Preventing Soil Inoculum of Calonectria pseudonaviculata from Splashing onto Healthy Boxwood Foliage by Mulching.

Boxwood blight, caused by Calonectria pseudonaviculata, is an emerging disease of great concern to horticulturists in the United States and other affected countries. The objective of this study was to evaluate the efficacy of mulching as a physical barrier to prevent soil inoculum from splashing onto healthy boxwood foliage. A field trial consisting of two treatments, mulched and nonmulched, was conducted under field conditions in Lowgap, North Carolina, and in a residential landscape setting near Richmond, Virginia, for 2 years at each site. Mulching efficacy was assessed by monitoring and comparing boxwood blight development on detector plants: containerized 'Justin Brouwers' boxwood, which were rotated through mulched and nonmulched plots at 1- and 2-week intervals in the Lowgap and Richmond sites, respectively. Boxwood blight was observed on detector plants in a combined 55 of the 88 monitoring periods during this study at the two sites. Mulching provided complete protection of Justin Brouwers boxwood from infection by C. pseudonaviculata soil inoculum during 33 of the 55 positive monitoring periods (60%) and good to excellent protection during 13 monitoring periods (24%). The potential applications of mulching for boxwood blight mitigation are discussed.

Effects of Inoculum Dose, Temperature, Cultivar, and Interrupted Leaf Wetness Period on Infection of Boxwood by Calonectria pseudonaviculata.

Boxwood blight is an emerging disease of great concern for the ornamental horticulture industry, historic garden managers, landscapers, and homeowners. Controlled-environment experiments were conducted to determine the effects of conidial concentration, temperature, interrupted leaf wetness period, cultivar, and leaf age on infection of boxwood leaves by Calonectria pseudonaviculata. Boxwood blight incidence (BBI) increased with increasing concentration up to 2.0 × 104 spores/ml. BBI also increased as temperature increased from 18 to 25°C, then declined gradually to zero at 29°C. Similar infection effects of inoculum concentration were observed in an experiment with four boxwood cultivars ('Justin Brouwers', 'John Baldwin', 'Green Mound', and 'Nana') of various degrees of susceptibility. The hypothesis that younger leaves are more susceptible than older leaves was supported for Justin Brouwers and Nana but not for Green Mound; and younger leaves of John Baldwin were less susceptible than older leaves. When inoculated plants ('Suffruticosa') were exposed to dry interruptions of 3 h or longer between 5 or 8 h of initial wetness and 12 h of additional wetness, these plants had significantly lower BBI compared with those exposed to continuous wetness for 20 h, and similar or at most slightly more infection than plants exposed to only the 5- or 8-h initial wetness. Continuous wetness durations beyond 20 h did not increase infection in these experiments. These results advanced our understanding of the environmental requirements of the infection process in boxwood blight development and they are essential for refining disease forecasting models.

A unique species in Phytophthora clade 10, Phytophthora intercalaris sp. nov., recovered from stream and irrigation water in the eastern USA.

A novel species of the genus Phytophthora was recovered during surveys of stream and nursery irrigation water in Maryland, Massachusetts, North Carolina, Virginia and West Virginia in the USA. The novel species is heterothallic, and all examined isolates were A1 mating type. It produced rare ornamented oogonia and amphigynous antheridia when paired with A2 mating type testers of Phytophthora cinnamomi and Phytophthora cryptogea. Sporangia of this novel species were non-papillate and non-caducous. Thin-walled intercalary chlamydospores were abundant in hemp seed agar and carrot agar, while they were produced only rarely in aged cultures grown in clarified V8 juice agar. Phylogenetic analyses based on sequences of the internal transcribed spacer region and the ß-tubulin and mitochondrial cytochrome-c oxidase 1 (cox1) genes indicated that the novel species is phylogenetically close to Phytophthora gallica in Phytophthora clade 10. The novel species has morphological and molecular features that are distinct from those of other species in Phytophthora clade 10. It is formally described here as Phytophthora intercalaris sp. nov. Description of this unique clade-10 species is important for understanding the phylogeny and evolution of Phytophthora clade 10.

Heat treatment induced bacterial changes in irrigation water and their implications for plant disease management.

A new heat treatment for recycled irrigation water using 48 °C for 24 h to inactivate Phytophthora and bacterial plant pathogens is estimated to reduce fuel cost and environmental footprint by more than 50 % compared to current protocol (95 °C for 30 s). The objective of this study was to determine the impact of this new heat treatment temperature regime on bacterial community structure in water and its practical implications. Bacterial communities in irrigation water were analyzed before and after heat treatment using both culture-dependent and -independent strategies based on the 16S ribosomal DNA. A significant shift was observed in the bacterial community after heat treatment. Most importantly, bacteria with biological control potential--Bacillus and Paenibacillus, and Pseudomonas species became more abundant at both 48 and 42 °C. These findings imply that the new heat treatment procedure not only controls existing plant pathogens but also may make the heat-treated irrigation water a more antagonistic environment against plant pathogens, promoting sustainable disease management.

