Comparative Efficacy of Chondrosterum purpureum and Chemical Herbicides for Control of Resprouts in Tanoak and Bay Laurel.

The invasive Oomycete pathogen Phytophthora ramorum has killed millions of susceptible oak and tanoak trees in California and southern Oregon forests and is responsible for losses in revenue to the nursery industry through mitigation activities. In addition, infestation of forests in the United Kingdom by this organism has resulted in the destruction of many hectares of larch plantations. Resprouting stumps can be a reservoir for the inoculum of P. ramorum persisting on a site. In areas where the application of herbicides is not permitted, a biocontrol treatment would be an indispensable alternative. Treatment of stumps with the sap-rotting fungus Chondrostereum purpureum (Pers.) Pouzar has been shown to be an effective tool for the suppression of resprouting on several species, most notably red alder. In this project, the ability of C. purpureum to suppress resprouting was evaluated on stumps of two host species, tanoak (Notholithocarpus densiflorus) and California bay laurel (Umbellularia californica). Laboratory testing of three California isolates of C. purpureum indicated that the fungus can colonize bay laurel stems. Field trials were established near Brookings, Oregon, on tanoak and on bay laurel near Soquel, California. Early results of field testing showed that C. purpureum was able to colonize the stumps of tanoak following treatment and was found to occur naturally on tanoak logs and stumps. Formulations of C. purpureum appear to have some effect on reducing sprout survival in tanoak, but the most effective and rapid treatment for this host is the hack and squirt method of applying the herbicide imazapyr. Sprayed herbicide prevents sprouting on bay laurel, and there was evidence that resprouting was inhibited on stumps treated with C. purpureum. Over time, applications of C. purpureum may be a more permanent solution as the stumps begin to decay.

A global genetic analysis of herbarium specimens reveals the invasion dynamics of an introduced plant pathogen.

The introduction, spread, and impact of fungal plant pathogens is a critical concern in ecological systems. In this study, we were motivated by the rather sudden appearance of Acermacrophyllum heavily infected with powdery mildew. We used morphological and genetic analyses to confirm the pathogen causing the epidemic was Sawadaea bicornis. In subsequent field studies, this pathogen was found in several locations in western North America, and in greenhouse studies, A. macrophyllum was found to be significantly more susceptible to S. bicornis than nine other Acer species tested. A genetic analysis of 178 specimens of powdery mildew from freshly collected and old herbarium specimens from 15 countries revealed seven different haplotypes. The high diversity of haplotypes found in Europe coupled with sequence results from a specimen from 1864 provides evidence that S. bicornis has a European origin. Furthermore, sequence data from a specimen from 1938 in Canada show that the pathogen has been present in North America for at least 82 years revealing a considerable lag time between the introduction and current epidemic. This study used old herbarium specimens to genetically hypothesize the origin, the native host, and the invasion time of a detrimental fungal plant pathogen.

First report of leaf blight caused by Phytophthora ramorum on periwinkle (Vinca minor) in Washington State, USA.

