Phylogeography, origin and population structure of the self-fertile emerging plant pathogen Phytophthora pseudosyringae.

Phytophthora pseudosyringae is a self-fertile pathogen of woody plants, particularly associated with tree species from the genera Fagus, Notholithocarpus, Nothofagus and Quercus, which is found across Europe and in parts of North America and Chile. It can behave as a soil pathogen infecting roots and the stem collar region, as well as an aerial pathogen infecting leaves, twigs and stem barks, causing particular damage in the United Kingdom and western North America. The population structure, migration and potential outcrossing of a worldwide collection of isolates were investigated using genotyping-by-sequencing. Coalescent-based migration analysis revealed that the North American population originated from Europe. Historical gene flow has occurred between the continents in both directions to some extent, yet contemporary migration is overwhelmingly from Europe to North America. Two broad population clusters dominate the global population of the pathogen, with a subgroup derived from one of the main clusters found only in western North America. Index of association and network analyses indicate an influential level of outcrossing has occurred in this preferentially inbreeding, homothallic oomycete. Outcrossing between the two main population clusters has created distinct subgroups of admixed individuals that are, however, less common than the main population clusters. Differences in life history traits between the two main population clusters should be further investigated together with virulence and host range tests to evaluate the risk each population poses to natural environments worldwide.

Cataloging Phytophthora Species of Agriculture, Forests, Horticulture, and Restoration Outplantings in California, U.S.A.: A Sequence-Based Meta-Analysis.

California contains a diverse flora, and knowledge of the pathogens that threaten those plants is essential to managing their long-term health. To better understand threats to California plant health, a meta-analysis of Phytophthora detections within the state was conducted using publicly available sequences as a primary source of data rather than published records. Accessions of internal transcribed spacer (ITS) ribosomal DNA were cataloged from 800 Californian Phytophthora isolates, analyzed, and determined to correspond to 80 taxa, including several phylogenetically distinct provisional species. A number of Phytophthora taxa not previously reported from California were identified, including 20 described species. Pathways of introduction and spread were analyzed by categorizing isolates' origins, grouped by land-use: (i) agriculture, (ii) forests and other natural ecosystems, (iii) horticulture and nurseries, or (iv) restoration outplantings. The pooled Phytophthora metacommunities of the restoration outplantings and horticulture land-use categories were the most similar, whereas the communities pooled from forests and agriculture were least similar. Phytophthora cactorum, P. pini, P. pseudocryptogea, and P. syringae were identified in all four land-use categories, while 13 species were found in three. P. gonapodyides was the most common species by number of ITS accessions and exhibited the greatest diversity of ITS haplotypes. P. cactorum, P. ramorum, and P. nicotianae were associated with the greatest number of host genera. In this analysis, the Phytophthora spp. most prevalent in California differ from those compiled from the scientific literature.

First Record of Colletotrichum anthrisci Causing Anthracnose on Avocado Fruits in Chile.

Anthracnose caused by Colletotrichum species is one of the most frequent and damaging fungal diseases affecting avocado fruits (Persea americana Mill.) worldwide. In Chile, the disease incidence has increased over the last decades due to the establishment of commercial groves in more humid areas. Since 2018, unusual symptoms of anthracnose have been observed on Hass avocado fruits, with lesions developing a white to gray sporulation. Morphological features and multi-locus phylogenetic analyses using six DNA barcodes (act, chs-1, gapdh, his3, ITS, and tub2) allowed the identification of the causal agent as Colletotrichum anthrisci, a member of the dematium species complex. Pathogenicity was confirmed by inoculating healthy Hass avocado fruits with representative isolates, reproducing the same symptoms initially observed, and successfully reisolating the same isolates from the margin of the necrotic pulp. Previously, several Colletotrichum species belonging to other species complexes have been associated with avocado anthracnose in other countries. To our knowledge, this is the first record of C. anthrisci and of a species of the dematium species complex causing anthracnose on avocado fruits in Chile and worldwide.

A Barcode-Based Phylogenetic Characterization of Phytophthora cactorum Identifies Two Cosmopolitan Lineages with Distinct Host Affinities and the First Report of Phytophthora pseudotsugae in California.

A collection of 30 Phytophthora cactorum and 12 P. pseudotsugae (subclade 1a) strains isolated from several recent surveys across California was phylogenetically compared to a worldwide collection of 112 conspecific strains using sequences from three barcoding loci. The surveys baited P. cactorum from soil and water across a wide variety of forested ecosystems with a geographic range of more than 1000 km. Two cosmopolitan lineages were identified within the widespread P. cactorum, one being mainly associated with strawberry production and the other more closely associated with apple orchards, oaks and ornamental trees. Two other well-sampled P. cactorum lineages, including one that dominated Californian restoration outplantings, were only found in the western United States, while a third was only found in Japan. Coastal California forest isolates of both Phytophthora species exhibited considerable diversity, suggesting both may be indigenous to the state. Many isolates with sequence accessions deposited as P. cactorum were determined to be P. hedraiandra and P. ×serendipita, with one hybrid lineage appearing relatively common across Europe and Asia. This study contains the first report of P. pseudotsugae from the state of California and one of the only reports of that species since its original description.

Identification and Characterization of Phytophthora Species Associated with Crown and Root Rot of Pistachio Trees in California.