Root and Stem Rot of Begonia Caused by Phytopythium helicoides in Virginia.

In the summer of 2011, severe root and stem rot of begonia (Begonia × semperflorens-cultorum cv. Vodka Dark Red) was observed during a field trial. Seventy-eight percent of the plants had symptoms included foliar blight, blackened and rotting roots, rotting stems, and collapsing crown, often leading to plant death. Isolation from the diseased plant roots consistently recovered a Pythium-like species and 41 isolates were subcultured for identification. These isolates produced very similar single-strand conformation polymorphism (SSCP) fingerprints (2), which were distinct from those of other oomycete pathogens known to attack begonia (1). These isolates produced proliferous, ovoid to globose, terminal, and papillate sporangia which were 30.6 to 45.4 µm (av. 38.7 µm) in length and 20.5 to 35.4 µm (av. 28.2 µm) in width. Oogonia were produced in single culture grown in clarified V8 juice agar. These smooth-walled oogonia were mostly aplerotic and 28.9 to 36.8 µm (av. 33.1 µm) in diameter. Each contained a single oospore with a diameter of 23.7 to 34.4 µm (av. 26.9 µm). Single to multiple antheridia were attached lengthwise to each oogonium. These morphological characteristics match the description of Phytopythium helicoides (= Pythium helicoides) (3). The identity of these isolates was confirmed by sequencing the rDNA internal transcribed spacer (ITS) 1 and 2 regions. ITS sequence of the representative isolate 55C7 (GenBank Accession No. KC907734) had 97 to 99% homology with P. helicoides sequences in GenBank. Two isolates, 55C7 and 56A7, were tested for pathogenicity to begonia in the summer of 2012. Twelve plants per isolate were inoculated by injecting ground P. helicoides-colonized rice grains into the root soil using a long-neck funnel. Sterile rice grains were used on control plants. Aboveground symptoms including foliar blight, stem rot, and collapsing crown were observed 7 days after inoculation and the disease progressed for additional 6 weeks. At 7 weeks, all inoculated plants showed different symptom levels. Four and 10 plants inoculated with 55C7 and 56A7, respectively, were already dead. Begonia roots showed severe symptoms including blackening, stunted growth, and rotting. Seven of 12 control plants also had notable symptoms due to cross contamination. Isolates recovered from all symptomatic plants had identical SSCP fingerprints to those of isolates 55C7 and 56A7. To our knowledge, this is the first report of P. helicoides attacking begonia plants. The avenue of this pathogen entering the 2011 field trial remains unknown. The field trial in 2011 and pathogenicity test in 2012 indicate that this pathogen is potentially destructive to begonia. Additional research is warranted to identify the origin and dissemination of this pathogen to mitigate the risk to begonia production. References: (1) C. X. Hong et al. Plant Dis. 92: 1201, 2008. (2) P. Kong et al. FEMS Microbiol. Lett. 240:229, 2003. (3) A. J. van der Plaats-Niterink. Monograph of the Genus Pythium. Centraalbureau voor Schimmelcultures, Baarn, the Netherlands, 1981.

Diversity and Mefenoxam Sensitivity of Phytophthora spp. Associated with the Ornamental Horticulture Industry in the Southeastern United States.

Phytophthora isolates associated with ornamental plants or recovered from irrigation water in six states in the southeastern United States (Georgia, North Carolina, South Carolina, Tennessee, Texas, and Virginia) were identified and screened for sensitivity to mefenoxam. Isolates from forest and suburban streams in Georgia and Virginia were included for comparison. A new in vitro assay, utilizing 48-well tissue culture plates, was used to screen for mefenoxam sensitivity; this assay allowed high throughput of isolates and used less material than the traditional petri plate assay. In total, 1,483 Phytophthora isolates were evaluated, and 27 species were identified with Phytophthora nicotianae, P. hydropathica, and P. gonapodyides, the most abundant species associated with plants, irrigation water, and streams, respectively. Only 6% of isolates associated with plants and 9% from irrigation water were insensitive to mefenoxam at 100 µg a.i./ml. Approximately 78% of insensitive isolates associated with plants were P. nicotianae, and most of these (67%) came from herbaceous annual plants. Most of the insensitive isolates recovered from irrigation water were P. gonapodyides, P. hydropathica, P. megasperma, and P. pini, and 83% of the insensitive isolates from streams were P. gonapodyides. Overall, this study suggests that mefenoxam should continue to be a valuable tool in the management of Phytophthora diseases affecting ornamental plants in the southeastern United States.