Phytophthora ramorum (Werres, De Cock & Man in't Veld) was recovered from symptomatic foliage of periwinkle at a botanical garden in WA in March 2015. Symptoms were tan colored lesions with a dark brown margin visible on both surfaces of the leaf and were found on wounds or around leaf margins. Periwinkle is native to Europe and is commonly used for ground cover in ornamental landscapes. It is known to be invasive in US forests near the urban/wildland interface. Potential spread of P. ramorum into WA forests is of regulatory concern, as well as long distance spread to other states via nursery stock (7 CFR §301.92-2). Phytophthora ramorum was isolated from symptomatic foliage by excising leaf pieces 4-6 mm in diameter and surface-sterilizing in 0.6% sodium hypochlorite followed by two rinses in sterile water. Leaf pieces were plated on PARP medium (Ferguson and Jeffers 1999) and after 2-3 days at 20°C, slow-growing dense colonies with coralloid hyphae were isolated onto V8 agar. Colony morphology and chlamydospore production were consistent with descriptions of P. ramorum (Werres et al. 2001), except that the isolate was slower growing and had irregular, non-wildtype morphology (Elliott et al. 2018) compared to other isolates of P. ramorum. ITS and COX1 regions of mycelial DNA was amplified and sequenced to confirm the identity of P. ramorum using primers ITS1/ITS4 (White et al. 1990) and COX1F1/COX1R1 (Van Poucke et al. 2012). Sequences were submitted to GenBank (accession nos. ITS MT031975, COX1 MT031974). BLAST results showed at least 98% similarity with sequences of P. ramorum (ITS, MN540640 [98%]; COX1, EU124920 [100%]), and belonged to the NA1 clonal lineage. Pathogenicity of P. ramorum to periwinkle was confirmed by completing Koch's Postulates. Inoculum was grown on V8 agar plates at 20°C for two weeks until sporangia were abundant. A zoospore suspension was produced by flooding plates with 7 ml sterile water, incubating for 2 hours at 5°C, then for an additional hour at 24°C. Zoospores were observed under the microscope and quantified with a hemocytometer, then diluted to 2 x 105 zoospores/ml. A 10 µl droplet of inoculum was placed at one wounded and one unwounded site on six leaves on each of four plants. In addition, a set of four plants was inoculated by dipping foliage on one branch per plant into the zoospore suspension for 30 seconds. A set of four control plants were mock inoculated in the same manner using sterile water. The trial was repeated once. Inoculated plant materials were incubated in a moist chamber for 3-5 days and free moisture was present on foliage upon removal. Plants were held in a biocontainment chamber (USDA-APHIS permit # 65857) at 20C and symptom development assessed after 7 days (Figure S1). . Symptoms developed on foliage inoculated using both methods in both trials. Phytophthora ramorum was isolated once from droplet inoculated foliage at a wounded site on one plant. Reisolation onto PARP and then V8 agar was conducted from surface-sterilized symptomatic tissue and the presence of P. ramorum confirmed by observation of colony morphology and chlamydospore production. The presence of P. ramorum was also confirmed with DNA extraction from symptomatic foliage from plants from each of the two trials followed by PCR and sequencing of the COX1 gene (EU124920, 100%) (Figure S2). None of the water-inoculated controls were positive for P. ramorum. Low isolation success could be attributed to reduced pathogenicity due to being a non-wildtype isolate. Acknowledgements This work was supported by the USDA National Institute of Food and Agriculture, McIntire-Stennis project 1019284 and USDA APHIS Cooperative Agreement AP17PPQS&T00C070.

First report of leaf blight caused by Phytophthora ramorum on cherry laurel (Prunus laurocerasus) in Washington State, USA.

In April 2014, Phytophthora ramorum (Werres, De Cock & Man in't Veld) was recovered from symptomatic foliage of cherry laurel (Prunus laurocerasus) at an ornamental plant nursery in Washington State. Cherry laurel, also known as English laurel, is widely propagated in WA because it is commonly used in landscaping. It is invasive in forests near the urban/wildland interface in the western US and in Europe (Rusterholz et al. 2018). Given its popularity as an ornamental species, the potential of this host to spread P. ramorum is of regulatory concern due to possible long distance spread to other states via nursery stock. Foliar symptoms consisted of dark brown lesions near wounds or around leaf margins where water collected. Shot-hole symptoms characterized by abscission zones and dropping of infected tissues were also observed. Lesions expanded beyond the margin of the shot-hole in some cases (Figure S1A). Phytophthora was isolated from symptomatic foliage by surface-sterilizing leaf pieces in 0.6% sodium hypochlorite and 2 rinses in sterile water. They were plated on PARP medium (Ferguson and Jeffers 1999). After 2-3 days, a slow-growing dense colony with coralloid hyphae was isolated onto V8 agar. P. ramorum was identified by observing morphological features (Figure S1B). Colony and spore morphology matched that of P. ramorum (Werres et al. 2001). The isolate was confirmed as P. ramorum by PCR and sequencing of ITS and COX1 regions using primers ITS1/ITS4 (White et al. 1990) and COX1F1/COX1R1 (Van Poucke et al. 2012). Sequences were submitted to GenBank (accession nos. ITS MT031969, COX1 MT031968). BLAST results showed at least 99% similarity with sequences of P. ramorum (ITS, KJ755124 [100%]; COX1, EU124926 [99%]). Multilocus genotyping with microsatellite markers placed the isolate in the EU1 clonal lineage. Pathogenicity of P. ramorum on cherry laurel was confirmed by completing Koch's Postulates using the isolate taken from this host. Two trials were done in a biocontainment chamber (USDA-APHIS permit # 65857) since P. ramorum is a quarantine pathogen and greenhouse trials could not be conducted, using detached stems from mature, visibly healthy cherry laurel plants growing in a landscape. Phytophthora ramorum inoculum was grown on V8A plates at 20®C for 2 weeks until sporangia were abundant. A zoospore suspension was produced by flooding plates with 7 ml sterile water, incubating for 2 hours at 5®C, then 1 hour at 24®C. Zoospores were observed with light microscopy, quantified with a hemocytometer and diluted to 1 x 104 zoospores/ml. A 10 µl droplet was placed at 3 wounded and 3 unwounded sites on 4 leaves per branch. In addition, a set of samples was inoculated by dipping foliage into the zoospore suspension for 30 seconds. A set of controls was mock inoculated using sterile water. Four branches per inoculation treatment were used and the trial was repeated once. Inoculated plant materials were incubated in moist chambers for 3-5 days at 20®C. Free moisture was present on foliage upon removal. Symptom development was assessed after incubation in the biocontainment chamber at 20®C for 7 days (Figure S1C). Phytophthora ramorum was reisolated from symptomatic tissue and the recovered culture was verified morphologically and by PCR and sequencing. It was isolated more often from foliage dipped in zoospore suspension than droplet inoculated, and more from wounded than unwounded sites. None of the water-inoculated controls were positive for P. ramorum. The presence of P. ramorum was also confirmed with DNA extraction from surface-sterilized symptomatic foliage followed by PCR and sequencing of the COX1 gene (EU124926, 100%) (Figure S2). To our knowledge, this is the first report of P. ramorum naturally infecting cherry laurel in the United States. Acknowledgements This work was supported by the USDA National Institute of Food and Agriculture, McIntire-Stennis project 1019284 and USDA APHIS Cooperative Agreement AP17PPQS&T00C070 Literature cited Ferguson and Jeffers, 1999. Plant Disease 83:1129-1136 Van Poucke, K. et al. 2012. Fungal Biology 116: 1178-1191. http://dx.doi.org/10.1016/j.funbio.2012.09.003 Werres, S. et al. 2001. Mycol. Res. 105:1155-1165. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

