C. L. Blomquist

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

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.

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.

Development of a Multiplex Assay for Genus- and Species-Specific Detection of Phytophthora Based on Differences in Mitochondrial Gene Order

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic assays were also validated against 130 environmental samples from a range of hosts. The only limitation observed was that primers for the 340 bp atp9-nad9 locus did not amplify Phytophthora bisheria or P. frigida. The identification of species present in a sample can be determined without the need for culturing by sequencing the genus-specific amplicon and comparing that with a reference sequence database of known Phytophthora spp.

Evaluation of Molecular Markers for Phytophthora ramorum Detection and Identification: Testing for Specificity Using a Standardized Library of Isolates

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as beta-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and β-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.

Leaf spot of Arugula, caused by Alternaria japonica, in California.

Arugula (Eruca vesicaria subsp. sativa (Mill.) Thell. is a Cruciferous plant used for culinary purposes. From 2012 to 2013, a foliar disease seriously impacted the growth and quality of about 0.1 ha of hydroponically grown arugula at a Santa Barbara County nursery. Samples of affected arugula seedlings exhibited adaxial and abaxial symptoms of mottling with circular to oval, water soaked, dark green leaf spots, each 1 to 3 mm in diameter, and some of which coalesced. Conidia of an Alternaria sp. were observed on the foliage. Symptomatic leaf pieces were disinfested with 0.6% NaOCl, blotted dry, and plated on acidified potato dextrose agar (APDA). Cultures were incubated under near-UV lights for 24 h/day. Olivaceous-grey colonies of the same Alternaria species observed on the leaves grew after 7 days. After 21 days on carrot-piece agar (3), the fungus produced beakless conidia with longitudinal and constricted transverse septa that measured 30.0 to 69.0 × 12.5 to 20.0 μm and were borne singly or in short chains of 2 to 3 conidia. In addition, knots of dark, thick-walled micro-chlamydospores were produced by the hyphae. The fungus was identified morphologically as Alternaria japonica Yoshii (2), and the species confirmed by sequence analysis. A portion of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified using ITS1 and ITS4 primers (4). The sequence (GenBank Accession No. KJ126846) was 100% identical to the ITS rDNA sequence of an isolate of A. japonica (KC584201) using a BLASTn query. A. japonica was also detected in seeds of the lot used to grow the affected arugula crop. Pathogenicity of a single isolate was tested by inoculating four 37-day-old plants each of arugula, cabbage (Brassica oleracea L. var. capitata), and broccoli (B. oleracea L. var. botrytis L.). Inoculum was obtained from 11-day-old cultures of the isolate grown at 24°C on half-strength APDA. Half of a 2.5 cm diameter agar plug containing hyphae and conidia was ground in 2 ml of sterilized water, and the volume of water increased to 45 ml. Leaves of four plants/host species were sprayed with 3.5 to 4.0 ml of inoculum. The inoculated plants and four control plants of each species treated similarly with sterilized water were immediately incubated in a dark dew chamber at 23°C. After 72 h in the dew chamber, inoculated plants of all three hosts produced similar symptoms of wilting, water soaking, and dark green leaf spotting as the original symptomatic field plants. Conidia formed in the leaf spots on both sides of inoculated leaves. A. japonica was re-isolated from all of the inoculated plants but from none of the symptomless control plants using the method previously described. Pathogenicity tests were repeated, with similar results. Although reported in Italy in 2013 (1), to our knowledge, this is the first report of A. japonica on arugula in the United States. References: (1) G. Gilardi et al. Acta Hort. 1005:569, 2013. (2) E. G. Simmons. Page 368 in: Alternaria, An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, 2007. (3) S. Werres et al. Z. Planzenkr. Pflanzensh. 108:113, 2001. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First report of Phytophthora ramorum infecting grand fir in California.

Phytophthora ramorum was detected on grand fir in 2003 and 2005 in a Christmas tree plantation near Los Gatos, CA, in association with infected California bay laurel. Isolates derived from stem lesions were used to inoculate grand fir seedlings in two tests. Isolations from lesions on inoculated plants were positive for P. ramorum in both tests. This work provides the completion of Kochs postulates to establish grand fir as a host of P. ramorum. The potential for grand fir to be infected within its native range is unknown.

