Krisp: A Python package to aid in the design of CRISPR and amplification-based diagnostic assays from whole genome sequencing data.

Recent pandemics like COVID-19 highlighted the importance of rapidly developing diagnostics to detect evolving pathogens. CRISPR-Cas technology has recently been used to develop diagnostic assays for sequence-specific recognition of DNA or RNA. These assays have similar sensitivity to the gold standard qPCR but can be deployed as easy to use and inexpensive test strips. However, the discovery of diagnostic regions of a genome flanked by conserved regions where primers can be designed requires extensive bioinformatic analyses of genome sequences. We developed the Python package krisp to aid in the discovery of primers and diagnostic sequences that differentiate groups of samples from each other, using either unaligned genome sequences or a variant call format (VCF) file as input. Krisp has been optimized to handle large datasets by using efficient algorithms that run in near linear time, use minimal RAM, and leverage parallel processing when available. The validity of krisp results has been demonstrated in the laboratory with the successful design of a CRISPR diagnostic assay to distinguish the sudden oak death pathogen Phytophthora ramorum from closely related Phytophthora species. Krisp is released open source under a permissive license with all the documentation needed to quickly design CRISPR-Cas diagnostic assays.

Open Access and Reproducibility in Plant Pathology Research: Guidelines and Best Practices.

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology.

Genetic Diversity, Evolution, and Diagnosis of Sugarcane Yellow Leaf Virus from 19 Sugarcane-Producing Locations Worldwide.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf, has been reported in an increasing number of sugarcane-growing locations since its first report in the 1990s in Brazil, Florida, and Hawaii. In this study, the genetic diversity of SCYLV was investigated using the genome coding sequence (5,561 to 5,612 nt) of 109 virus isolates from 19 geographical locations, including 65 new isolates from 16 geographical regions worldwide. These isolates were distributed in three major phylogenetic lineages (BRA, CUB, and REU), except for one isolate from Guatemala. Twenty-two recombination events were identified among the 109 isolates of SCYLV, thus confirming that recombination was a significant driving force in the genetic diversity and evolution of this virus. No temporal signal was found in the genomic sequence dataset, most likely because of the short temporal window of the 109 SCYLV isolates (1998 to 2020). Among 27 primers reported in the literature for the detection of the virus by RT-PCR, none matched 100% with all 109 SCYLV sequences, suggesting that the use of some primer pairs may not result in the detection of all virus isolates. Primers YLS111/YLS462, which were the first primer pair used by numerous research organizations to detect the virus by RT-PCR, failed to detect isolates belonging to the CUB lineage. In contrast, primer pair ScYLVf1/ScYLVr1 efficiently detected isolates of all three lineages. Continuous pursuit of knowledge of SCYLV genetic variability is therefore critical for effective diagnosis of yellow leaf, especially in virus-infected and mainly asymptomatic sugarcane plants.

Phylogeography and population structure of the global, wide host-range hybrid pathogen Phytophthora × cambivora.

Invasive, exotic plant pathogens pose a major threat to native and agricultural ecosystems. Phytophthora × cambivora is an invasive, destructive pathogen of forest and fruit trees causing severe damage worldwide to chestnuts (Castanea), apricots, peaches, plums, almonds and cherries (Prunus), apples (Malus), oaks (Quercus), and beech (Fagus). It was one of the first damaging invasive Phytophthora species to be introduced to Europe and North America, although its origin is unknown. We determined its population genetic history in Europe, North and South America, Australia and East Asia (mainly Japan) using genotyping-by-sequencing. Populations in Europe and Australia appear clonal, those in North America are highly clonal yet show some degree of sexual reproduction, and those in East Asia are partially sexual. Two clonal lineages, each of opposite mating type, and a hybrid lineage derived from these two lineages, dominated the populations in Europe and were predominantly found on fagaceous forest hosts (Castanea, Quercus, Fagus). Isolates from fruit trees (Prunus and Malus) belonged to a separate lineage found in Australia, North America, Europe and East Asia, indicating the disease on fruit trees could be caused by a distinct lineage of P. × cambivora, which may potentially be a separate sister species and has likely been moved with live plants. The highest genetic diversity was found in Japan, suggesting that East Asia is the centre of origin of the pathogen. Further surveys in unsampled, temperate regions of East Asia are needed to more precisely identify the location and range of the centre of diversity.

Openness and Computational Reproducibility in Plant Pathology: Where We Stand and a Way Forward.