Phytophthora hedraiandra Detected from Irrigation Water at a Perennial Ornamental Plant Nursery in Virginia.

Water survey for Phytophthora spp. by baiting with rhododendron leaves in April 2006 at a perennial ornamental plant nursery in Virginia detected five isolates showing a unique, previously unknown single-strand conformation polymorphism (SSCP) fingerprint (1). These cultures were isolated from two reservoirs at different depths of water column from surface to 2 m. They were homothallic and produced smooth-surfaced spherical oogonia with an average diameter of 27 µm on 10% V8 agar. Oospores were aplerotic. The paragynous antheridia were averaging 12 µm in diameter. Sporangia were papillate, spherical to ovoid, averaging 39 by 28 µm (length by width). They were caducous with short (<4 µm) pedicels. Chlamydospores and hyphal swellings were not observed. Two isolates were sequenced for rDNA internal transcribed spacer (ITS) 1 and 2 regions and cytochrome oxidase subunit 1 (Cox 1) gene. ITS sequences of both isolates (GenBank Accession Nos. JN376065 and JN376066) were identical to that of Phytophthora hedraiandra type culture (GenBank Accession No. AY707987). Also, the Cox 1 sequence of an isolate (Accession No. JN376067) had 99% homology with that of the type culture (GenBank Accession No. AY69115). Pathogenicity of both isolates was tested on Rhododendron catawbiense and Viburnum tinus, two known hosts of P. hedraiandra (2). For each isolate and host, five leaves and stems on potted plants were wounded by needles and then inoculated by placing over each wound a 5-mm2 mycelial plug from a 7-day-old culture and securing with Parafilm. V8 agar was used instead of mycelial plugs on control plants. After inoculation, each plant was enclosed in a plastic bag for 1 day and then incubated at 22°C with a 12-h photoperiod. Distilled water was sprayed daily for 5 days postinoculation (dpi) until disease symptoms were observed. At 15 dpi, 3 of the 10 inoculated rhododendron leaves and 6 of the 10 stems showed leaf lesions, wilting, dieback, and cankers, eventually leading to rhododendron death. Two of the 10 viburnum leaves and 4 of the 10 stems showed similar symptoms. Leaf lesions were approximately 3 to 5 cm in diameter. P. hedraiandra was recovered from diseased tissues and all resulting cultures showed an identical SSCP fingerprint to tested isolates as well as a P. hedraiandra isolate from Minnesota (3). No symptom developed on control plants. To our knowledge, this is the first report of P. hedraiandra in Virginia. Considering neither host plant has been grown or bought for resale by this nursery, this study indicates that P. hedraiandra may have a wider host range than is currently known. This possibility and the importance of water dispersal for P. hedraiandra in disease epidemiology warrant further investigation. References: (1) P. Kong et al. Fungal Genet. Biol. 39:238, 2003. (2) W. A. Man in't Veld et al. Eur. J. Plant Pathol. 117:25, 2007. (3) B. W. Schwingle et al. Plant Dis. 90:109, 2006.

Population Structure, Mating Type, and Mefenoxam Sensitivity of Phytophthora nicotianae in Virginia Tobacco Fields.

Black shank is an important disease of tobacco (Nicotiana tabacum) caused by the fungus-like organism, Phytophthora nicotianae. Three physiological races (0, 1, and 3) have been documented in the United States. Shifts in the pathogen population structure have become a concern due to the widespread use of cultivars possessing resistance to race 0 arising from a single gene (Php or Phl). A comprehensive statewide survey conducted throughout major tobacco-growing areas during summer 2006 and supplemented by additional isolates in 2007 and 2008 yielded 217 isolates from flue-cured, burley, and dark fire-cured tobacco fields. After determining species identity using a single-strand conformational polymorphism fingerprinting technique, the race identity of isolates was assessed via greenhouse tests using three differential cultivars (Hicks, L8, and NC1071). Approximately 76% of the isolates belonged to race 1, 21% to race 0, and the remaining 3% were race 3. This race structure was comparable with those in the other tobacco-producing states in the United States. Approximately 94% of isolates belonged to A2 mating type and merely 6% were A1. These data suggest that it is unlikely that sexual recombination serves as a major mechanism enhancing the genetic diversity of the pathogen in Virginia. All isolates were also evaluated against mefenoxam at 5 µg/ml. None were insensitive; 98% of isolates were either highly sensitive or sensitive and the remaining 2% were intermediately sensitive. These results indicate that mefenoxam remains effective for control of black shank in Virginia. The results of this study can assist breeders to develop cultivars possessing the most appropriate set of disease resistance traits, as well as extension specialists, county agents, and tobacco growers in their decision-making process to manage tobacco black shank in Virginia.