Phylogeny and taxonomy of the genus Tubakia s. lat.

The genus Tubakia is revised on the basis of morphological and phylogenetic data. The phylogenetic affinity of Tubakia to the family Melanconiellaceae (Diaporthales) was recently postulated, but new analyses based on sequences retrieved from material of the type species of Tubakia, T. dryina, support a family of its own, viz. Tubakiaceae fam. nov. Our phylogenetic analyses revealed the heterogeneity of Tubakia s. lat. which is divided into several genera, viz., Tubakia s. str., Apiognomonioides gen. nov. (type species: Apiognomonioides supraseptata), Involutiscutellula gen. nov. (type species: Involutiscutellula rubra), Oblongisporothyrium gen. nov. (type species: Oblongisporothyrium castanopsidis), Paratubakia gen. nov. (type species: Paratubakia subglobosa), Racheliella gen. nov. (type species: Racheliella wingfieldiana sp. nov.), Saprothyrium gen. nov. (type species: Saprothyrium thailandense) and Sphaerosporithyrium gen. nov. (type species: Sphaerosporithyrium mexicanum sp. nov.). Greeneria saprophytica is phylogenetically closely allied to Racheliella wingfieldiana and is therefore reallocated to Racheliella. Particular emphasis is laid on a revision and phylogenetic analyses of Tubakia species described from Japan and North America. Almost all North American collections of this genus were previously referred to as T. dryina s. lat., which is, however, a heterogeneous complex. Several new North American species have recently been described. The new species Sphaerosporithyrium mexicanum, Tubakia melnikiana and T. sierrafriensis, causing leaf spots on several oak species found in the North-Central Mexican state Aguascalientes and the North-Eastern Mexican state Nuevo León, are described, illustrated, and compared with similar species. Several additional new species are introduced, including Tubakia californica based on Californian collections on various species of the genera Chrysolepis, Notholithocarpus and Quercus, and T. dryinoides, T. oblongispora, T. paradryinoides, and Paratubakia subglobosoides described on the basis of Japanese collections. Tubakia suttoniana nom. nov., based on Dicarpella dryina, is a species closely allied to T. californica and currently only known from Europe. Tubakia dryina, type species of Tubakia, is epitypified, and the phylogenetic position and circumscription of Tubakia are clarified. A revised, supplemented key to the species of Tubakia and allied genera on the basis of conidiomata is provided.

Entyloma helianthi: identification and characterization of the causal agent of sunflower white leaf smut

ABSTRACT White leaf smut is a minor foliar disease of sunflower (Helianthus annuus) in the United States. The disease occurs primarily in greenhouse-grown sunflowers in California and causes leaf spot, defoliation, and a reduction in yield and crop value. Historically, many Entyloma specimens with similar morphological characters, but infecting diverse plant genera including Helianthus, were called Entyloma polysporum. Recent comparative morphological and molecular work has shown that Entyloma species infect hosts within a single genus or species, suggesting that the sunflower Entyloma species may not be E. polysporum. In 2015, sunflower leaf smut material was collected from ornamental sunflowers in a greenhouse in Santa Barbara County, California. Morphologically, this species differed from E. polysporum in having smaller, more regular-shaped teliospores and prominently developed conidiophores with cylindrical conidia. The rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS]) region of the sunflower leaf smut was phylogenetically distinct from all previously sequenced Entyloma species and found only on H. annuus. This study confirms that the sunflower leaf smut pathogen represents a novel species, Entyloma helianthi. Possible misidentification of the anamorphic stage of Entyloma helianthi as another leaf spot pathogen, Ramularia helianthi, is also discussed.

Powdery mildews on lilac in western North America include Phyllactinia syringae, sp. nov.

ABSTRACT Two powdery mildews, Erysiphe syringae and the previously undescribed Phyllactinia syringae, sp. nov., occur on lilac in western North America. Phyllactinia syringae is found on common lilac, whereas E. syringae is found on Chinese lilac and, occasionally, common lilac. Infection by P. syringae is extremely unobtrusive until formation of a hypophyllous mycelial mat with chasmothecia in late fall. Infection by E. syringae in late summer is conspicuous, with its thick, superficial mycelial mat on the leaf upper surface detracting from the aesthetic appearance of the bush.