Zoospore Suspension Research Articles

First Report of Taro (Colocasia esculenta) Leaf Blight Caused by Phytophthora colocasiae in Nigeria.

In November 2009, many farmers in Abia State were alarmed by complete destruction of their taro (Colocasia esculenta (L.) Schott.) crop. Symptoms, suggestive of leaf blight caused by Phytophthora colocasiae Raciborski (2), began as small, brown, water-soaked lesions that rapidly enlarged to form large, dark brown, coalescing lesions, sometimes with orange host exudations. White sporulation was evident on the lesion surface under wet conditions. The pathogen caused rapid defoliation and killed plants. The epidemic was widespread in 2010 during the rainy season (April to November) in all taro-growing areas of Nigeria. Diseased leaves were collected from taro in Iwo Village near Ibadan, cut into 4-cm2 pieces, washed in several changes of sterile water, and incubated in petri dishes lined with wet filter paper at 22°C. Newly produced sporangia were collected from the incubated leaves and plated on a selective medium (1). Sporangia were hyaline, papillate, and measured 25 to 55 × 15 to 30 μm. Zoospores encysted within 30 min after release; cysts were 9.7 to 19.5 μm in diameter. Sporangia and zoospore formation were induced in water and by chilling, respectively (1). Two leaves each of three 1-month-old taro and three Xanthosoma plants (both unknown clones) and six detached leaves of taro were inoculated with a 1 × 105/ml zoospore suspension of isolates PC01 and PC02. Detached leaves were incubated in moist chambers at 22°C. Plants were covered with polyethylene bags for 12 h after inoculation and maintained in a screenhouse. Water-soaked lesions appeared on detached leaves within 24 h postinoculation and the leaves were completely rotted 48 h later. All inoculated attached leaves of taro, but not Xanthosoma, showed typical leaf blight symptoms including abundant sporangial production. Noninoculated control detached leaves and plants were disease free. Sporangia from detached and attached inoculated leaves, when plated on selective medium, produced typical P. colocasiae colonies. The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1 and ITS4 primers (3). Amplicons (786 bp) were sequenced in both directions and submitted to GenBank (Accession Nos. HQ602756, HQ602757, HQ602758, and HQ602759). A BLASTn search revealed 99% similarity to a P. colocasiae strain of the Pacific Region (Accession No. GU111604), but only 94% similarity to a P. colocasiae strain from India (Accession No. GQ202149). The sequence analysis, morphological characteristics, and pathogenicity test confirmed the taro leaf blight pathogen as P. colocasiae. There are previous reports of occurrence of taro blight-like disease attributed to P. colocasiae in Ethiopia, Equatorial Guinea (1), and more recently in Cameroon, but comprehensive details on pathogen or disease are not available. To our knowledge, this is the first confirmed record in Nigeria of P. colocasiae causing taro blight. This disease poses a serious threat to the production and biodiversity of this important food crop. Urgent interventions are necessary to halt this emerging epidemic in West and Central Africa.

First Report of Phytophthora ramorum Causing Shoot Dieback on Bilberry (Vaccinium myrtillus) in Norway.

In the annual Norwegian Phytophthora ramorum survey in 2009, wild bilberry samples, collected during September and October in a semimanaged park (arboretum) in the southwest coast of Norway, tested positive in a P. ramorum-specific real-time PCR test (1). Necrotic lesions were observed in shoot tips, branching points, and around leaf abscission scars. The lesions were of variable dimensions. In the samples collected in October, some lesions were confluent and completely covered some shoots. After direct detection on plant material, P. ramorum was isolated from necrotic lesions of the stems on semiselective media PARP (corn meal agar amended with pimaricin, ampicillin, rifampicin, and pentacloronitrobenzene) (2). The isolates were identified by the production of abundant chlamydospores on agar and deciduous, semipapillate sporangia that is characteristic of P. ramorum (3). Sexual structures were not observed. Three pure cultures obtained from different plant samples also tested positive for P. ramorum by the specific real-time PCR test (1). All positive samples were found in close vicinity of infected rhododendron plants. In this location, P. ramorum had already been detected on rhododendron in 2005. A pathogenicity test was performed with two isolates from bilberry and one from rhododendron. Wild asymptomatic bilberry plants were collected at the end of June in the forest around Oslo. Two shoot tips with 6 to 10 leaves each and one small part of a branch with several shoots and immature berries were used for testing each isolate. The inoculations were made by dipping the shoots in a zoospore suspension (2 to 3 × 104 zoospores ml-1) for 1 min. Inoculated material was placed in moist incubation chambers and incubated at room temperature (19 to 24°C). Controls were obtained by dipping shoots in sterile water. After 2 days, lesions were observed on leaf laminae from all the shoots inoculated with the three different isolates. After 4 days, severe petiole necroses were observed and leaves abscised easily from the stems. Symptoms on the stems were observed in the apical part or areas around the nodes. Some shoots were almost completely necrotic. Heavy sporulation was observed on the berries. P. ramorum was reisolated from leaves and stems of inoculated shoots for all the isolates. P. ramorum was not recovered from control plants. To our knowledge, this is the first report of P. ramorum on bilberry in Norway.