Foliar Blight of Annual Vinca (Catharanthus roseus) Caused by Phytophthora tropicalis in Virginia.

Annual vinca (Catharanthus roseus), also known as Madagascar periwinkle, has been cultivated as a major color crop for landscape and as an herbal medicine. This plant performs well in dry, warm locations with full sun or partial shade. Two-month-old diseased plants (cv. First Kiss Blueberry) with blighted leaves and stems were received from a local nursery in Suffolk, VA in May 2009. The disease began with dark, greenish black lesions on young leaves. Lesions gradually became tan or brown and leaves wilted and curled and finally turned necrotic. Brown, sunken lesions beginning at the branching points were typical symptoms on blighted stems and shoots. Blighted areas and spots were approximately 30 to 90 mm long and 20 to 40 mm wide on leaves and 40 mm long on stems. A Phytophthora species previously unknown to attack this plant was consistently isolated from diseased leaves and stems, and resultant isolates were grown on PARP-V8 agar. These isolates produced papillate sporangia on umbellate sympodium. Sporangia were mostly ellipsoid with a length/breadth ratio of >1.8 and tapered base; they were caducous with a long pedicel (usually >50 µm). These isolates also produced chlamydospores that averaged 31 µm in diameter. The isolates were identified as Phytophthora tropicalis by morphology. The identity was confirmed by DNA fingerprinting (1) and sequence analysis of ribosomal DNA internal transcribed spacers (GenBank Accession No. GQ478707). For pathogenicity studies, a zoospore suspension of 1,100 spores per milliliter was sprayed onto the foliage of 50-day-old healthy plants of the same cultivar grown in pine bark medium in six 10-cm-diameter plastic containers with a hand mister in the evening until runoff. Control plants were sprayed with tap water. Plants were placed in a tray containing a small amount of water and enclosed in plastic tents overnight to facilitate infection. The tents were removed the following morning, and plants were kept in a greenhouse under natural light and watered as needed. Within 4 days, all six inoculated plants developed foliar symptoms similar to what was observed on the diseased plant samples from the production nursery. The pathogen was reisolated from infected leaves and stems and its identity was confirmed by colony PCR-single-strand conformation polymorphism (3). The pathogenicity test was repeated once with the same results. Phytophthora aerial blight is a common destructive disease of annual vinca, which is usually caused by P. nicotianae. To our knowledge, this is the first report of foliar blight caused by P. tropicalis on annual vinca in Virginia. According to the head grower who submitted the disease samples, this new disease caused 10% crop loss of annual vinca this past spring. P. tropicalis was previously reported to attack ornamental shrubs, including Pieris japonica and Rhododendron catawbiense (2). This study indicates that P. tropicalis could be a potential threat to herbaceous annual crops as well. References: (1) M. E. Gallegly and C. X. Hong. Phytophthora: Identifying Species by Morphology and DNA Fingerprints. The American Phytopathological Society, St. Paul, MN, 2008. (2) C. Hong et al. Plant Dis. 90:525, 2006. (3) P. Kong et al. J. Microbiol. Methods 61:25, 2005.

The avocado subgroup of Phytophthora citricola constitutes a distinct species, Phytophthora mengei sp. nov.

Isolates from avocado tree cankers have been recognized as a distinct subgroup within the P. citricola complex since 1974, both morphologically and molecularly (isozyme and amplified fragment length polymorphism [AFLP] analyses). This subgroup is formally separated from P. citricola after comparative DNA fingerprinting and sequence analyses of the ITS region, as well as by morphological examinations. This new taxon is homothallic, produces plerotic oospores with paragynous antheridia and noncaducous semipapillate sporangia. Morphologically it differs from other species of Waterhouse group III by producing many large bizarre-shaped sporangia and smaller oogonia with asymmetric capitate antheridia. It belongs to clade 2 and is phylogenetically closer to P. siskiyouensis, P. capsici and P. tropicalis than to P. citricola. P. mengei can be easily differentiated from its relatives in the same clade and other species of this morpho-group by DNA fingerprints and sequence analysis. This new taxon is named Phytophthora mengei sp. nov.

A novel indicator plant to test the hypersensitivity of phytopathogenic bacteria.

Hypersensitive response is an important, definitive test to separate plant pathogenic bacteria from saprophytes. The current standard protocol uses tobacco and four o'clock plants as indicators for Gram negative and Gram positive phytopathogenic bacteria, respectively, and inoculation is accomplished by infiltrating bacterial suspensions into intact leaves. Both plants, especially the four o'clock, have thin leaves which make inoculation difficult, sometimes leading to inaccurate tests. Here we propose the use of Sedum hybridum as an alternative indicator plant. Sedum plants are readily available, easy to propagate and fast growing. Their leaves are much thicker, thus infiltration of a bacterial suspension is easier compared to tobacco and four o'clock. Additionally, sedum plants can be used universally to test both Gram negative and Gram positive phytopathogenic bacteria.