A Novel Phagomyxid Parasite Produces Sporangia in Root Hair Galls of Eelgrass (Zostera marina).

The objective of this study was to identify the parasite causing the formation of root hair galls on eelgrass (Zostera marina) in Puget Sound, WA. Microscopic and molecular analyses revealed that a novel protist formed plasmodia that developed into sporangia in root hair tip galls and released biflagellate swimming zoospores. Root hair galls were also observed in the basal section of root hairs, and contained plasmodia or formed thick-walled structures filled with cells (resting spores). Phylogenetic analyses of 18S rDNA sequence data obtained from cells in sporangia indicated that the closest relative of the parasite with a known taxonomic identification was Plasmodiophora diplantherae (86.9% sequence similarity), a phagomyxid parasite that infects the seagrass Halodule spp. To determine the local geographic distribution of the parasite, root and soil samples were taken from four eelgrass populations in Puget Sound and analyzed for root hair galls and parasite DNA using a newly designed qPCR protocol. The percent of root hairs with galls and amount of parasite DNA in roots and sediment varied among the four eelgrass populations. Future studies are needed to establish the taxonomy of the parasite, its effects on Z. marina, and the factors that determine its distribution and abundance.

Characterization of phenotypic variation and genome aberrations observed among Phytophthora ramorum isolates from diverse hosts.

BACKGROUND: Accumulating evidence suggests that genome plasticity allows filamentous plant pathogens to adapt to changing environments. Recently, the generalist plant pathogen Phytophthora ramorum has been documented to undergo irreversible phenotypic alterations accompanied by chromosomal aberrations when infecting trunks of mature oak trees (genus Quercus). In contrast, genomes and phenotypes of the pathogen derived from the foliage of California bay (Umbellularia californica) are usually stable. We define this phenomenon as host-induced phenotypic diversification (HIPD). P. ramorum also causes a severe foliar blight in some ornamental plants such as Rhododendron spp. and Viburnum spp., and isolates from these hosts occasionally show phenotypes resembling those from oak trunks that carry chromosomal aberrations. The aim of this study was to investigate variations in phenotypes and genomes of P. ramorum isolates from non-oak hosts and substrates to determine whether HIPD changes may be equivalent to those among isolates from oaks. RESULTS: We analyzed genomes of diverse non-oak isolates including those taken from foliage of Rhododendron and other ornamental plants, as well as from natural host species, soil, and water. Isolates recovered from artificially inoculated oak logs were also examined. We identified diverse chromosomal aberrations including copy neutral loss of heterozygosity (cnLOH) and aneuploidy in isolates from non-oak hosts. Most identified aberrations in non-oak hosts were also common among oak isolates; however, trisomy, a frequent type of chromosomal aberration in oak isolates was not observed in isolates from Rhododendron. CONCLUSION: This work cross-examined phenotypic variation and chromosomal aberrations in P. ramorum isolates from oak and non-oak hosts and substrates. The results suggest that HIPD comparable to that occurring in oak hosts occurs in non-oak environments such as in Rhododendron leaves. Rhododendron leaves are more easily available than mature oak stems and thus can potentially serve as a model host for the investigation of HIPD, the newly described plant-pathogen interaction.