Meristem culture for the elimination of the strawberry crown rot pathogen Phytophthora cactorum

Two methods to induce a symptomless infection of crown rot (Phytophthora cactorum) in stolons of three strawberry (Fragaria×ananassa) cultivars were evaluated. Spraying a 10 −4 ml zoospore suspension was the most effective method, producing a mean infection rate of 12%. Meristem cultures were subsequently established from all of the inoculated stolons and tested eight weeks later for the presence of P. cactorum using a PCR-based assay. A total of 784 meristems were excised and DNA was successfully extracted from 505 viable meristem cultures, 46 of which had been excised from stolons that were shown to have a symptomless infection of crown rot. No viable meristems were found to be infected with the pathogen. This investigation confirmed the suitability and reliability of meristem culture for the production of strawberry plants free from crown rot.

First Report of Phytophthora ramorum Infecting Mistletoe in California

In 2005 and 2006, white fir and Douglas-fir growing in a Christmas tree plantation near Los Gatos, CA, under a black walnut tree infected with mistletoe tested positive for Phytophthora ramorum, the cause of Sudden Oak Death. Isolation from a symptomatic mistletoe inflorescence stalk was positive for P. ramorum. In 2007, fresh mistletoe leaves, stems, inflorescence stalks, and berries were inoculated with a zoospore suspension of the mistletoe isolate. All of the plant parts developed symptoms, and P. ramorum was isolated from each of them. This is the first report of an infection of a hemiparasite with P. ramorum. Accepted for publication 20 January 2011. Published 9 February 2011.

Diplanetism and microcyclic sporulation in Phytophthora ramorum

SummaryThe zoosporic phase of the pathogen Phytophthora ramorum plays a crucial role in the process of plant infection, yet little is known about the fate of zoospores failing to target their hosts. Here, we describe new stages in the life cycle of P. ramorum concerning the in vitro development of monomorphic diplanetism and microcyclic sporulation in free water. Papillate cysts were formed after zoospore suspensions of isolates of the EU1 and NA1 clonal lineages were vortexed. Cysts usually germinated directly forming an emerging tube, or indirectly by releasing a secondary zoospore, which leaves behind an empty cyst with a short evacuation tube. Germinate cysts frequently developed either an appressorium or a microsporangium both terminally. We also observed microcyclic sporulation, i.e. sporangia indirectly germinated by forming a microsporangium, as in microcyclic conidiation of true fungi. Temporal progress of encysted zoospores in solution showed that percentage of germination varied significantly among and within isolates as well as between experiments, suggesting that germination is partly ruled by internal mechanisms. Diplanetism and microcyclic sporulation in P. ramorum may provide a second opportunity for host infection and may increase the chance of long dispersal in moving water.

First Report of Phytophthora ramorum Infecting California Red Fir in California.

