Phytophthora : taxonomic and phylogenetic revision of the genus.

Many members of the Oomycota genus Phytophthora cause economic and environmental impact diseases in nurseries, horticulture, forest, and natural ecosystems and many are of regulatory concern around the world. At present, there are 223 described species, including eight unculturable and three lost species. Twenty-eight species need to be redescribed or validated. A lectotype, epitype or neotype was selected for 20 species, and a redescription based on the morphological/molecular characters and phylogenetic placement is provided. In addition, the names of five species are validated: P. cajani, P. honggalleglyana (Synonym: P. hydropathica), P. megakarya, P. pisi and P. pseudopolonica for which morphology and phylogeny are given. Two species, P. ×multiformis and P. uniformis are presented as new combinations. Phytophthora palmivora is treated with a representative strain as both lecto- and epitypification are pending. This manuscript provides the updated multigene phylogeny and molecular toolbox with seven genes (ITS rDNA, ß-tub, COI, EF1α, HSP90, L10, and YPT1) generated from the type specimens of 212 validly published, and culturable species (including nine hybrid taxa). The genome information of 23 types published to date is also included. Several aspects of the taxonomic revision and phylogenetic re-evaluation of the genus including species concepts, concept and position of the phylogenetic clades recognized within Phytophthora are discussed. Some of the contents of this manuscript, including factsheets for the 212 species, are associated with the "IDphy: molecular and morphological identification of Phytophthora based on the types" online resource (https://idtools.org/tools/1056/index.cfm). The first version of the IDphy online resource released to the public in September 2019 contained 161 species. In conjunction with this publication, we are updating the IDphy online resource to version 2 to include the 51 species recently described. The current status of the 223 described species is provided along with information on type specimens with details of the host (substrate), location, year of collection and publications. Additional information is provided regarding the ex-type culture(s) for the 212 valid culturable species and the diagnostic molecular toolbox with seven genes that includes the two metabarcoding genes (ITS and COI) that are important for Sanger sequencing and also very valuable Molecular Operational Taxonomic Units (MOTU) for second and third generation metabarcoding High-throughput sequencing (HTS) technologies. The IDphy online resource will continue to be updated annually to include new descriptions. This manuscript in conjunction with IDphy represents a monographic study and the most updated revision of the taxonomy and phylogeny of Phytophthora, widely considered one of the most important genera of plant pathogens. Taxonomic novelties: New species: Phytophthora cajani K.S. Amin, Baldev & F.J. Williams ex Abad, Phytophthora honggalleglyana Abad, Phytophthora megakarya Brasier & M.J. Griffin ex Abad, Phytophthora pisi Heyman ex Abad, Phytophthora pseudopolonica W.W. Li, W.X. Huai & W.X. Zhao ex Abad & Kasiborski; New combinations: Phytophthora ×multiformis (Brasier & S.A. Kirk) Abad, Phytophthora uniformis (Brasier & S.A. Kirk) Abad; Epitypifications (basionyms): Peronospora cactorum Lebert & Cohn, Pythiacystis citrophthora R.E. Sm. & E.H. Sm., Phytophthora colocasiae Racib., Phytophthora drechsleri Tucker, Phytophthora erythroseptica Pethybr., Phytophthora fragariae Hickman, Phytophthora hibernalis Carne, Phytophthora ilicis Buddenh. & Roy A. Young, Phytophthora inundata Brasier et al., Phytophthora megasperma Drechsler, Phytophthora mexicana Hotson & Hartge, Phytophthora nicotianae Breda de Haan, Phytophthora phaseoli Thaxt., Phytophthora porri Foister, Phytophthora primulae J.A. Toml., Phytophthora sojae Kaufm. & Gerd., Phytophthora vignae Purss, Pythiomorpha gonapodyides H.E. Petersen; Lectotypifications (basionym): Peronospora cactorum Lebert & Cohn, Pythiacystis citrophthora R.E. Sm. & E.H. Sm., Phytophthora colocasiae Racib., Phytophthora drechsleri Tucker, Phytophthora erythroseptica Pethybr., Phytophthora fragariae Hickman, Phytophthora hibernalis Carne, Phytophthora ilicis Buddenh. & Roy A. Young, Phytophthora megasperma Drechsler, Phytophthora mexicana Hotson & Hartge, Phytophthora nicotianae Breda de Haan, Phytophthora phaseoli Thaxt., Phytophthora porri Foister, Phytophthora primulae J.A. Toml., Phytophthora sojae Kaufm. & Gerd., Phytophthora vignae Purss, Pythiomorpha gonapodyides H.E. Petersen; Neotypifications (basionym): Phloeophthora syringae Kleb., Phytophthora meadii McRae Citation: Abad ZG, Burgess TI, Bourret T, Bensch K, Cacciola S, Scanu B, Mathew R, Kasiborski B, Srivastava S, Kageyama K, Bienapfl JC, Verkleij G, Broders K, Schena L, Redford AJ (2023). Phytophthora: taxonomic and phylogenetic revision of the genus. Studies in Mycology 106: 259-348. doi: 10.3114/sim.2023.106.05.