Open research practices have been highlighted extensively during the last 10 years in many fields of scientific study as essential standards needed to promote transparency and reproducibility of scientific results. Scientific claims can only be evaluated based on how protocols, materials, equipment, and methods were described; data were collected and prepared; and analyses were conducted. Openly sharing protocols, data, and computational code is central to current scholarly dissemination and communication, but in many fields, including plant pathology, adoption of these practices has been slow. We randomly selected 450 articles published from 2012 to 2021 across 21 journals representative of the plant pathology discipline and assigned them scores reflecting their openness and computational reproducibility. We found that most of the articles did not follow protocols for open science and failed to share data or code in a reproducible way. We propose that use of open-source tools facilitates computationally reproducible work and analyses, benefitting not just readers but the authors as well. Finally, we provide ideas and suggest tools to promote open, reproducible computational research practices among plant pathologists. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Whole Genome Sequencing-Based Tracing of a 2022 Introduction and Outbreak of Xanthomonas hortorum pv. pelargonii.

Globalization has made agricultural commodities more accessible, available, and affordable. However, their global movement increases the potential for invasion by pathogens and necessitates development and implementation of sensitive, rapid, and scalable surveillance methods. Here, we used 35 strains, isolated by multiple diagnostic laboratories, as a case study for using whole genome sequence data in a plant disease diagnostic setting. Twenty-seven of the strains were isolated in 2022 and identified as Xanthomonas hortorum pv. pelargonii. Eighteen of these strains originated from material sold by a plant breeding company that had notified clients following a release of infected geranium cuttings. Analyses of whole genome sequences revealed epidemiological links among the 27 strains from different growers that confirmed a common source of the outbreak and uncovered likely secondary spread events within facilities that housed plants originating from different plant breeding companies. Whole genome sequencing data were also analyzed to reveal how preparatory and analytical methods can impact conclusions on outbreaks of clonal pathogenic strains. The results demonstrate the potential power of using whole genome sequencing among a network of diagnostic labs and highlight how sharing such data can help shorten response times to mitigate outbreaks more expediently and precisely than standard methods.

Comparative Genomic Analysis of 31 Phytophthora Genomes Reveals Genome Plasticity and Horizontal Gene Transfer.

Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Chromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families.

Phytophthora species are notorious plant pathogens, with some causing devastating tree diseases that threaten the survival of their host species. One such example is Phytophthora agathidicida, the causal agent of kauri dieback - a root and trunk rot disease that kills the ancient, iconic and culturally significant tree species, Agathis australis (New Zealand kauri). A deeper understanding of how Phytophthora pathogens infect their hosts and cause disease is critical for the development of effective treatments. Such an understanding can be gained by interrogating pathogen genomes for effector genes, which are involved in virulence or pathogenicity. Although genome sequencing has become more affordable, the complete assembly of Phytophthora genomes has been problematic, particularly for those with a high abundance of repetitive sequences. Therefore, effector genes located in repetitive regions could be truncated or missed in a fragmented genome assembly. Using a combination of long-read PacBio sequences, chromatin conformation capture (Hi-C) and Illumina short reads, we assembled the P. agathidicida genome into ten complete chromosomes, with a genome size of 57 Mb including 34% repeats. This is the first Phytophthora genome assembled to chromosome level and it reveals a high level of syntenic conservation with the complete genome of Peronospora effusa, the only other completely assembled genome sequence of an oomycete. All P. agathidicida chromosomes have clearly defined centromeres and contain candidate effector genes such as RXLRs and CRNs, but in different proportions, reflecting the presence of gene family clusters. Candidate effector genes are predominantly found in gene-poor, repeat-rich regions of the genome, and in some cases showed a high degree of duplication. Analysis of candidate RXLR effector genes that occur in multicopy gene families indicated half of them were not expressed in planta. Candidate CRN effector gene families showed evidence of transposon-mediated recombination leading to new combinations of protein domains, both within and between chromosomes. Further analysis of this complete genome assembly will help inform new methods of disease control against P. agathidicida and other Phytophthora species, ultimately helping decipher how Phytophthora pathogens have evolved to shape their effector repertoires and how they might adapt in the future.

Genomic biosurveillance detects a sexual hybrid in the sudden oak death pathogen.