In May 2005, branches originating from five separate whorls below the terminal on a single California red fir (Abies magnifica) in a mixed grand fir (Abies grandis) and Douglas-fir (Pseudotsuga menziesii) Christmas tree plantation near Los Gatos, CA displayed wilting and dieback of new shoot growth. Brown dieback, delineated by needle loss, extended 6 to 8 cm into 1-year-old and sometimes 2-year-old growth. The ~7-year-old, 1-m tall tree was located near the edge of the plantation, beneath an overstory of California bay laurel (Umbellularia californica) trees that were infected with Phytophthora ramorum. Isolations from dieback margins onto corn meal agar amended with ampicillin, rifamycin, and pimaricin (CARP) yielded hyphae and large, dark brown chlamydospores that were morphologically consistent with P. ramorum (1). Microsatellite analysis confirmed that isolates were of the NA1 lineage of P. ramorum. Isolates were deposited in the Washington State University Puyallup Phytophthora Master Collection. Dormant bareroot California red fir seedlings were obtained from the USDA Forest Service Placerville Nursery (Camino, CA) in February 2006 and planted in SC-10 super cell cones (Stuewe & Sons, Inc., Tangent, OR) in a standard greenhouse potting mix. Seedlings (average height 11 cm) were then forced to initiate bud break and new shoot elongation (0.5 to 1.5 cm) in a greenhouse at 21°C. Eight unwounded seedlings were inoculated with a zoospore suspension (4.185 × 105 zoospores/ml of sterile water) produced from 3- to 4-week-old V8 juice agar cultures of isolate WSU#106-0021 using an artist's airbrush powered by Badger Propel canned propellant. Eight control seedlings were sprayed with water alone. Seedlings were placed in plastic tubs with ~2.5 cm of warm water in the bottom to provide humidity. A plastic bag supported by a wire frame was used to cover each tub. Tubs were placed in a biocontainment unit at 15 to 16°C under 24 h of fluorescent light. The plastic was removed after 5 days and seedlings were left under the same conditions. Seven days after inoculation, 25 to 100% (average 68%) of the new shoots on each of the eight inoculated seedlings were wilted and 100% of these seedlings exhibited dark brown dieback into the 1-year-old stems (range of 1.0 to 2.3 cm, average 1.6 cm). Tissues from shoots and dieback edges were plated onto CARP media. All of these attempts resulted in successful isolation of P. ramorum, and cultures exhibited the same hyphal morphology and chlamydospore characteristics when compared with the isolate tested. Control plants did not develop symptoms. This trial completes Koch's postulates to establish California red fir as a host of Phytophthora ramorum. To our knowledge, this site contains the only reported infection of California red fir by P. ramorum. The potential for infection within its native range is unknown. Reference: (1) S. Werres et al. Mycol. Res. 105:1155, 2001.

Aggressiveness of Phytophthora cactorum, P. citricola I, and P. plurivora from European Beech.

Phytophthora cactorum, P. citricola I, and P. plurivora cause bleeding cankers on mature European beech (Fagus sylvatica) trees in the northeastern United States. Inoculation experiments were conducted to compare the aggressiveness of the three Phytophthora spp. on stems, leaf disks, and roots of European beech and common lilac (Syringa vulgaris) seedlings. Isolates were obtained from bleeding cankers on European beech from five cities in New York (Albany, Ithaca, Oyster Bay, Plainview, and Rochester) and from a bleeding canker on sugar maple in Ithaca, NY. Stems were inoculated with colonized agar plugs, leaf disks with a zoospore suspension, and roots via infested soil at three inoculum levels. All organs of inoculated beech and lilac developed disease except for lilac roots inoculated with zoospores of P. cactorum. Disease incidence, severity, and plant survival were dependent on isolate and were also influenced by the tissue inoculated and host. Isolates of P. cactorum were the least aggressive and caused less necrosis than isolates of P. citricola I and P. plurivora. Results emphasize the utility of stem and root inoculation for evaluation of this canker disease and underscore critical differences in species aggressiveness.

Zoospore Density-Dependent Behaviors of Phytophthora nicotianae Are Autoregulated by Extracellular Products

