First Report of the European Lineage of Phytophthora ramorum on Viburnum and Osmanthus spp. in a California Nursery

Phytophthora ramorum S. Werres & A.W.A.M. de Cock is the causal agent of sudden oak death in California and Oregon forests and ramorum blight on a broad range of host species in wildlands and nurseries. It is thought to be an introduced pathogen and only three clonal lineages are known (3). The North American lineage (lineage NA1, mating type A2) is responsible for infections in California and Oregon forests. The European lineage (lineage EU1, predominantly A1) is responsible for infections in Europe, but has also been found in nurseries in Oregon and Washington. A third lineage (NA2) has only been isolated in a few instances from nurseries in Washington and California. In June 2006, P. ramorum was isolated from diseased Viburnum tinus, Osmanthus heterophyllus, and O. fragrans cultivars from a Humboldt County retail nursery in northern California. We genotyped isolates and placed them into clonal lineages using microsatellite markers developed for P. ramorum (3,4). Genomic DNA was extracted from mycelia with the FastDNA SPIN kit (Q-Biogene, Morgan, Irvine, CA). Primers used were PrMS6, Pr9C3, PrMS39, PrMS43a, PrMS43b, and PrMS45 (3) and 18, 64, and 82 (4). We sized fluorescently labeled amplicons using capillary electrophoresis (3100 Avant Genetic Analyzer, Applied Biosystems, Foster City, CA). Isolate genotypes were compared with control isolates of known clonal lineage, including BBA9/95 (EU1), Pr102 (NA1), and WSDA3765 (NA2). Three of four isolates belonged to genotype EU1. The fourth isolate, obtained from O. fragrans, belonged to genotype NA1. We repeated genotyping on independent genomic DNA extractions and obtained identical results. Two EU1 isolates and the single NA1 isolate were tested for mating type (1) and found to be of A1, A1, and A2 mating type, respectively. The coexistence of A1 and A2 mating types in the same retail nursery suggests the potential for sexual reproduction, as is the case in P. infestans where clonal and sexual populations exist (2), although to date, sexual reproduction in nature has not been documented in P. ramorum. The California retail nursery infestation highlights the risks associated with the unintentional transport of host nursery stock infested with P. ramorum. References: (1) C. M. Brasier and S. Kirk. Mycol. Res. 108:823, 2004. (2) N. J. Grünwald and W. G. Flier. Ann. Rev. Phytopathol. 43:171, 2005. (3) K. Ivors et al. Mol. Ecol. 15:1493, 2006. (4) S. Prospero et al. Mol. Ecol. 16:2958, 2007.

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First reports of Phytophthora ramorum clonal lineages NA1 and EU1 causing Sudden Oak Death on tanoaks in Del Norte County, California

Plant Disease ◽

10.1094/pdis-12-20-2633-pdn ◽

2021 ◽

Author(s):

Matteo Garbelotto ◽

Francesco Dovana ◽

Douglas Schmidt ◽

Cameron Chee ◽

Chris Lee ◽

...

Keyword(s):