Invasive exotic pathogens pose a threat to trees and forest ecosystems worldwide, hampering the provision of essential ecosystem services such as carbon sequestration and water purification. Hybridization is a major evolutionary force that can drive the emergence of pathogens. Phytophthora ramorum, an emergent pathogen that causes the sudden oak and larch death, spreads as reproductively isolated divergent clonal lineages. We use a genomic biosurveillance approach by sequencing genomes of P. ramorum from survey and inspection samples and report the discovery of variants of P. ramorum that are the result of hybridization via sexual recombination between North American and European lineages. We show that these hybrids are viable, can infect a host and produce spores for long-term survival and propagation. Genome sequencing revealed genotypic combinations at 54,515 single nucleotide polymorphism loci not present in parental lineages. More than 6,000 of those genotypes are predicted to have a functional impact in genes associated with host infection, including effectors, carbohydrate-active enzymes and proteases. We also observed post-meiotic mitotic recombination that could generate additional genotypic and phenotypic variation and contribute to homoploid hybrid speciation. Our study highlights the importance of plant pathogen biosurveillance to detect variants, including hybrids, and inform management and control.

Repeated Emergence of Sudden Oak Death in Oregon: Chronology, Impact, and Management.

It has been two decades since the first detection of the sudden oak death pathogen Phytophthora ramorum in Oregon forests. Although the epidemic was managed since its first discovery in 2001, at least three invasions of three separate variants (clonal lineages), NA1, EU1, and NA2, are documented to have occurred to date. Control of this epidemic has cost over US$32 million from 2001 to 2020. This is dwarfed by the predicted cost of the closure to the Coos Bay export terminal, estimated at $58 million per year, if the epidemic was allowed to spread unchecked. Management efforts in Oregon have reduced inoculum and limited the spread of the pathogen. An outreach and citizen scientist program has been piloted to help in early detection efforts and search for disease-resistant tanoak. This feature article documents the repeated emergence, impact, costs, and lessons learned from managing this devastating invasive pathogen.

First report of the NA2 clonal lineage of the sudden oak death pathogen, Phytophthora ramorum, infecting tanoak in Oregon forests.

Phytophthora ramorum Werres, de Cock & Man in't Veld, causal agent of sudden oak death (SOD) and ramorum leaf blight, is comprised of four clonal lineages in its invasive ranges of North America and Europe (Grünwald et al. 2012, Van Poucke et al. 2012). Of these, three - the NA1, NA2, and EU1 lineages - are found in U.S. nurseries, but only two, the NA1 and EU1 lineages, have been found infecting trees in North American forests (Grünwald et al. 2012, 2016). In the spring of 2021, tanoak (Notholithocarpus densiflorus Manos, Cannon & Oh) displaying symptoms consistent with SOD were detected north of Port Orford (Curry County, Oregon). Symptoms were canopy dieback and blackened petiole and stem lesions on tanoak sprouts. The pathogen isolated on PAR (CMA plus 200 ml/L ampicillin, 10 mg/L rifamycin, 66.7 mg/L PCNB) selective media was determined to be P. ramorum based on characteristic morphology of hyphae, sporangia, and chlamydospores (Werres et al. 2001). Positive identification as P. ramorum was obtained with a lineage-specific LAMP assay targeting an NA2 orphan gene, indicating the presence of the NA2 lineage. NA2 was confirmed by sequencing a portion of the cellulose binding elicitor lectin (CBEL) gene using CBEL5U and CBEL6L primers (Gagnon et al. 2014). Sequences (GenBank accessions MZ733981 and MZ733982) were aligned against reference sequences for all lineages (Gagnon et al. 2014) confirming the presence of NA2. Lineage determination as NA2 was further confirmed at eleven SSR loci (ILVOPrMS145, PrMS39, PrMS9C3, ILVOPrMS79, KI18, KI64, PrMS45, PrMS6, ILVOPrMS131, KI82ab, and PrMS43) using the methods of Kamvar et al. (2015). We completed Koch's postulates using potted tanoaks, wound-inoculated at the midpoint of 1-year old stems with either hyphal plugs or non-colonized agar (n=4 per treatment). Tanoaks were maintained in a growth chamber (20°C-day / 18°C-night temperatures) with regular watering and an 18-photoperiod using F32T8 fluorescent bulbs (Phillips, Eindhoven, The Netherlands). After 7 days, brown to black lesions 1.2 to 2.9 cm in length were observed on the inoculated stems, from which P. ramorum was subsequently re-isolated; no symptoms were observed on the controls, and no pathogens were recovered when plating the wound sites in PAR. This is the first detection of the NA2 lineage causing disease in forests worldwide. The outbreak was found on private and public lands in forests typical to the SOD outbreak in Oregon (mixed conifer and tanoak), and was 33 km north of the closest known P. ramorum infestation. Follow-up ground surveys on adjacent lands have identified over 100 P. ramorum-positive tanoak trees, from which additional NA2 isolates have been recovered from bole cankers. NA2 is thought to be more aggressive than the NA1 lineage (Elliott et al. 2011), which has been present in Curry County since the mid-1990s (Goheen et al. 2017). Eradication of the NA2 lineage is being pursued to slow its further spread and prevent overlap with existing NA1 and EU1 populations. The repeated introductions of novel lineages into the western United States native plant communities highlights the vulnerability of this region to Phytophthora establishment, justifying continued monitoring for P. ramorum in nurseries and forests. References • Elliott, M, et al. 2011. For. Path. 41:7. https://doi.org/10.1111/j.1439-0329.2009.00627.x • Gagnon, M.-C., et al. 2014. Can. J. Plant Pathol. 36:367. https://doi.org/10.1080/07060661.2014.924999 • Goheen, E.M., et al. 2017. For. Phytophthoras 7:45. https://doi: 10.5399/osu/fp.7.1.4030 • Grünwald, N. J., et al. 2012. Trends Microbiol. 20:131. https://doi.org/10.1016/j.tim.2011.12.006 • Grünwald, N. J., et al. 2016. Plant Dis. 100:1024. https://doi.org/10.1094/PDIS-10-15-1169-PDN • Kamvar, Z.N. et al. 2015. Phytopath. 105:982. https://doi.org/10.1094/PHYTO-12-14-0350-FI • Van Poucke, K., et al. 2012. Fungal Biol. 116:1178. https://doi.org/10.1016/j.funbio.2012.09.003 • Werres, S., et al. 2001. Mycol. Res. 105: 1155. https://doi.org/10.1016/S0953-7562(08)61986-3.