Pistachio is one of the most widely cultivated nut crops in California, with approximately 115,000 ha of bearing pistachio trees. In recent years, several orchards were identified, with declining trees leading to substantial tree losses. Symptoms included trees with poor vigor, yellowing and wilting of leaves, crown rot, and profuse gumming on the lower portion of trunks. Thirty-seven Phytophthora-like isolates were obtained from crown rot tissues in the rootstock of grafted pistachio trees and characterized by means of multilocus phylogeny comprising internal transcribed spacer rDNA, beta-tubulin, and mt cox1 sequence data. The analysis provided strong support for the delineation and identification of three Phytophthora species associated with declining pistachio trees, including P. niederhauserii, P. mediterranea, and Phytophthora taxon walnut. Pathogenicity studies in potted University of California Berkeley I (UCBI) rootstocks (clonal and seeded) confirmed that all three Phytophthora species can cause crown and root rot of pistachio, thus fulfilling Koch's postulates. The widespread occurrence of Phytophthora crown rot in recently planted pistachio orchards and the susceptibility of UCBI rootstocks suggest this disease constitute an emerging new threat to the pistachio industry of California. To the best of our knowledge, this study is the first to report P. niederhauserii, P. mediterranea, and Phytophthora taxon walnut as causal agents of crown and root rots of pistachio.

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.

The population of oomycetes in a recycled irrigation water system at a horticultural nursery in southern California.

Recapture and recycling of irrigation water is often required to meet enormous water demands at horticultural nurseries. We tested four water types associated with a recycled irrigation system at a commercial container nursery in southern California for presence of oomycete plant pathogens from July 2015 to December 2017. These water types included: the main source of water originating from a reservoir, retention water from an on-site collection pond, irrigation water received by different growing areas within the nursery, and irrigation runoff captured in polyethylene sheet-lined runoff channels. The genera Phytophthora, Pythium, and Phytopythium together contributed more than 85% of the total oomycete population detected in the recycled irrigation system. The Phytophthora and Pythium genera were represented by member species from nine (1-4, 6-10) and eight (A, B, D-F, H-J) different sub-generic clades, respectively. Incoming water sourced from the reservoir was found to harbor known plant pathogens such as Phytophthora citricola-complex, P. capsici-cluster, P. tropicalis,P citrophthora-cluster, P. nemorosa-cluster, P. riparia, P. cryptogea-complex, P. parsiana-cluster, P. sp. nov. aff. kernoviae, Pythium dissotocum-complex, Py. oligandrum-cluster, Py. irregulare, and Phytopythium litorale. Runoff water showed the highest oomycete species richness and frequency of detection with both filtration and leaf baiting methods. In addition to plant pathogens, oomycete fish pathogens such as Aphanomyces laevis, Pythium chondricola-complex, Pythium flevoense-complex, and Saprolegnia diclina-complex were also detected in greater abundance in the recycled irrigation water. The oomycete species richness in the runoff water was correlated with several environmental parameters such as soil temperature. Greater oomycete richness in incoming water was associated with higher soil temperatures, whereas richness in runoff declines with increasing soil temperature, likely suggesting connections to weather-dependent nursery operations.

Genetic and phenotypic variation of Phytophthora crassamura isolates from California nurseries and restoration sites.

Phenotypic and sequence data were used to characterize 28 isolates resembling Phytophthora megasperma from 14 host species in 2 plant production facilities and 10 restoration sites across the San Francisco Bay Area (California; USA). Size of the oogonia and DNA sequences (nuclear internal transcribed spacer (ITS) and mitochondrial cytochrome c oxidase subunit 1 (COX 1)) were compared, and sensitivity to mefenoxam and pathogenicity were measured. Based on ITS 61 % of isolates matched ex-type sequences of Phytophthora crassamura from Italy, and the remainder matched or were close to the P. megasperma ex-type. However, all California P. crassamura genotypes belonged to four unique COX 1 haplotype lineages isolated from both nurseries and restoration sites. Although lineages were sensitive to mefenoxam, a significant difference in sensitivity was identified, and all continued growth in-vitro. These results suggested previous mefenoxam exposure in plant production facilities resulting in tolerance. In conclusion, all evidence pointed to a nursery origin of novel P. crassamura lineages found in California restoration sites. In this study, COX 1 sequences and oogonia size provided information relevant to identify geographic and evolutionary intraspecific variation within P. crassamura, and was additionally used to track the spread of this species from nurseries into wildlands.

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.

Characterizing the Brown Spot Pathosystem in Late-Harvest Table Grapes (Vitis vinifera L.) in the California Central Valley.

Brown spot, caused by Cladosporium spp., is becoming a problematic postharvest disease of late season table grape (Vitis vinifera) in the California central valley, and management is hindered by knowledge gaps in disease etiology and epidemiology. Brown spot is herein described as a pre- and postharvest dry rot typified by an external brown to black spot or black mycelium which encases the placenta. Isolates in the Cladosporium herbarum and C. cladosporioides species complexes were recovered from 85 and 5% of brown-spot affected berries, respectively. Five isolates in the C. herbarum species complex, representing three phylogenetically distinct species (C. limoniforme, C. ramotenellum, and C. tenellum), and one C. cladosporioides isolate all caused brown spot symptoms under cold-storage conditions, with and without mechanical wounding. Isolate virulence was similar (P > 0.05) based on disease incidence and severity on intact berries but severity varied on wounded berries (P < 0.001). Surface disinfestation reduced severity of cluster rot development following 2 weeks in cold storage (P = 0.027) but incidence was not affected (P = 0.17). This work provides foundational information on brown spot pathosystem etiology and biology in late-harvest table grape, which can be used to improve management.