Phytophthora species are destructive fungus-like plant pathogens that use asexual single-celled flagellate zoospores for dispersal and plant infection. Many of the zoospore behaviors are density-dependent although the underlying mechanisms are poorly understood. Here, we use P. nicotianae as a model and demonstrate autoregulation of some zoospore behaviors using signal molecules that zoospores release into the environment. Specifically, zoospore aggregation, plant targeting, and infection required or were enhanced by threshold concentrations of these signal molecules. Below the threshold concentration, zoospores did not aggregate and move toward a cauline leaf of Arabidopsis thaliana (Col-0) and failed to individually attack annual vinca (Catharanthus roseus cv. Little Bright Eye). These processes were reversed when supplemented with zoospore-free fluid (ZFF) prepared from a zoospore suspension above threshold densities but not with calcium chloride at a concentration equivalent to extracellular Ca(2+) in ZFF. These results suggest that Ca(2+) is not a primary signal molecule regulating these communal behaviors. Zoospores coordinated their communal behaviors by releasing, detecting, and responding to signal molecules. This chemical communication mechanism raises the possibility that Phytophthora plant infection may not depend solely on zoospore number in the real world. Single zoospore infection may take place if it is signaled by a common molecule available in the environment which contributes to the destructiveness of these plant pathogens.

First Report of Phytophthora Blight of Lily Caused by Phytophthora nicotianae in China.

Lily (Lilium spp.) is an economically important cut flower in China. In August 2009, 30 to 40% of plants of lily cv. Siberia in a greenhouse for cut flower production in Yunnan, China were severely diseased. Infected plants developed water-soaked lesions and soft rot on the base of stems and leaves near the soil surface. As the disease progressed, stems bent and plants collapsed. Soft rot symptoms were observed on some bulbs and roots of severely diseased plants. Small, diseased tissue fragments (approximately 3 mm) were surface disinfected with 0.5% NaOCl and then plated to Phytophthora selective medium (10% V8 juice agar) (4). Inoculated dishes were incubated at 25°C in the dark. After 5 days, white colonies with abundant aerial mycelia developed from all plated tissue samples. The fungus had aseptate hyphae. Sporangia were papillate, both caducous and noncaducous, and the shape ranged from ovoid to spherical. The dimensions of sporangia were 30 to 62 × 21 to 46 μm. On the basis of morphological features, isolates were identified as Phytophthora nicotianae Breda de Haan. The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1/ITS4 and sequenced. BLAST analysis of the 835-bp fragment showed a 99% homology with the sequence of P. nicotianae AY833527. The nucleotide sequence has been assigned GenBank No. GU299778. PCR amplification of genomic DNAs using the P. nicotianae-specific primer pair ITS3-PNIC1 generated a 455-bp sequence (3). The result further confirmed the identity of P. nicotianae. Pathogenicity tests were conducted in the greenhouse on lily cv. Siberia grown in pots. Ten 3-month-old plantlets were inoculated by watering the wounded stem bases and soil surface with 30 ml of zoospore suspensions (105 spores per ml). Five uninoculated plantlets were used as controls. All plantlets were covered with plastic bags and incubated at room temperature (22 to 26°C) for 48 h. Inoculated plants developed initial symptoms of slight chlorosis and wilting of lower leaves. Within a 3-week period, all plants died due to soft rot of stem bases and leaves. The pathogen was reisolated from inoculated plants but not from control plants that were symptomless. P. nicotianae has been reported as the causal agent of Phytophthora blight on lily in Korea, Japan, and Hungary (1,2). To our knowledge, this is the first report of Phytophthora blight of lily in China.

Effects of hydrostatic pressure, agitation and CO2 stress on Phytophthora nicotianae zoospore survival

Phytophthora nicotianae Breda de Haan is a common pathogen of ornamental plants in recycled irrigation systems. In a previous study, annual vinca (Catharanthus roseus Don) inoculated with zoospore suspensions using a CO(2)-pressurized sprayer had less foliage blight than plants inoculated using a hand sprayer. Here, the impact of hydrostatic pressure, agitation and aeration with CO(2) on the survival of P. nicotianae zoospores was examined. Exposure of zoospores to 840 kPa hydrostatic pressure for 8 min or agitation at a mixing intensity (G) of 6483 s(-1) for 4 min at 22-23 degrees C did not kill zoospores, but resulted in viable cysts. Motile and forcefully encysted zoospores of P. nicotianae were equally infectious on vinca or lupine (Lupinus polyphylus Lindl.). Bubbling CO(2) into zoospore-infested water at 110.4 mL (0.2 g) min(-1) for 5 min caused 81% reduction in the number of germinated zoospores. Pressure at 630 kPa (16.3 g CO(2)) or 70 kPa (3.85 g CO(2)) facilitated CO(2) injection and shortened the zoospore inactivation time to 30 s. When air was bubbled through the suspension, germination was similar to the control. Exposure to CO(2) killed P. nicotianae zoospores in water. Neither pressure nor agitation had an effect on zoospore viability or infectivity. Based on results of this study, the authors designed a recycling CO(2) water treatment system that is currently under evaluation.