Risk of Epidemic Development in Nurseries from Soil Inoculum of Phytophthora ramorum.

Soilborne inoculum arising from buried, infested leaf debris may contribute to the persistence of Phytophthora ramorum at recurrently positive nurseries. To initiate new epidemics, inoculum must not only survive but also produce sporangia during times conducive to infection at the soil surface. To assess this risk, we performed two year-long experiments in a soil plot at the National Ornamentals Research Site at Dominican University of California. Inoculated rhododendron leaf disks were buried at a depth of 5 or 15 cm in the early summer of 2014 or 2015. Inoculum was baited at the soil surface with noninfested leaf disks (2014 only) and then retrieved to assess pathogen viability and sporulation capacity every 5 weeks. Two 14-week-long trials were conducted in 2016. We were able to consistently culture P. ramorum over all time periods. Soil incubation rapidly reduced the capacity of inoculum to sporulate, especially at 5 cm; however, sporulation capacity increased with the onset of seasonally cooler temperatures. P. ramorum was baited most frequently between November and January, especially from inoculum buried at 5 cm 1 day before the baiting period; in January we also baited P. ramorum from inoculum buried at 15 cm the previous June. We validate prior observations that P. ramorum poses a greater risk after exposure to cooler temperatures and provide evidence that infested leaf debris plays a role in the perpetuation of P. ramorum in nurseries. This work provides novel insights into the survival and epidemic behavior of P. ramorum in nursery soils.

Diversification of plasmids in a genus of pathogenic and nitrogen-fixing bacteria.

Members of the agrobacteria-rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Is Disease Induced by Flooding Representative of Nursery Conditions in Rhododendrons Infected with Phytophthora cinnamomi or P. plurivora?