Movement of Phytophthora spp. in Maryland's Nursery Trade.

Phytophthora spp. cause major losses in the nursery industry worldwide. However, a clear demonstration of the route of movement has not been previously shown. A survey of 10 Maryland nurseries was conducted over a 3-year period to investigate the presence of Phytophthora spp. on newly arrived plants, mainly from West Coast suppliers. Local nursery plants, irrigation water, and potting media were also sampled for Phytophthora spp. Isolates were identified using a combination of morphological characteristics and DNA sequencing. Species identified included Phytophthora cactorum, P. cambivora, P. cinnamomi, P. citrophthora, P. drechsleri, P. elongata, P. gonapodyides, P. hydropathica, P. irrigata, P. lacustris, P. multivora, P. nicotianae, P. pini, P. plurivora, and P. syringae. P. taxon pgchlamydo was also isolated from irrigation water. Eight of the abovementioned Phytophthora spp. were isolated in association with incoming material, indicating that the movement of these pathogens continues to occur. Asymptomatic plant material was the main route of introduction of Phytophthora spp. to Maryland nurseries. Results also indicated that several Phytophthora spp. could be found in Maryland nurseries in association with infested potting media of asymptomatic plants. Although P. ramorum was not detected, our surveys underscore the significance of nursery practices that allow introductions of these significant plant pathogens to new geographic locations.

Phomopsis Blight: A New Disease of Pieris japonica Caused by Phomopsis amygdali in the United States.

Pieris japonica, also known as Japanese andromeda, is an economically valuable broadleaf evergreen used in landscapes across the United States. From spring 2010 to 2012, P. japonica 'Mountain Fire' plants growing in Maryland nurseries were observed with a high incidence of stem canker, shoot dieback, and blight symptoms. Necrosis was evident on shoot tips and often advanced into lateral shoots, as well as to the crowns, leading to plant death. Phomopsis amygdali, known as a destructive pathogen of peach and almond, was consistently isolated from symptomatic plants. P. amygdali also caused similar symptoms on Mountain Fire test plants following inoculations. P. amygdali was consistently recovered and its identity was confirmed with both morphological and molecular tools, thus fulfilling Koch's postulates. In addition, nursery sampling in 2012 revealed that P. amygdali could also be isolated from asymptomatic plants. In all instances, infected plants were shipped from a West Coast nursery, indicating that this pathogen was inadvertently introduced to new locations. P. amygdali may be emerging as an important pathogen in nurseries because this is the first known association of this pathogen with an ornamental plant species.

Symptomatic and Asymptomatic Host Range of Fusarium virguliforme, the Causal Agent of Soybean Sudden Death Syndrome.

Sudden death syndrome, caused by Fusarium virguliforme, is an important disease of soybean in the United States. Fifteen species of crops, weeds, or prairie plants were evaluated for their potential as hosts of F. virguliforme. Root and foliar symptoms and plant biomass were assessed following greenhouse inoculation studies. Root colonization of F. virguliforme was determined with isolations and with polymerase chain reaction assays. Soybean, alfalfa, pinto and navy bean, white and red clover, pea, and Canadian milk vetch developed root necrosis. Soybean, alfalfa, and red clover also developed foliar symptoms following inoculation. Sugar beet and canola did not develop symptoms but had significant reductions in biomass, suggesting that they are also hosts of F. virguliforme. Corn, wheat, ryegrass, pigweed, and lambsquarters did not develop symptoms. However, these species appeared to be asymptomatic hosts because quantities of pathogen DNA detected in inoculated roots were similar to quantities detected in inoculated soybean roots. These results suggest that the number and diversity of hosts for F. virguliforme are greater than previously reported. The likely broad host range limits the efficacy of crop rotation and indicates that crops other than soybean can be damaged by F. virguliforme and maintain or increase inoculum in soil.

First Report of Clonostachys rosea Causing Root Rot of Soybean in the United States.