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.

Effects of Temperature, Concentration, Age, and Algaecides on Phytophthora capsici Zoospore Infectivity.

Controlled laboratory studies were undertaken to determine the effects of water temperature (2, 9, 12, 19, 22, and 32°C), inoculum concentration (1 × 102, 1 × 103, 5 × 103, 1 × 104, 2 × 104, and 4 × 104 zoospores/ml), and zoospore suspension age (0, 1, 3, and 5 days old) on infection of pickling cucumbers (Cucumis sativus) by Phytophthora capsici. Zoospore motility and mortality in response to commercial algaecides were also investigated. Cucumbers became infected at all temperatures tested, except 2°C, and the highest infection incidence was observed for cucumbers incubated in suspensions held at ≥19°C. Fewer fruit (<40% at ≥19°C, 0% at ≤12°C) became infected when water contained 1 × 102 zoospores/ml. Almost 100% of fruit were infected when water contained ≥5 × 103 zoospores/ml at temperatures ≥12°C. While the incidence of fruit infection declined with the zoospore suspension age, infection still occurred when 5-day-old suspensions were used. Commercial algaecides inhibited zoospore motility and caused significant zoospore mortality in laboratory assays, and show promise for treatment of infested irrigation water. Avoidance of infested irrigation water throughout the growing season is warranted until effective and economically acceptable water treatments are developed for field use.

Resistance of Pepper to Phytophthora Crown, Root, and Fruit Rot Is Affected by Isolate Virulence.

Greenhouse and laboratory experiments were conducted to determine the virulence of four Phytophthora capsici isolates from Michigan to 31 bell and hot pepper cultivars and breeding lines. Resistance to crown and root rot was assessed following the inoculation of soilless media with P. capsici-infested millet seed. In a detached fruit assay, fruit rot resistance was evaluated following inoculation with zoospore suspensions of 1.75 × 106 zoospores/ml. The four isolates differed in virulence to pepper lines screened for crown and root rot resistance and were considered to be four different physiological races. The pepper lines CM334, NY07-8001, NY07-8006, and NY07-8007 were resistant to the isolates tested. None of the commercial cultivars were resistant to P. capsici isolate 12889, but several cultivars were resistant to the other isolates screened. The isolates varied in their ability to cause infection on the fruits of the different cultivars. Overall, pepper fruit were more susceptible to P. capsici than the roots and crowns. Management of Phytophthora crown and root rot of pepper can be improved through the use of resistant cultivars. However, since isolate virulence affects resistance, cultivar resistance will need to be utilized on a local scale accordingly.

Zoosporic plant pathogens produce bacterial autoinducer-2 that affectsVibrio harveyiquorum sensing

The frequent coisolation of bacteria with Phytophthora and Pythium species suggests possible interspecies communication. Zoospore-free fluids (ZFF) from bacteria-free and nutrient-depleted zoospore suspensions were examined to investigate the production of autoinducer-2 (AI-2), a bacterial interspecies signal molecule, by zoosporic oomycetes. ZFF from Phytophthora nicotianae, Phytophthora sojae, and Pythium aphanidermatum triggered luminescence of the Vibrio harve7yi AI-2 reporter, indicating the presence of AI-2 in zoospore extracellular products and the potential of cross-kingdom communication between oomycetes and bacteria. The production of AI-2 by zoospores was confirmed by chemical assays. These results provide a new insight into the physiology and ecology of oomycetes.

Zoospores of three Arctic laminariales under different UV radiation and temperature conditions: exceptional spectral absorbance properties and lack of phlorotannin induction.

