First Report of Phytophthora ramorum Causing Leaf Spots on Cornus capitata (Evergreen Dogwood) in United States.

Cornus capitata Wall. ex Roxb. (evergreen dogwood) is a bushy evergreen tree or shrub native to East Asia grown for its showy creamy bracts in late spring followed by attractive red fruit. In Feb 2023, a sample of foliage with leaf spots and tip dieback from C. capitata 'Mountain Moon' was submitted from a Humboldt Co. nursery as part of a CDFA inspection program for Phytophthora ramorum. The leaf spots were medium to dark brown, irregularly shaped, and ranged from 5 to 8 mm in diameter. They were located primarily along the leaf midrib and covered up to 1/4 of the leaf surface. Six 6-mm-diameter leaf discs taken from the margins of brown lesions and tip dieback were plated on Phytophthora selective CMA-PARP (PARP) media (Jeffers and Martin 1986). After 6 to 10 days, colonies resembling P. ramorum, with coralloid coenocytic hyphae, chlamydospores, ellipsoidal semi-papillate and caducous sporangia, and a relatively slow growth rate were recovered. Abundant sporangia formed on agar singly or in clusters on sympodially branched sporangiophores (n = 50), varying in size from 35 to 60 µm × 20 to 30 µm (mean 45.6 × 24.8 µm) with a length/breadth ratio ranging from 1.3 to 2.3 (mean 1.8). Chlamydospores (n = 50) ranged from 35 to 62 µm in diameter (mean 51.9 µm) on 14-day-old PARP cultures. The internal transcribed spacer region (ITS) using primers ITS5/ITS4 (White et al. 1990; accession no. OR636225) and cytochrome oxidase subunit 1 region (cox1) using primers OomCox1Levup/Fm85mod (Robideau et al. 2011; OR635665) of one isolate (0254-32A) were amplified and sequenced. BLAST analysis showed 100% identity of both regions to P. ramorum ex-type strain (MG865581 and MH136973). Microsatellite loci placed the P. ramorum isolate in the NA2 clonal lineage (Goss et al, 2011). Pathogenicity of P. ramorum isolate 0254-32A was tested using five C. capitata plants (2.5-year-old, 28-cm-tall, 3.78-liter pot). Zoospore inoculum was produced as described in Blomquist et al. (2021). Above ground parts of each plant were sprayed with inoculum (15 ml, 1.3 × 105 zoospores/ml). Inoculated plants were incubated in a dew chamber in the dark at 23°C for 72 h and then placed in a 23±1°C growth chamber with a 12-h photoperiod. Five control plants were treated as above but with sterile water instead of the zoospore suspension. Two days after inoculation, brown spots were visible on leaves on all inoculated plants, initiating from where the drops of inoculum had persisted. After 3 days, brown lesions, from water drop- to majority of entire leaf-sized, were observed on approximately 75% of inoculated leaves. After 6 days, lesions expanded to the edges of leaves, causing leaf curling and defoliation. Lesions stopped expanding after 3 weeks, and by 4 weeks, most infected leaves had abscised, with no new infections observed. Phytophthora ramorum was consistently isolated from foliar lesions of inoculated plants on PARP. It was not isolated from leaf or stem tissues of control plants, which remained asymptomatic during the 4-week experiment period. Phytophthora ramorum was detected on C. capitata in the UK in 2015 (DEFRA 2015). To our knowledge, this is the first report of P. ramorum infecting C. capitata in the United States and the completion of Koch's postulates on any Cornus spp. Incidence on C. capitata in the California nursery was low. However, their proximity to other infected foliar hosts suggests Cornus spp. may present a potential risk for the spread of P. ramorum.

Genetic Analysis of the Emerging Citrus Yellow Vein Clearing Virus Reveals a Divergent Virus Population in American Isolates.

Citrus yellow vein clearing virus is a previously reported citrus virus from Asia with widespread distribution in China. In 2022, the California Department of Food and Agriculture conducted a multipest citrus survey targeting multiple citrus pathogens including citrus yellow vein clearing virus (CYVCV). In March 2022, a lemon tree with symptoms of vein clearing, chlorosis, and mottling in a private garden in the city of Tulare, California, tested positive for CYVCV, which triggered an intensive survey in the surrounding areas. A total of 3,019 plant samples, including citrus and noncitrus species, were collected and tested for CYVCV using conventional reverse transcription polymerase chain reaction, reverse transcription quantitative polymerase chain reaction, and Sanger sequencing. Five hundred eighty-six citrus trees tested positive for CYVCV, including eight citrus species not previously recorded infected under field conditions. Comparative genomic studies were conducted using 17 complete viral genomes. Sequence analysis revealed two major phylogenetic groups. Known Asian isolates and five California isolates from this study made up the first group, whereas all other CYVCV isolates from California formed a second group, distinct from all worldwide isolates. Overall, the CYVCV population shows rapid expansion and high differentiation indicating a population bottleneck typical of a recent introduction into a new geographic area.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Multiple origins of downy mildews and mito-nuclear discordance within the paraphyletic genus Phytophthora.