Downy Mildew of Rudbeckia fulgida cv. Goldsturm by Plasmopara halstedii in Virginia.

A severe foliage disease was observed on black-eyed Susan (Rudbeckia fulgida cv. Goldsturm) at a commercial nursery in Virginia Beach, VA in early June of 2005 and 2006. In both years, disease symptoms began after plants were transferred from 1-liter (1quart) to 4-liter (1-gallon) containers. Several hundred 'Goldsturm' plants were grown at this nursery but none was marketable because of this disease. Typical symptoms were dark, blotchy necrotic areas on the upper leaf surface. Fuzzy, grayish white zoosporangiophores and zoosporangia were commonly produced on the underside of affected leaves. These disease signs also were observed on both sides of new leaves in severely affected plants. Zoosporangiophores were monopodially branched at right angles with acutely tapering termini. Zoosporangia were ovoid to elliptical, hyaline, and measured 19 to 28 × 17 to 21 µm. On the basis of these morphological characters and host specificity, the organism was identified as Plasmopara halstedii (Farl.) Berl. & De Toni in Sacc. Inoculum at 1.4 × 104 zoosporangia/ml was prepared by washing diseased leaves with deionized reverse osmosis water and applied with a hand sprayer onto the foliage of test plants until runoff. Control plants were treated with deionized reverse osmosis water only. Inoculated and control plants were placed in plastic bags to keep moist for 48 h at 20°C. The inoculated plants developed the same symptoms with fresh crops of the zoosporangiophores and zoosporangia of P. halstedii on the underside of leaves, while the control plants remained healthy 14 days after treatment. The source of the primary inoculum is not known. Interestingly, none of 12 other Rudbeckia spp. and cultivars (R. hirta cvs. Autumn Colors, Becky, Cherokee Sunset, Indian Summer, Irish Eye, Prarie Sun, and Sonora, R. laciniata, R. maxima, R. missouriensis, R. occidentalis cv. Green Wizard, and R. speciosa cv. Viette's Little) grown at the same nursery adjacent to those severely diseased 'Goldsturm' plants was affected. Although downy mildew was reported on rudbeckias in north Florida previously (1), to my knowledge, this is the first report from Virginia. This pathogen attacks a number of plants in the genera of Centaurea, Coreopsis, Erigeron, Helianthus, and Verbena in addition to Rudbeckia spp. (2). Thus, it could significantly impact the ornamental plant nursery industry. References: (1) H. Dankers et al. Plant Health Progress. Online publication. doi:10.1094/PHP-2004-0119-01-HN, 2004. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989.

Effects of Zoospore Concentration and Application Pressure on Foliage Blight of Catharanthus roseus Caused by Phytophthora nicotianae.

This study investigated the effects of inoculum concentration, application pressure, use of carbon dioxide (CO2), and agitation associated with passage through a spray nozzle on zoospore survival and on foliage blight caused by Phytophthora nicotianae. In a greenhouse experiment, plants of Catharanthus roseus were inoculated by spraying zoospore suspensions at pressures of 210, 420, and 630 kPa (30, 60, and 90 lbs/in2 [psi]). A low-pressure 35-kPa (5-psi) control inoculation was provided with a hand-operated aspirator-type mister. There was a consistent reduction in level of disease with increased application pressure, regardless of the zoospore concentration. To determine the source of this disease reduction, laboratory assays were conducted. Zoospore suspensions were pressurized to 210, 420, and 630 kPa with CO2 or air, then transferred from the pressure bottle into a flask by either spraying or pouring. From the flask, the suspensions were spread over plates of PARP-V8 agar and incubated for 72 h, at which time total colony numbers were recorded. CO2 significantly reduced zoospore survival. Pressure strength and method of spore transfer out of pressure bottles also impacted survival to lesser extents. There were significant interactions between pressure source and means of spore suspension transfer, and between pressure strength and means of spore transfer. These results may lead to development of alternative methods of water decontamination to prevent inoculum from entering crop systems through irrigation water.

Phytophthora tropicalis Isolated from Diseased Leaves of Pieris japonica and Rhododendron catawbiense and Found in Irrigation Water and Soil in Virginia.