Multiple fungal species have been associated with root rot of soybean (Glycine max) in the United States, but root rot in Minnesota (MN) also occurs in plants not known to be infected with previously reported pathogens (1). Soybean plants that lacked foliar symptoms, but exhibited taproot and lateral root necrosis were observed in 15 fields from nine counties in MN during 2007 and 2008. Plants were arbitrarily dug up at the R3 growth stage in July as part of a root rot study. Roots were washed, surface disinfested in 0.5% NaOCl for 3 min, rinsed in deionized water, dried, and embedded in potato dextrose agar (PDA). Thirty isolates with morphological characteristics consistent with those of Clonostachys rosea were recovered in total from necrotic lesions on different plants from all fields (3). For further morphological characterization, cultures were grown on PDA for 1 week at 24°C in the dark. Colonies were 39 to 46 mm in diameter, yellowish-white, and the surface was felty to tomentose with thick aerial hyphae. Primary conidiophores were Verticillium-like with two to three levels. The stipe length measured 65 to 105 µm and the base width was 5 µm. Primary conidia were smooth, hyaline, slightly curved, with an average length and width of 7 to 9 × 2.6 to 3 µm. Secondary conidiophores were penicillate with two or three whorls of phialides. The stipe length measured 50 to 75 µm, base width was 5 µm, and penicillus height was 25 to 35 µm. Secondary conidia were 5 to 6 × 2.5 µm. Perithecia were not produced. The identity of isolates was confirmed by sequencing the internal transcribed spacer (ITS) locus using the primers ITS1F/ITS4. BLAST analysis of the sequences in the NCBI database resulted in a 99.8 to 100% match for both C. rosea and its teleomorph Bionectria ochroleuca (e.g., HM751081, GU256766). Each isolate was tested for pathogenicity on soybean by initially growing it on sterile sorghum grain for 2 weeks at 23°C. Sterile sorghum was used for control plants. Seeds of soybean 'AG2107' were planted in 11.4-cm square pots containing pasteurized potting mix and a 25-cm3 layer of infested or sterile sorghum placed ~1 cm below the seeds. Two replicate pots containing four plants each were used per treatment and the experiment was repeated once. Root rot was assessed 28 days after planting in a greenhouse at 23°C day and 18°C night with a 14-h photoperiod. Twenty-eight of 30 C. rosea isolates caused taproot necrosis on inoculated plants in both experiments, whereas control roots did not exhibit necrosis. Approximately 6% of inoculated plants also developed interveinal chlorosis and marginal necrosis on trifoliates. Isolations were attempted from roots of all plants, and the isolates recovered from inoculated plants were identified as C. rosea based on morphology and ITS sequences. This fungus was not isolated from control plants. C. rosea was also isolated from petioles of symptomatic trifoliates, indicating systemic colonization of the plants. To our knowledge, this is the first report of C. rosea causing root rot of soybean and systemically colonizing soybean. This fungus may have been previously isolated from asymptomatic soybean plants and identified as Gliocladium roseum (2). The impact of this fungus on soybean production is unknown. References: (1) G. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathological Society, St. Paul, MN, 1999. (2) J. D. Mueller and J. B. Sinclair. Trans. Brit. Mycol. Soc. 86:677, 1986. (3) H.-J. Schroers et al. Mycologia 91:365, 1999.

First Report of Fusarium redolens Causing Root Rot of Soybean in Minnesota.