San Francisco ◽

Perfect Match ◽

Ornamental Plants ◽

Forest Health ◽

Phytophthora Ramorum ◽

Sudden Oak Death ◽

Clonal Lineage ◽

Humboldt County ◽

The North ◽

Cox 1

A year of forest health surveys has led to the first detection of Phytophthora ramorum in Del Norte County followed by the first wildland detection of the EU1 clonal lineage (Grunwald et al. 2009) of this pathogen in California. In July 2019, leaves were sampled from two tanoaks (Notholithocarpus densiflorus) and 16 California bay laurels (Umbellularia californica) in Jedediah Smith State Park in Del Norte County, the northernmost coastal County of California. Leaves displayed lesions normally associated with Sudden Oak Death (SOD) caused by P. ramorum and were discovered during the citizen science-based survey known as SOD Blitz (Meentemeyer et al. 2015). Samples were surface sterilized using 75% Ethanol and plated on PARPH-V8 agar (Jeffers and Martin 1986). After plating, DNA was extracted and amplified using two P. ramorum-specific assays (Hayden et al. 2006, Kroon et al. 2004). Leaves from two tanoaks exhibiting twig die-back had typical SOD lesions along the midvein, gave positive PCR results and yielded cultures with colony morphology, sporangia and chlamydospores typical of the NA1 lineage of P. ramorum originally isolated in California from tanoaks and coast live oaks (Quercus agrifolia) (Rizzo et al. 2002). The ITS locus and a portion of the Cox-1 locus were sequenced from DNA extracts of each culture using primers DC6-ITS4 (Bonants et al. 2004) and COXF4N-COXR4N (Kroon et al. 2004), respectively. ITS sequences (GB MN540639-40) were typical of P. ramorum and Cox-1 sequences (GB MN540142-3) perfectly matched the Cox-1 sequence of the NA1 lineage (GB DQ832718) (Kroon et al. 2004). Microsatellite alleles were generated as described in Croucher et al. (2013) for the two Del Norte cultures and for eight P. ramorum cultures, representative of the four main multilocus genotypes (MLGs) present in California, namely c1 (Santa Cruz/Commercial Nurseries), c3 (San Francisco Bay Area), c2 (Monterey County), and c4 (Humboldt County) (Croucher et al. 2013). The two Del Norte MLGs were identical to one another and most similar to MLG c1, with a single repeat difference at a single locus. SSR results suggest the inoculum source may not be from Humboldt County, neighboring to the South, but from a yet unidentified outbreak, possibly associated with ornamental plants. Jedediah Smith State Park was surveyed for 12 months following the initial detection, however the pathogen has yet to be re-isolated in that location. In July 2020, SOD symptomatic leaves from two tanoak trees exhibiting twig cankers were collected 8 Km north of Jedediah Smith State Park, where three additional tanoak trees displayed rapidly browned dead canopies consistent with late stage SOD. Leaves were processed as above. Colonies from these samples produced chlamydospores and sporangia typical of P. ramorum on PARPH-V8 agar, but displayed a growth rate faster than that of NA1 genotypes and were characterized by aerial hyphae, overall resembling the morphology of EU1 lineage colonies (Brasier 2003). The EU1 lineage was confirmed by the perfect match of the sequence of a portion of the Cox-1 gene (GB MW349116-7) with the Cox-1 sequence of EU1 genotypes (GB EU124926). The EU1 clonal lineage has been previously isolated from tanoaks in Oregon forests, approximately 55 Km to the North (Grünwald et al. 2016), but this is the first report for California wildlands and will require containment and government regulations. It is unknown whether the EU1 strains in Del Norte County originated from Oregon forests or elsewhere.

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Disease on Nursery Stock as Affected by Environmental Factors and Seasonal Inoculum Levels of Phytophthora ramorum in Stream Water Used for Irrigation

Plant Disease ◽

10.1094/pdis-92-11-1566 ◽

2008 ◽

Vol 92 (11) ◽

pp. 1566-1573 ◽

Cited By ~ 26

Author(s):

S. A. Tjosvold ◽

D. L. Chambers ◽

S. T. Koike ◽

S. R. Mori

Keyword(s):

Stream Water ◽

Disease Incidence ◽

Phytophthora Ramorum ◽

Sudden Oak Death ◽

Nursery Stock ◽

Stream Sampling ◽

Flow Through ◽

High Concentrations ◽

Electronic Sensors ◽

Native Host

A pear bait monitoring system was used to detect and quantify Phytophthora ramorum propagules in streams that flow through woodland areas with sudden oak death in Santa Cruz County, CA from 2001 to 2007. Stream propagules were detected most frequently or occurred in highest concentrations in winter and spring. The stream propagule concentration was characterized with statistical models using temperature and rainfall variables from 2004 to 2007. The highest concentrations of propagules occurred when stream sampling was preceded by about 2 months with low maximum daily temperatures and by 4 days with high rainfall. The occurrence of propagules in streams in the summer was mostly associated with infected leaves from the native host Umbellaria californica that prematurely abscised and fell into the water. When the stream water was used for irrigating rhododendron nursery stock from 2004 to 2007, disease occurred only three times in the two wettest springs (2005 and 2006) on plants sprinkler irrigated with stream water with relatively high concentrations of propagules. Disease incidence was described with a statistical model using the concentration of infective propagules as measured by pear baiting and consecutive hours of leaf wetness measured by electronic sensors at rhododendron height. The concentration of infective propagules was significantly reduced after water was pumped from the stream and applied through sprinklers.