The degree of flooding commonly used to induce disease in Phytophthora root rot studies rarely occurs in container nurseries. Instead, over-irrigation and poor drainage result in plants periodically sitting in shallow pools of water. Rhododendron plants were grown in a noninfested substrate or substrate infested with Phytophthora cinnamomi or P. plurivora to determine whether root rot induced by flooding represents disease that occurs under simulated nursery conditions when plants are in a shallow pool of water (saucers), or are allowed to freely drain and maintained at ∼75% container capacity (CC). Generally P. cinnamomi caused more disease than P. plurivora, and all water treatments were conducive to root rot. In experiment 1, the amount of disease caused by flooding was similar to that in the saucer treatment (75% CC not tested) while in experiment 2, flooding often caused more rapid and severe disease than the saucer or 75% CC treatment. Pathogens differed in their response to water treatments. P. cinnamomi caused more disease in treatments with >90% substrate moisture for either a short (flood) or long duration (saucer), while P. plurivora was less capable of causing disease when soil moisture was maintained >90% than when substrate moisture was maintained at a more moderate level (flood, 75% CC). Our results indicate that it is not necessary to flood plants to induce disease under experimental conditions and that disease induced by flooding can represent disease in container nurseries when containers are in pools of water or maintained at ∼75% CC. In addition, our results suggest that P. cinnamomi is a more aggressive pathogen than P. plurivora in nursery conditions where drainage is poor; however, both species are capable of causing a similar amount of disease under more typical irrigation management.

Phylogeography of the wide-host range panglobal plant pathogen Phytophthora cinnamomi.

Various hypotheses have been proposed regarding the origin of the plant pathogen Phytophthora cinnamomi. P. cinnamomi is a devastating, highly invasive soilborne pathogen associated with epidemics of agricultural, horticultural and forest plantations and native ecosystems worldwide. We conducted a phylogeographic analysis of populations of this pathogen sampled in Asia, Australia, Europe, southern and northern Africa, South America, and North America. Based on genotyping-by-sequencing, we observed the highest genotypic diversity in Taiwan and Vietnam, followed by Australia and South Africa. Mating type ratios were in equal proportions in Asia as expected for a sexual population. Simulations based on the index of association suggest a partially sexual, semi-clonal mode of reproduction for the Taiwanese and Vietnamese populations while populations outside of Asia are clonal. Ancestral area reconstruction provides new evidence supporting Taiwan as the ancestral area, given our sample, indicating that this region might be near or at the centre of origin for this pathogen as speculated previously. The Australian and South African populations appear to be a secondary centre of diversity following migration from Taiwan or Vietnam. Our work also identified two panglobal, clonal lineages PcG1-A2 and PcG2-A2 of A2 mating type found on all continents. Further surveys of natural forests across Southeast Asia are needed to definitively locate the actual centre of origin of this important plant pathogen.

Genomic Approaches to Plant-Pathogen Epidemiology and Diagnostics.

Diseases have a significant cost to agriculture. Findings from analyses of whole-genome sequences show great promise for informing strategies to mitigate risks from diseases caused by phytopathogens. Genomic approaches can be used to dramatically shorten response times to outbreaks and inform disease management in novel ways. However, the use of these approaches requires expertise in working with big, complex data sets and an understanding of their pitfalls and limitations to infer well-supported conclusions. We suggest using an evolutionary framework to guide the use of genomic approaches in epidemiology and diagnostics of plant pathogens. We also describe steps that are necessary for realizing these as standard approaches in disease surveillance.

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

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.

Phytophthora Species Differ in Response to Phosphorous Acid and Mefenoxam for the Management of Phytophthora Root Rot in Rhododendron.

Phytophthora root rot, caused by many soilborne Phytophthora spp., is a significant disease affecting the $42 million rhododendron nursery industry. Rhododendron growers have increasingly reported failure by two systemic fungicides, phosphorous acid and mefenoxam, to adequately control root rot. Both fungicides may be applied as a foliar spray or soil drench but it is unknown how application method, fungicide chemistry, or pathogen diversity affects disease control. Therefore, two experiments were conducted to (i) determine whether differences in application method or fungicide chemistry affect control of root rot caused by P. cinnamomi and P. plurivora and (ii) evaluate the sensitivity of Phytophthora spp. and isolates from the rhododendron industry to each fungicide. Results demonstrated that soil drenches of either fungicide were more effective than foliar sprays for control of P. cinnamomi but were ineffective for P. plurivora. Furthermore, Phytophthora spp. and isolates varied in sensitivity to phosphorous acid and mefenoxam, and there were multiple fungicide-insensitive isolates, especially within P. plurivora. Differences in sensitivity were also observed among isolates from different nurseries and production systems, with some nurseries having less sensitive isolates than others and with container systems generally having less sensitive isolates than field systems. Our results provide three potential reasons for why fungicide control of Phytophthora root rot might fail: (i) the fungicide can be applied to the wrong portion of the plant for optimal control, (ii) there are differences in fungicide sensitivity among soilborne Phytophthora spp. and isolates infecting rhododendron, and (iii) fungicide-insensitive isolates are present in the rhododendron nursery industry.