Multiple Fusarium species have been found in association with soybean (Glycine max) plants exhibiting root rot in the United States (3). Soybean plants that lacked apparent foliar symptoms, but exhibited 2- to 5-mm brown, necrotic taproot lesions and lateral root necrosis were observed in Minnesota in one field each in Marshall and Otter Tail counties in July of 2007, as well as in one field in Marshall County in July of 2008. Sampling was conducted as part of a study investigating root rot in major soybean-production areas of Minnesota. Plants were arbitrarily dug up at the R3 growth stage. Root systems were washed, surface disinfested in 0.5% NaOCl for 3 min, rinsed in deionized water, and dried. Fusarium isolates were recovered from root sections with necrotic lesions embedded in modified Nash-Snyder medium (1). One resulting Fusarium colony from one plant per county was transferred to half-strength acidified potato dextrose agar (PDA) and carnation leaf agar (CLA) to examine morphological characteristics (4). Culture morphology on PDA consisted of flat mycelium with sparse white aerial mycelium. On CLA, thick-walled macroconidia with a hooked apical cell and a foot-shaped basal cell were produced in cream-colored sporodochia. Macroconidia ranged from 32.5 to 45.0 µm long. Microconidia were oval to cylindrical with 0 to 1 septa, ranged from 7.5 to 11.25 µm long, and were produced on monophialides. Chlamydospores were produced abundantly in chains that were terminal and intercalary in the hyphae of 4-week-old cultures. Morphological characteristics of the three isolates were consistent with descriptions of F. redolens (2,4). The identity of each isolate was confirmed by sequencing the translation elongation factor 1-α (TEF) locus (4). BLAST analysis of the TEF sequences from each isolate against the FUSARIUM-ID database resulted in a 100% match for 17 accessions of F. redolens (e.g., FD 01103, FD 01369). Each F. redolens isolate was tested for pathogenicity on soybean. Sterile sorghum grain was infested with each isolate and incubated for 2 weeks. Sterile sorghum was used for control plants. Soybean seeds of cv. AG2107 were planted in 11.4-cm pots ~1 cm above a 25-cm3 layer of infested sorghum or sterile sorghum. Two replicate pots containing four plants each were used per treatment and the experiment was repeated once. Root rot was assessed 28 days after planting. Each F. redolens isolate consistently caused taproot necrosis on inoculated plants, whereas control plants did not exhibit root necrosis. Isolations were made from roots of inoculated and control plants and the isolates recovered from inoculated plants were identified as F. redolens based on morphological characteristics and TEF sequences. Fusarium species were not isolated from control plants. To our knowledge, this is the first report of F. redolens causing root rot of soybean; however, it is possible F. redolens has been found previously and misidentified as F. oxysporum (2,4). Results from inoculations suggest that F. redolens may be an important root rot pathogen in Minnesota soybean fields. References: (1) J. C. Bienapfl et al. Acta Hortic. 668:123, 2004. (2) C. Booth and J. M. Waterston. No. 27 in: CMI Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, England, 1964. (3) G. L. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathological Society, St. Paul, MN, 1999. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

First Report of Infection of Maiden-Hair Fern (Adiantum jordanii and A. aleuticum) by Phytophthora ramorum in California.

During July 2005, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from nine native Adiantum jordanii plants growing at two forest sites (Samuel P. Taylor State Park, Marin County and Peachland Road, Mendocino County) and from seven A. aleuticum plants at one forest site (Peachland Road) in California. At both locations, symptomatic plants were distributed close to rivers and roads and in association with infected bay laurel trees (Umbellularia californica), toyon (Heteromeles arbutifolia), and tanoaks (Lithocarpus densiflorus). Symptomatic leaflets showed brown spots that sometimes coalesced, killing entire leaves, but the disease did not appear to be fatal to the ferns. Necrotic tissues were plated on PARP and maintained in the dark at 18°C for 1 to 2 weeks. Isolates were identified as P. ramorum on the basis of colony morphology, the presence of chlamydospores and caducous, semipapillate sporangia, and the internal transcribed spacer (ITS) rDNA sequences (1,2). The P. ramorum isolates, Pr-419 from A. jordanii and Pr-422 from A. aleuticum, have been deposited in the American Type Culture Collection (ATCC MYA-3677 and MYA-3679, respectively) and a region of the ITS rDNA sequence deposited in the NCBI database (GenBank Accession No. DQ173082 and DQ219821, respectively). To test the pathogenicity, the tips of freshly detached leaves of A. jordanii and A. aleuticum were dipped into a solution of 1 × 103 zoospores per ml of Pr-419 and Pr-422 for 1 min. The wounded end of the leaves was not exposed to the inoculum. The zoospores were produced by flooding agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar with sterile deionized water. After 3 days of incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then at room temperature for 60 min. For each Adiantum species and P. ramorum isolate, 15 leaves collected from five potted nursery plants were tested. Control leaves were dipped in sterile deionized water. Leaves were maintained in a moist chamber at 19°C with 13 h of natural light for 9 days. Brown lesions similar to those seen in the forest developed on approximately 60 and 33% of the A. jordanii and A. aleuticum leaves, respectively, inoculated with Pr-419 and on approximately 73 and 40% of the leaves inoculated with Pr-422. Under these experimental conditions, A. aleuticum appeared to be slightly more susceptible than the A. jordanii, with a necrotic leaf area of approximately 38% compared with 20%. The pathogen was reisolated on PARP after surface sterilization from all symptomatic leaves. Control leaves did not develop symptoms and P. ramorum was not recovered. A. jordanii and A. aleuticum have already been listed as associated hosts for P. ramorum on the APHIS (USDA Animal and Plant Health Inspection Service) website ( http://www.aphis.usda.gov/ ). To our knowledge, this is the first report of ferns as natural hosts of P. ramorum. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol Res. 105:1155, 2001.