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First Report of Phytophthora ramorum Infecting Camellia Flower Buds in North America

Plant Health Progress ◽

10.1094/php-2006-0825-01-br ◽

2006 ◽

Vol 7 (1) ◽

pp. 52

Author(s):

Steve A. Tjosvold ◽

David L. Chambers ◽

Samantha L. Thomas ◽

Cheryl L. Blomquist

Keyword(s):

North America ◽

North American ◽

Phytophthora Ramorum ◽

Sudden Oak Death ◽

Flower Buds ◽

Flower Bud ◽

First Report ◽

The North ◽

Landscape Plants ◽

North American Genotype

Camellias are important nursery and landscape plants and are known to be highly susceptible hosts of Phytophthora ramorum, the pathogen that causes Sudden Oak Death. This is the first report of camellia flower bud infection in the field with the North American genotype of P. ramorum Accepted for publication 31 May 2006. Published 25 August 2006.

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Susceptibility of Camellia to Phytophthora ramorum

Plant Health Progress ◽

10.1094/php-2006-0315-01-rs ◽

2006 ◽

Vol 7 (1) ◽

pp. 28 ◽

Cited By ~ 3

Author(s):

Nina Shishkoff

Keyword(s):

Phytophthora Ramorum ◽

Sudden Oak Death ◽

Stem Tissue ◽

Relative Susceptibility ◽

Stem Blight ◽

Nursery Stock ◽

The U.S

Camellia is a known host of Phytophthora ramorum, the “sudden oak death” pathogen. During 2003-2004, the organism was shipped from California throughout the U.S. on infected nursery stock, leading to a nationwide effort to recover the infected plants. This paper describes the symptoms on Camellia and the relative susceptibility of nine species and four hybrids. Camellias varied widely in susceptibility, with Camellia × ‘Roni gaki’ showing the worst overall symptoms, while some other cultivars showed little or none. Obvious symptoms include leaf lesions and stem blight; defoliation, while more difficult to observe, was also characteristic in camellias. The pathogen persisted in diseased plants, notably in stem tissue, for at least a month as demonstrated by isolation. One month after inoculation, roots remained Accepted for publication 23 December 2005. Published 15 March 2006.

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First Report of A1 Mating Type of Phytophthora ramorum in North America

Plant Disease ◽

10.1094/pdis.2003.87.10.1267a ◽

2003 ◽

Vol 87 (10) ◽

pp. 1267-1267 ◽

Cited By ~ 53

Author(s):

E. M. Hansen ◽

P. W. Reeser ◽

W. Sutton ◽

L. M. Winton ◽

N. Osterbauer

Keyword(s):