An Overview of Canadian Research Activities on Diseases Caused by Phytophthora ramorum: Results, Progress, and Challenges.

International trade and travel are the driving forces behind the spread of invasive plant pathogens around the world, and human-mediated movement of plants and plant products is now generally accepted as the primary mode of their introduction, resulting in huge disturbance to ecosystems and severe socio-economic impact. These problems are exacerbated under the present conditions of rapid climatic change. We report an overview of the Canadian research activities on Phytophthora ramorum. Since the first discovery and subsequent eradication of P. ramorum on infected ornamentals in nurseries in Vancouver, British Columbia, in 2003, a research team of Canadian government scientists representing the Canadian Forest Service, Canadian Food Inspection Agency, and Agriculture and Agri-Food Canada worked together over a 10-year period and have significantly contributed to many aspects of research and risk assessment on this pathogen. The overall objectives of the Canadian research efforts were to gain a better understanding of the molecular diagnostics of P. ramorum, its biology, host-pathogen interactions, and management options. With this information, it was possible to develop pest risk assessments and evaluate the environmental and economic impact and future research needs and challenges relevant to P. ramorum and other emerging forest Phytophthora spp.

Comparison of Five Detection and Quantification Methods for Phytophthora ramorum in Stream and Irrigation Water.

Propagules of Phytophthora ramorum, the causal agent of sudden oak death (SOD) and ramorum blight, can be recovered from infested stream and nursery irrigation runoff using baiting and filtration methods. Five detection methods, including pear and rhododendron leaf baits, Bottle O' Bait, filtration, and quantitative polymerase chain reaction (qPCR) performed on zoospores trapped on a filter were compared simultaneously in laboratory assays using lab or creek water spiked with known quantities of P. ramorum zoospores. The detection threshold for each method was determined and methods that could be used to quantify zoospore inoculum were identified. Filtration and qPCR were the most sensitive at detecting low levels of zoospores, followed by wounded rhododendron leaves, rhododendron leaf disks, and pear baits. Filtration, qPCR, and leaf disks were able to quantify P. ramorum zoospores ranging from 2 to 451 direct-plate CFU/liter while wounded leaves and pear baits appeared to be better at detection rather than quantification. The ability to detect and quantify P. ramorum inoculum in water will assist scientists, regulatory agencies, and nursery personnel in assessing the risk of spreading P. ramorum in nurseries and landscape sites where untreated infested water is used for irrigation.

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.

Fusicoccum arbuti sp. nov. causing cankers on pacific madrone in western North America with notes on Fusicoccum dimidiatum, the correct name for Scytalidium dimidiatum and Nattrassia mangiferae.

Pacific madrone (Arbutus menziesii) is a broadleaf evergreen tree native to western North America that has been in decline for the past 30 years. A fungus has been isolated and was verified as the cause of cankers on dying trees. It was determined to belong in the genus Fusicoccum, an asexual state of Botryosphaeria. This genus in both its sexual and asexual states commonly causes canker diseases of deciduous woody plants. Using morphological and molecular data the fungus causing cankers on Pacific madrone is characterized, described and illustrated as a new species of Fusicoccum, F. arbuti D.F. Farr & M. Elliott sp. nov. No sexual state is known for F. arbuti. Evidence from the literature, cultures and specimens suggests that F. arbuti, often mistakenly identified as Nattrassia mangiferae, has been causing madrone canker since at least 1968. Authentic isolates of Nattrassia mangiferae as the synanamorph Scytalidium dimidiatum were sequenced and determined to be different from Fusicoccum arbuti and to belong in Botryosphaeria/Fusicoccum. In addition to molecular sequence data, the morphology of the pycnidial and arthric conidial states of Nattrassia mangiferae/ Scytalidium dimidiatum resembles that of Fusicoccum. Therefore the correct name for Nattrassia mangiferae and its numerous synonyms (Dothiorella mangiferae, Torula dimidata, Scytilidium dimidiatum, Fusicoccum eucalypti, Hendersonula toruloidea, H. cypria, Exosporina fawcetii, H. agathidia, and S. lignicola) is Fusicoccum dimidiatum (Penz.) D.F. Farr, comb. nov.