An unidentified species of Phytophthora was isolated from irrigation water at a production nursery in Suffolk, VA in 2000 and 2001. Water samples were assayed using a filtration method (3). A similar species was recovered from soil samples collected in two mixed-hardwood forests in Fairfax County in 2002. Soil samples were air dried, remoistened, flooded, and then baited with rhododendron and camellia leaf pieces at room temperature (22 to 24°C) (2). A Phytophthora sp. was recovered from bait pieces cultured on PARPH-V8 selective medium (2). This same species also was isolated from symptomatic leaves of Pieris japonica cv. Temple Bells and Rhododendron catawbiense cv. Maximum Roseum at a garden center in Virginia Beach in 2004. On P. japonica, symptoms appeared as water-soaked, necrotic lesions and marginal necrosis on leaves and necrosis of shoot tips; on R. catawbiense, symptoms were wilting, dieback, and death of shoots. Representative isolates produced semipapillate to papillate sporangia with tapered bases that were caducous and had long pedicels (16 to 120 µm). Sporangia on four isolates were measured: mean lengths were 40.6 to 48.4 µm, mean widths were 26.9 to 31.4 µm, and length/width ratios consistently were 1.5. Sporangia occasionally were distorted and had dual apices, and they often contained a large globule after zoospore release. Chlamydospores ranged from 25 to 32 µm in diameter. All isolates were heterothallic; four isolates paired with known isolates of P. nicotianae were found to be mating type A1. Optimum temperature for mycelium growth on cornmeal agar was 25°C with slight growth at 35°C by some isolates and no growth at 4°C. These morphological characteristics were mostly consistent with those of P. tropicalis (1). P. tropicalis is reported to have sporangia that are papillate, have lengths of 40 to 55 µm, widths of 19 to 27 µm, and length/width ratios of 1.8 to 2.4 (1). The identity of these isolates as P. tropicalis was confirmed using single-strand conformational polymorphism analysis with comparison to a reference isolate (4). These isolates have been retained in permanent collections in the Hong and Jeffers labs. One isolate from each host plant and one isolate from irrigation water were tested for pathogenicity; agar blocks of mycelium (4 × 4 mm) were placed on wounded and nonwounded leaves of P. japonica cv. Mountain Fire and R. catawbiense cv. Olga plants and wrapped with Parafilm to prevent desiccation. Lesions formed on wounded and nonwounded leaves after 4 days at 20 to 30°C, and P. tropicalis was reisolated; no lesions formed on noninoculated control leaves. To our knowledge, this is the first report of P. tropicalis in the continental United States, in irrigation water systems, and as a cause of Phytophthora foliage blight on P. japonica and R. catawbiense (1). This study suggests that the host range of this pathogen is not limited to tropical plants. Although this pathogen did not cause significant economic loss in the garden center surveyed, it was isolated in irrigation water at the production nursery from late spring through fall. An investigation of its impact on nursery crops is warranted. References: (1) M. Aragaki and J. Y. Uchida. Mycologia 93:137, 2001. (2) A. J. Ferguson and S. N. Jeffers. Plant Dis. 83:1129, 1999. (3) C. X. Hong et al. Phytopathology 92:610, 2002. (4) P. Kong et al. Fungal Genet. Biol. 39:238, 2003.

Shoot Blight of Forsythia × intermedia in Virginia Nurseries Caused by Phytophthora nicotianae.

A severe blighting of shoots on Forsythia × intermedia cv. Lynwood Gold plants was observed at several commercial nurseries in Virginia from 2001 to 2004. Crop losses ranged from 10 to 35%. Symptoms first occurred at the tips of shoots, including those that were trimmed and not trimmed, and then progressed downward. Diseased shoots wilted quickly and usually turned black, and foliage on these shoots withered and became necrotic. With PARP-V8 selective medium (2), a species of Phytophthora was isolated consistently from symptomatic shoots (including tissues from shoot tips, leaves, and stems) as well as from apparently healthy roots. These isolates produced arachnoid mycelia and numerous noncaducous, papillate sporangia but did not produce sexual structures on isolation plates; these morphological characters are consistent with those of Phytophthora nicotianae. All isolates produced a single-strand conformation polymorphism pattern typical of P. nicotianae (3). To test pathogenicity, 1-year-old, healthy-appearing cv. Lynwood Gold forsythia plants (canopy size = 100 cm × 60 cm) in four 12-liter containers were sheared. Two plants were inoculated by spraying each plant with 200 ml of a zoospore suspension (1.6 × 104 spores per ml, prepared from one isolate), and the other two plants were not treated and served as controls. Plants were covered with plastic bags overnight to encourage infection and then were grown in a field (temperature range = 20 to 33°C). Severe blight developed on trimmed shoots and new shoot tips of inoculated plants within 1 week after inoculation. The same pathogen was isolated from all blighted leaf and stem pieces assayed. Blight symptoms were not observed on control plants during a 1-month observation period. Phytophthora nicotianae has been reported to attack F. viridissima in Italy (1) causing root and collar rot but not shoot blight. To our knowledge, this is the first report of shoot blight on Forsythia spp. caused by P. nicotianae and the first report of P. nicotianae on Forsythia spp. in the United States. References: (1) S. O. Cacciola et al. Plant Dis. 78:525, 1994. (2) A. J. Ferguson and S. N. Jeffers. Plant Dis. 83:1129, 1999. (3) P. Kong et al. Fun. Gen. Biol. 39:238, 2003.