United States ◽

Mating Type ◽

North American ◽

The United States ◽

Phytophthora Ramorum ◽

United States Department ◽

Jackson County ◽

Nursery Stock ◽

Clackamas County ◽

Microsatellite Alleles

Phytophthora ramorum is known in Europe and the western United States (1). In Europe, it is found in nurseries and landscape plantings. In the United States, it has been confined to coastal forests, and in California, it is found in a few horticultural nurseries. All European isolates tested have been A1 mating type, while all North American isolates were A2 mating type (2). Amplified fragment length polymorphism markers also indicated that the populations on the two continents are distinct, and nearly all North American isolates are from one clone (Kelly Ivors, unpublished). In June 2003, P. ramorum was isolated from diseased Viburnum and Pieris spp. cultivars from a Clackamas County nursery in northern Oregon and diseased Camellia sp. cultivar from a Jackson County nursery in southern Oregon. Representative isolates were submitted to the American Type Culture Collection, Manassas, VA. As part of the effort to determine the origin of these new infestations, we tested the nursery isolates for mating type. Seven Oregon nursery isolates, three Oregon forest isolates (from the predominant North American clone), and two European isolates were paired. Agar plugs from 3-day-old colonies were placed in close proximity on carrot agar plates, and then the plates were examined for oogonia after 3 and 10 days as advised by C. M. Brasier (personal communication). Oogonia and antheridia typical of P. ramorum (2) formed when isolates from the Clackamas County nursery were paired with the Oregon forest isolates and also when isolates from the Jackson County nursery were paired with the European isolates. Gametangia also formed in pairings between Oregon forest isolates and European isolates, but not in any other combinations. We developed polymerase chain reaction (PCR) primers for four microsatellite loci and determined allele sizes for the same set of isolates (unpublished). Microsatellite alleles of the Clackamas County isolates were identical to the European tester isolates, and alleles of the Jackson County isolates were identical to the Oregon forest isolates. These results indicate that the recent Oregon nursery infestations are of separate origins. The Clackamas County isolates are A1 mating type and have microsatellite alleles like the European testers, but according to shipping records, the nursery has received no host nursery stock directly from Europe. However, host nursery stock has been received from a Canadian nursery. The Jackson County isolates are of A2 mating type and have microsatellite alleles like the forest isolates of Oregon, which is consistent with the reported origin of these plants from a California nursery. These preliminary microsatellite results need to be validated against a larger isolate set but are congruent with the mating type results. The Oregon nursery infestations highlight the dangers of unregulated or underregulated transport of host nursery stock from infested areas to noninfested areas. All host plants from infested nursery blocks at the affected Oregon nurseries have been destroyed by incineration, and a monitoring program has been implemented. Other host nursery stock on site has been taken “off-sale” pending verification that it is disease free, per the United States Department of Agriculture, APHIS requirements. References: (1) J. M. Davidson et al. On-line publication. doi:10.1094/PHP-2003-0707-01-DG. Plant Health Progress, 2003. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.

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Standardizing the Nomenclature for Clonal Lineages of the Sudden Oak Death Pathogen, Phytophthora ramorum

Phytopathology ◽

10.1094/phyto-99-7-0792 ◽

2009 ◽

Vol 99 (7) ◽

pp. 792-795 ◽

Cited By ~ 72

Author(s):

Niklaus J. Grünwald ◽

Erica M. Goss ◽

Kelly Ivors ◽

Matteo Garbelotto ◽

Frank N. Martin ◽

...

Keyword(s):

North America ◽

Molecular Marker ◽

Mating Type ◽

Recent Literature ◽

Research Community ◽

Phytophthora Ramorum ◽

Causal Agent ◽

Sudden Oak Death

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

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Spatial and Temporal Analysis of Populations of the Sudden Oak Death Pathogen in Oregon Forests

Phytopathology ◽

10.1094/phyto-12-14-0350-fi ◽

2015 ◽

Vol 105 (7) ◽

pp. 982-989 ◽

Cited By ~ 27

Author(s):

Z. N. Kamvar ◽

M. M. Larsen ◽

A. M. Kanaskie ◽

E. M. Hansen ◽

N. J. Grünwald

Keyword(s):

American West ◽

Temporal Analysis ◽

Phytophthora Ramorum ◽

Sudden Oak Death ◽

Clonal Lineage ◽

Early Introduction ◽

North American West ◽

Genetic History ◽

The North ◽

Spatial And Temporal Analysis

Sudden oak death caused by the oomycete Phytophthora ramorum was first discovered in California toward the end of the 20th century and subsequently emerged on tanoak forests in Oregon before its first detection in 2001 by aerial surveys. The Oregon Department of Forestry has since monitored the epidemic and sampled symptomatic tanoak trees from 2001 to the present. Populations sampled over this period were genotyped using microsatellites and studied to infer the population genetic history. To date, only the NA1 clonal lineage is established in this region, although three lineages exist on the North American west coast. The original introduction into the Joe Hall area eventually spread to several regions: mostly north but also east and southwest. A new introduction into Hunter Creek appears to correspond to a second introduction not clustering with the early introduction. Our data are best explained by both introductions originating from nursery populations in California or Oregon and resulting from two distinct introduction events. Continued vigilance and eradication of nursery populations of P. ramorum are important to avoid further emergence and potential introduction of other clonal lineages.