Phylogenetic relationships between thirteen species of downy mildew and 103 species of Phytophthora (plant-pathogenic oomycetes) were investigated with two nuclear and four mitochondrial loci, using several likelihood-based approaches. Three Phytophthora taxa and all downy mildew taxa were excluded from the previously recognized subgeneric clades of Phytophthora, though all were strongly supported within the paraphyletic genus. Downy mildews appear to be polyphyletic, with graminicolous downy mildews (GDM), brassicolous downy mildews (BDM) and downy mildews with colored conidia (DMCC) forming a clade with the previously unplaced Phytophthora taxon totara; downy mildews with pyriform haustoria (DMPH) were placed in their own clade with affinities to the obligate biotrophic P. cyperi. Results suggest the recognition of four additional clades within Phytophthora, but few relationships between clades could be resolved. Trees containing all twenty extant downy mildew genera were produced by adding partial coverage of seventeen additional downy mildew taxa; these trees supported the monophyly of the BDMs, DMCCs and DMPHs but suggested that the GDMs are paraphyletic in respect to the BDMs or polyphyletic. Incongruence between nuclear-only and mitochondrial-only trees suggests introgression may have occurred between several clades, particularly those containing biotrophs, questioning whether obligate biotrophic parasitism and other traits with polyphyletic distributions arose independently or were horizontally transferred. Phylogenetic approaches may be limited in their ability to resolve some of the complex relationships between the "subgeneric" clades of Phytophthora, which include twenty downy mildew genera and hundreds of species.

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

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.

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.

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.

Phytophthora foliorum sp. nov., a new species causing leaf blight of azalea.

A previously unknown Phytophthora was recovered more than 60 times from evergreen hybrid azalea leaves collected during surveys for the sudden oak death pathogen Phytophthora ramorum in California and Tennessee. The novel Phytophthora was discovered when genomic DNA from this species cross-reacted with the ITS-based diagnostic PCR primers used to screen plants for the presence of P. ramorum. This species had caducous, semi-papillate sporangia, was homothallic with both paragynous and amphigynous antheridia, and was pathogenic on both wounded and intact azalea leaves. Nuclear and mitochondrial sequence data indicate that this species is related to, but distinct from, P. ramorum. AFLP analysis indicates that the isolates of this species have limited genotypic diversity and share no markers with P. ramorum. This paper presents the formal description of P. foliorum as a new species and underscores the need for caution when relying solely on DNA-based diagnostic tools.

Characterization of homologues of the apple proliferation immunodominant membrane protein gene from three related phytoplasmas.

SUMMARY Homologues of the immunodominant membrane protein gene from apple proliferation (AP) phytoplasma have been cloned and sequenced for three further members of the AP subclade, namely European stone fruit yellows, peach yellow leaf roll and a European isolate of pear decline (PD). The putative translation products of all three were similar in size to that of AP and all had a transmembrane region towards the N-terminus and a large C-terminal hydrophilic domain, probably held on the outside of the cell membrane in vivo. Sequence similarities for the putative proteins were compared with interrelationships of the phytoplasmas as measured by rRNA gene sequence similarity. The proteins from AP and PD were more similar (57% identical in the major hydrophilic domain) than those for any other pair (31-34%), but these two phytoplasmas were not more closely related by rRNA gene sequences than other pairs. The possibility that the relative similarities of these proteins is related to the host is discussed. It is suggested that the similarity of the AP and PD proteins may reflect the fact that these two proteins have narrow plant host ranges in two closely related genera in the tribe Maloideae (family Rosaceae), whilst the other two have broader host ranges, mainly in the tribe Prunoideae.

An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas.

Membrane proteins mediate several important processes, including attachment, in several Mollicute species. Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors. Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission. A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced. PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots. This PCR product was used to identify a 2.5 kbp ECO:RI-HIN:dIII fragment that was cloned and sequenced. A complete 864 bp ORF (idpA) was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers. Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point. The amino acid sequence had no significant homology with other known phytoplasma IDP genes. The idpA ORF was cloned into an Escherichia coli expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum. A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the E. coli-expressed and native WX phytoplasma proteins. This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.