Crown Rot of Abies balsamea var. phanerolepis Caused by Phytophthora cactorum in Virginia.

In early July 2004, a severe crown rot of Canaan fir (Abies balsamea var. phanerolepis Fern.) was reported to the Virginia Cooperative Extension, Frederick County Office, and subsequently to the Virginia Tech Disease Clinic in Virginia Beach. One thousand five-year-old Canaan fir transplants (approximately 11 mm in caliper and 31 cm high) had been purchased from a tree nursery in Oregon and planted in the field in Frederick County, VA, in April of 2004. The field site had not been cultivated for 4 years after an apple orchard had been removed in 2000. By mid-May, needle browning had become serious, affecting the lower crown first. By August, transplants had suffered 40% mortality. Basal stems of affected plants were obviously discolored. Root and basal stem samples from several infected plants were then cultured on PARP-V8 agar on three different dates. Phytophthora sp. isolates were recovered from all stem samples but none from the roots. These isolates produced a large number of papillate sporangia that were caducous with short pedicels. Abundant oogonia with paragynous antheridia formed oospores directly on isolation plates within 7 days. The isolates were keyed as P. cactorum (2). This identification was confirmed using a single-strand-conformation polymorphism analysis of ribosomal DNA internal transcribed spacer (ITS)-1 (4). It appears that the source of inoculum was P. cactorum associated with the previous apple crop, since Canaan fir from the same transplant lot planted in a nearby field without a history of apples remained healthy. P. cactorum has been reported to cause root rot of noble fir (A. procera Rhedo), Pacific silver fir (A. amabilis (Dougl.) Forbes), and Shasta red fir (A. magnifica var. shastensis Lemm.) in the Pacific Northwest (3). It has also caused crown rot of Fraser fir (A. fraseri (Pursh) Poir.), noble fir, white fir (A. concolor (Gord. & Glend.) Lindl.), and balsam fir (A. balsamea (L.) Mill.) in Michigan (1). To our knowledge, this is the first report of P. cactorum attacking Canaan fir. Canaan fir currently is a recommended Christmas tree species for areas where Fraser fir does not do well due to root rot caused by Phytophthora cinnamomi. This study suggests that such a recommendation must be used with caution. Growing Canaan fir trees in P. cactorum-infested soil could result in devastating crop losses as reported in this note. References: (1) G. C. Adams, Jr. and A. Bielenin. Plant Dis. 72:79, 1988. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) P. B. Hamm and E. M. Hansen. Eur. J. For. Pathol. 12:167, 1982. (4) P. Kong et al. Fungal Genet. Biol. 39:238, 2003.

Rapid identification of Phytophthora ramorum using PCR-SSCP analysis of ribosomal DNA ITS-1.

AIMS: The primary objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis can be used for rapid identification of Phytophthora ramorum, an important quarantine plant pathogen worldwide, and to further assess the potential of the SSCP technique as a taxonomic tool for the genus Phytophthora. METHODS AND RESULTS: SSCP of ribosomal DNA internal transcribed spacer 1 was characterized for 12 isolates of P. ramorum, using a recently reported protocol. The SSCP patterns of this species then were compared with those of 18 closely related Phytophthora species. Phytophthora ramorum had a unique pattern and was easily distinguished from genetically, morphologically and ecologically close relatives. CONCLUSION: An immediate benefit of this study is provision of a highly effective and efficient identification tool for P. ramorum in the quarantine process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study also provides additional evidence demonstrating that the SSCP is an ideal DNA marker for species differentiation within the genus Phytophthora.

First Report of Phytophthora cinnamomi on Ilex glabra in Virginia.