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Detection of Phytophthora ramorum Blight in Oregon Nurseries and Completion of Koch's Postulates on Pieris, Rhododendron, Viburnum, and Camellia

Plant Disease ◽

10.1094/pdis.2004.88.1.87a ◽

2004 ◽

Vol 88 (1) ◽

pp. 87-87 ◽

Cited By ~ 28

Author(s):

J. L. Parke ◽

R. G. Linderman ◽

N. K. Osterbauer ◽

J. A. Griesbach

Keyword(s):

Mating Type ◽

Culture Collection ◽

Selective Medium ◽

Phytophthora Ramorum ◽

Coastal Forests ◽

Koch’S Postulates ◽

The North ◽

Plastic Bags ◽

New Hosts ◽

Koch's Postulates

Phytophthora ramorum, the cause of sudden oak death in California and Oregon coastal forests and ramorum blight in European nurseries and landscapes (1), was detected in six Oregon nurseries in Jackson, Clackamas, and Washington counties from May to June 2003. The pathogen was isolated from: Viburnum bodnantense ‘Dawn’, V. plicatum var. tomentosum ‘Mariesii’, Pieris japonica × formosa ‘Forest Flame’, P. japonica ‘Variegata’ and ‘Flaming Silver’, P. floribunda × japonica ‘Brouwer's Beauty’, Camellia sasanqua ‘Bonanza’ and other cultivars, C. japonica, and Rhododendron × ‘Unique’. Samples of symptomatic tissues were plated on a Phytophthora-selective medium (PARP) and tested by polymerase chain reaction (PCR) (3). All samples positive for P. ramorum with PCR yielded P. ramorum isolates in culture. The isolates have the European genotype, mating type A1, except for the Camellia spp. isolates, which have the North American genotype, mating type A2 (2). Isolates are deposited in the American Type Culture Collection. Koch's postulates for this pathogen have been completed on V. bodnantense and C. japonica (1). To confirm pathogenicity on the new hosts, isolates from V. plicatum var. tomentosum ‘Mariesii’, Pieris × ‘Forest Flame’, Pieris × ‘Brouwer's Beauty’, and P. japonica ‘Variegata’ and ‘Flaming Silver’ were used to inoculate healthy plants of the same cultivars. For isolates from Rhododendron × ‘Unique’ and C. sasanqua ‘Bonanza’, pathogenicity was tested on Rhododendron × ‘Nova Zembla’ and C. sasanqua ‘Sutsugekka’ and ‘Kanjiro’. Three to five plants of each cultivar were inoculated and three to five were noninoculated. Zoospore inoculum was prepared on dilute V8 agar for one isolate from each host. Foliage of plants growing in 10-cm pots was dipped for 5 sec in a zoospore suspension (3 × 104 zoospores per ml) or sprayed to runoff with a hand mister (6 × 104 zoospores per ml). Control plants were dipped in or sprayed with sterile water. C. sasanqua plants were also inoculated by placing 6-mm mycelial plugs on individual leaves that had been wounded by piercing with a pin. Control leaves were wounded but not inoculated. Foliage was enclosed in plastic bags to retain humidity and the pathogen, and plants were incubated in a locked growth chamber (21 to 23°C). After 21 days, plants were examined for symptoms, and isolations onto PARP were made. All inoculated plants showed foliar symptoms, and P. ramorum was consistently isolated from inoculated plants, but not from asymptomatic control plants. On Rhododendron × ‘Nova Zembla’, nearly all leaves were wilted and dead, as were terminal buds and stems. Pieris spp. cultivars exhibited leaf and stem necrosis and defoliation. On V. plicatum var. tomentosum ‘Mariesii’, necrotic leaf lesions and defoliation of the lower leaves were observed. On C. sasanqua, necrotic lesions developed only on wounded leaves inoculated with mycelial plugs; these leaves abscised. Our results confirm the pathogenicity of Oregon nursery isolates of P. ramorum on V. plicatum var. tomentosum ‘Mariesii’, P. japonica × formosa ‘Forest Flame’, P. japonica ‘Variegata’ and ‘Flaming Silver’, P. floribunda × japonica ‘Brouwer's Beauty’, C. sasanqua and Rhododendron and complete Koch's postulates for several new hosts. References: (1) J. M. Davidson et al. Online publication. doi:10.1094/PHP-2003-0707-01-DG. Plant Health Progress, 2003. (2) E. M. Hansen et al. Plant Dis. 87:1267, 2003. (3) L. M. Winton and E. M. Hansen. For. Pathol. 31:275, 2001.

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