were inoculated with P. kernoviae and percent lesion area was determined after 6 days. Leaves were then surface sterilized and misted to stimulate sporulation and after 24 h sporangia production was assessed. The incidence of symptomless infections and sporulation were recorded. All NA native plants tested were susceptible to P. kernoviae and supported sporangia production; roots of U. californica and R. occidentale were both susceptible to P. kernoviae and supported sporangia production. Oospore production was also observed in U. californica roots. The results highlight the vulnerability of select NA native plants to infection by P. kernoviae, suggest that symptomless infections may thwart pathogen detection, and underscore the importance of implementing a proactive and adaptive biosecurity plan.     

Effects of temperature on germination of sporangia,infection and protein secretion by Phytophthora kernoviae

Phytophthora kernoviae is a pathogen on a wide range of plants, but little is known of optimal infection conditions. Rhododendron ponticum leaves were inoculated with six different isolates of P. kernoviae sporangia and incubated at different temperatures from 10 to 28 °C. After 1 week, lesion development and pathogen recovery were only observed from all isolates at 15 and 20 °C and a few isolates at 10 °C. In an experiment with temperatures ranging from 20 to 25 °C, lesion development and pathogen recovery on R. ponticum, Magnolia stellata and Viburnum tinus occurred consistently at 20 and 21 °C, was limited at 22 °C, and did not occur at 23 °C and above. There was no difference in sporangia and zoospore germination at 20–25 °C. In a temperature fluctuation experiment, the necrotic area of inoculated R. ponticum leaves increased with longer incubation at 20 °C and decreased with longer incubation at 24 °C. Crude extracts of secreted proteins from P. kernoviae cultures grown at 20 and 24 °C were compared to determine any effects of temperature on pathogenicity. When spot tested on R. ponticum leaves, crude protein suspensions from cultures grown at 20 °C induced necrosis, while proteins from cultures grown at 24 °C did not. Proteomic analysis confirmed that a 10 kDa protein secreted at both 20 and 24 °C shared sequence homology to the conserved domains of known elicitins of other Phytophthora spp. The protein secreted at 20 °C that was responsible for necrosis has not been identified.     

Inactivation of soil-borne plant pathogens during small-scale composting of crop residues

Samples of heavily infested crop residues were incorporated in static compost heaps (2.5–4.6 m³) of the Indore type. Temperature increased to 50–70°C within 6 days depending on the type of crop residues used and the location within the heap. The heat phase (>40 °C) lasted 2–3 weeks and was followed by a c. 5-months maturation phase (<40 °c).=" among=" the=" 17=" pathogens=" tested,=">Olpidium brassicae and one of the four formae speciales ofFusarium oxysporum that were tested survived composting, but also their inoculum was greatly reduced.Survival during specific phases of composting was studied by incorporation and retrieval of samples at various stages of the process.F. oxysporum f. sp.melonis was completely inactivated andO. brassicae andPlasmodiophora brassicae were almost completely inactivated during the short heat phase. The three pathogens survived the long-lasting maturation phase without loss of viability. Heat evolved during composting was found to be the most important factor involved with sanitation of crop residues. The possible involvement of fungitoxic conversion products and microbial antagonism is discussed.     

Effect of host factors on the susceptibility of Rhododendron to Phytophthora ramorum

Phytophthora ramorum causes sudden oak death (SOD) in western coastal forests of the USA. In Europe, the pathogen is mainly present in the nursery industry, particularly on Rhododendron. Because of the primary role of Rhododendron as a host and potentially as a vector, the effect of Rhododendron host factors on P. ramorum susceptibility and sporulation was investigated. Inoculation methods using either wounded or non‐wounded detached leaves were applied to 59 Rhododendron cultivars and 22 botanical species, replicated in three separate years. All Rhododendron species and cultivars were susceptible when using wounded leaves, but not when using non‐wounded leaves, suggesting a resistance mechanism operating at the level of leaf penetration. Using a regression tree analysis, the cultivars and species were split into four susceptibility classes. Young leaves were more susceptible than mature leaves when wounded, but less susceptible when non‐wounded. This effect was not correlated with leaf hydrophobicity or the number of leaf hairs. The presence or the type of rootstock did not affect the cultivar susceptibility level. Sporangia and chlamydospore production in the leaf lesions varied widely among Rhododendron cultivars and was not correlated with the susceptibility level. The susceptibility to P. ramorum correlated well with the susceptibility to P. citricola and P. hedraiandra × cactorum, suggesting that the resistance mechanisms against these species are non‐specific. Susceptibility to P. kernoviae was low for most cultivars. These findings have implications for detection, spread and disease control, and are therefore important in pest risk assessment.     