During 2002, two nurseries in southeastern Virginia reported losses exceeding 75% of container-grown inkberry holly (Ilex glabra) cv. Shamrock. The development of necrotic leaf spots and blotches followed initial symptoms of leaf yellowing and wilting. Affected leaves rapidly turned brown and fell. Dark brown-to-black roots were washed and plated on agar media. Phytophthora cinnamomi Rands was consistently isolated and identified on the basis of its morphology (2) and single-stranded conformational polymorphism fingerprint (1). The organism had nonpapillate, internally proliferating, noncaducous, ovoid to ellipsoid sporangia that formed only in water. It did not grow at 35°C and had abundant botryose hyphal swellings, coralloid hyphae, and grape-like clusters of chlamydospores. The isolate, determined to be the A2 mating type, produced elongate cylindrical, amphigynous antheridia and oogonia with a tapered base. A pine bark potting mix amended with V8 juicetreated vermiculite colonized by the suspected pathogen was placed in 12-liter containers. Two inkberry holly cv. Shamrock liners were planted in each of three containers and two 1-yr-old plants were planted in each of three additional containers during April 2004. An identical set of six containers of noninoculated plants was also established. During June 2004, inoculated plants exhibited symptoms identical to those observed in nurseries, and P. cinnamomi was isolated. Noninoculated check plants did not develop symptoms. Japanese holly (I. crenata) was previously known as a host, but to our knowledge, this is the first report of inkberry holly (I. glabra) susceptibility. References: (1) P. Kong et al. Fungal Genet. Biol. 39:238, 2003. (2) D. J. Stamps et al. Mycol. Pap. No. 162. CAB International Mycological Institute, Wallingford, UK, 1990.

Efficacy of Chlorine on Multiple Species of Phytophthora in Recycled Nursery Irrigation Water.

Recycled irrigation water is a primary source of inoculum for Phytophthora spp. and is capable of spreading propagules throughout nursery production. Chlorination commonly is used by the industry to disinfest recycled irrigation water; however, chlorine has not been fully researched as a disinfestant for this purpose. In this study, zoospores of seven species and eight isolates of Phytophthora were exposed for 2 min to free available chlorine at 0.25, 0.5, 1.0, 2.0, and 4.0 mg/liter. Zoospores, mycelial fragments, and culture plugs of P. nicotianae also were exposed to chlorine concentrations ranging from 0.25 to 8.0 mg/liter for periods ranging from 15 s to 8 min. In addition, chlorinated water was assayed monthly in 2000 and 2001 at two commercial nurseries, and quarterly in the first year at four other nurseries in Virginia, for chlorine and survival of pythiaceous species using a selective medium. No zoospores of any species tested survived endpoint free chlorine at 2 mg/liter, while limited mycelial fragments of P. nicotianae survived at 8 mg/liter, and mycelial plugs treated at the same level of chlorine were able to produce few sporangia. Phytophthora spp. were recovered only from nursery irrigation water with levels of free chlorine at 0.77 mg/liter or lower. The results of this study are essential for improving current chlorination protocols.

First Report on White Smut of Gaillardia × grandiflora Caused by Entyloma polysporum in Virginia.

Disease samples of Gaillardia × grandiflora cvs. Goblin and Baby Cole were received at the Hampton Roads Agricultural Research and Extension Center in Virginia Beach in early April 2002. Samples were from a nursery in eastern Virginia, and most diseased plants had several to more than a dozen, round, flat, white to tan spots with indistinct margins up to 1 cm in diameter on their leaves. The spots later turned brown and necrotic, followed by necrosis of the entire leaf. Leaves of 'Baby Cole' were beginning to wilt and were more spotted than those of 'Goblin'. Fungal fruiting bodies were not observed on the surface of diseased leaves. However, microscopic examination of internal leaf tissues revealed masses of round, double-walled, pale green-to-yellow spores approximately 12 µm in diameter and typical of the ustilospores of Entyloma polysporum (2,3). Inoculum for pathogenicity tests was prepared by blending 10 diseased leaves in 200 ml of sterile distilled water (SDW) for 2 min in a blender at low speed. The spore suspension was adjusted to 5 × 105 spores per ml with SDW. Healthy 'Goblin' gaillardia plants were obtained from a nursery where smut symptoms had never been seen. Four plants in one-gallon containers were inoculated by spraying them to runoff with the spore suspension. Four control plants were sprayed with SDW only. All plants were maintained in a greenhouse (15 to 35°C) and covered with a clean polyethylene plastic sheet overnight (14 h) to maintain high humidity and separated to avoid potential cross contamination. Inoculated and uninoculated plants were hand-watered separately, with application of water to the foliage to enhance spread of the disease. Typical white smut symptoms were observed on inoculated plants 2 weeks after inoculation, and numerous spores of E. polysporum were observed in the diseased tissues. No disease symptoms were seen on control plants. White smut has been reported on gaillardia in a few other states (1), but to our knowledge, this is the first report of the disease on gaillardia in Virginia. Growers at the affected nursery reported observing white smut symptoms on gaillardia in previous years, but in the spring of 2002, almost the entire gaillardia crop was destroyed. The disease has not been seen on gaillardia in any other nurseries, but it could have significant impact on production if it spreads. References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (2) W. Fischer. Manual of the North American Smut Fungi. Ronald Press, New York, 1953. (3) D. B. O. Savile. Can. J. Res. 25(C):109,1947.