接种体密度、土壤水分基质势和土壤温度对辣椒疫病死苗率的影响

 在生长箱内控制条件下分析测定了接种体密度、土壤水分基质势和土壤温度对辣椒疫病死苗率的影响。结果表明,在每克干土中接种1个辣椒疫霉菌孢子囊就能造成侵染,引致辣椒死苗,随着接种体密度升高死苗率增大,直至接种体孢子囊密度达到50~80个/g干土时死苗率达到最高。土壤温度和土壤水分状况是决定辣椒疫病死苗率的重要因子,病菌侵染的最适土温是22~28℃,土壤水分接近饱和,即土壤水分基质势(Ψ_m值)为0时最容易侵染发病,土壤过于干燥和过饱和都不利于侵染发病。辣椒疫病死苗率与土壤温度、土壤水分基质势及其互作之间可用数学模式描述。     

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.     

大豆疫霉卵孢子致死温度的测定

大豆疫霉根腐病是由大豆疫霉菌(Phytophthora sojae)侵染引起的、危害极其严重的最具毁灭性的大豆病害之一.病原菌以卵孢子在土壤中越冬,在条件适宜时导致下一年大豆发生根茎腐烂.病原菌卵孢子也可以存在于患病种皮内,但其在病害循环中的作用还不清楚.卵孢子是大豆疫霉的主要传播方式.本研究中对大豆疫霉卵孢子的致死条件进行了筛选.结果表明,使用60℃湿热处理大豆疫霉卵孢子20min或80℃干热处理大豆疫霉卵孢子20min,其萌发率为0,且死亡率为100%.     

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.     

Pathogenicity and infectivity of Phytophthora ramorum vary depending on host species,infected plant part,inoculum potential,pathogen genotype,and temperature

A total of 25 ornamental plant species representing 10 families were inoculated using three genotypes, each representing one of the genetic lineages NA1, NA2, and EU1 of the pathogen Phytophthora ramorum. Leaves were inoculated using suspensions with two zoospore concentrations and exposure at three temperatures, while stems were inoculated using agar plugs colonized by mycelia. Susceptibility was determined by measuring either the success of pathogen reisolation or lesion length caused by the pathogen. Infectivity was determined by counting sporangia in washes of inoculated leaves or stems. Results from all three pathogen genotypes combined were used to rank each of the 25 plant species for susceptibility and infectivity, while pooled results per genotype from all 25 hosts combined were employed for a preliminary comparison of pathogenicity and infectivity among genotypes. Statistical analyses showed that leaf results were affected by the concentration of zoospores, temperature, plant host, pathogen genotype, and by the interaction between host and pathogen genotype. Stem results were mostly affected by host and by the interaction between host and pathogen genotype. Hosts ranked differently when looking at the various parameters, and differences in rankings were also significant when comparing stem and leaf results. Differences were identified among the 25 hosts and the three pathogen genotypes for all parameters: results can be used for decision-making regarding regulations or selection of plants to be grown where infestations by P. ramorum are an issue.     

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.     

Control of Phytophthora species in plant stock for habitat restoration through best management practices

Emergent plant pathogens represent one of the most significant threats to biodiversity, and exotic Phytophthora species have recently emerged as a serious problem in restored habitats in California and in nurseries producing the plant stock. It is hypothesized that ‘best management practices’ prescribed through a Phytophthora Prevention Programme (PPP) could be useful in minimizing phytophthora disease incidence. To understand the magnitude of the problem and the efficacy of the PPP, plants in restoration nurseries were evaluated for (i) the Phytophthora species assemblage present in the absence of the PPP, and (ii) the effectiveness of the PPP to reduce them. Sampling included 203 plants grown in the absence of the PPP, and 294 grown implementing the PPP. Only samples collected in the absence of the PPP were Phytophthora-positive, and cumulatively yielded 55 isolates from 13 different taxa, including 1 putative interspecific hybrid genotype. There were 21 novel Phytophthora–plant species combinations. The most common Phytophthora species was P. cactorum. Four plant species had the highest disease incidence, namely: Diplacus aurantiacus (50 ± 11.2%), Heteromeles arbutifolia (33 ± 9.6%), Ceanothus thyrsiflorus (30 ± 8.4%), and Frangula californica (30 ± 8.4%). Disease incidence in nurseries after the implementation of the PPP dropped to zero (P < 0.001), and was unaffected to any significant degree by nursery differences, or plant species tested. This study identifies a large number of novel ‘plant species × Phytophthora species’ combinations, and provides for the first time strong evidence that the PPP significantly reduced Phytophthora in plant stock for habitat restoration.