Phlorotannins have often been considered to act as UV-protective compounds in zoospores of brown algae. However, only the absorption characteristics of zoospores under UV exposure have been determined and no data are available on the actual content of phlorotannins or on temperature-UV interactions. Therefore, we determined the absorbance spectra and the phlorotannin contents in zoospore suspensions of three Arctic species (Saccharina latissima, Laminaria digitata, Alaria esculenta), and in the media surrounding zoospores after exposure to different radiation (400-700, 320-700, 295-700 nm) and temperature (2-18 degrees C) conditions for 8 h. Absorption typical of phlorotannins with a maximum at 276 nm was monitored in zoospore suspensions as well as in the media surrounding zoospores, but the results depended strongly on radiation treatments and on zoospore densities. Surprisingly, the content of UV-absorbing phlorotannins subsequent to different exposures did not change in any of the three species. The observed exceptional absorption properties could, therefore, not be related to phlorotannin contents. These findings are discussed in light of a strong phlorotannin investment from sporophytes during spore release and a minor UV-protective role of phlorotannins for zoospores of Arctic kelp species.

Interactive effects of radiation, temperature and salinity on different life history stages of the Arctic kelp Alaria esculenta (Phaeophyceae).

To estimate the potential effects of climate change on polar marine macroalgae, studies on interactive stress effects of multiple climate-related parameters are essential. Interactions of temperature, radiation and salinity on two different life history stages of Alaria esculenta (L.) Greville from the Kongsfjord (Spitsbergen) were investigated for the first time within this study. Adult macroscopic sporophytes of A. esculenta were exposed to different temperatures between 4 and 21 degrees C combined with artificial irradiation conditions [photosynthetically active radiation, ultraviolet (UV) radiation: UV-A/UV-B, first experiment] and with different salinities [34, 28, 20 practical salinity units (p.s.u.) second experiment]. Effects of photosynthetic activity were determined by measuring variable chlorophyll fluorescence of photosystem II. Germination success of young microscopic zoospores of A. esculenta was studied under multifactorial stress. Zoospore suspensions were exposed to the three different salinities and irradiation conditions at four temperatures between 2 and 16 degrees C. Overall, A. esculenta exhibited a highly stage-specific susceptibility towards the experimental treatments. In both experiments using sporophytes, photosynthetic activity showed significant temperature effects and only very few significant radiation and salinity effects. Microscopic stages of A. esculenta were shown to be more sensitive than the adult macroscopic stages, since germination capacity of zoospores was significantly affected by temperature and salinity changes, and interactions of both. These results suggest that multiple stress factors interact synergistically. Temperature seems to be a predominant environmental parameter for the kelp A. esculenta. Overall, A. esculenta proved to be relatively tolerant and adaptable to increasing temperature and UV radiation, as well as to diluted salinities, but only up to a specific limit.

Susceptibility of Fraser Fir to Phytophthora capsici.

Phytophthora cinnamomi, P. drechsleri, P. citricola, and P. cactorum limit Fraser fir production, whereas P. capsici affects Solanaceous, Cucurbitaceous, and Fabaceous crops. Some vegetable growers in Michigan plant conifers for the Christmas tree market in fields infested with P. capsici. To determine the susceptibility of Fraser fir to P. capsici, stems (no wound or 1- or 3-mm-diameter wound) or roots (2 or 4 g of infested millet seed or 2 or 5 × 103 zoospores/ml of a zoospore suspension) of seedlings were inoculated with each of four P. capsici isolates and incubated in growth chambers (20 or 25°C). In addition, Fraser fir seedlings were planted in two commercial fields naturally infested with P. capsici. All P. capsici isolates tested incited disease in the seedlings regardless of incubation temperature or inoculation method. Seedlings (72%) planted in P. capsici-infested fields developed disease symptoms and died. Most of the P. capsici isolates obtained from the Fraser fir seedlings infected while in the field were recovered from root tissue. Identification was confirmed by species-specific direct colony polymerase chain reaction. The pathogen was successfully recovered from stems of all stem-inoculated seedlings, and from roots and stems of all root-inoculated seedlings; the phenotype of the recovered isolate matched the phenotype of the inoculum. This study suggests that planting Fraser fir in fields infested with P. capsici could result in infection and that adjustments in current rotational schemes are needed.

First Report of Mefenoxam Sensitivity and Pathogenicity of Phytophthora citricola Isolated from American Ginseng (Panax quinquefolium).

In March of 2004, stratified ginseng seeds from commercial Wisconsin gardens were planted in sterilized silica sand in a research greenhouse at Michigan State University. Following emergence, seedlings exhibiting wilting, damping off, and black stem lesions were observed. In the laboratory, symptomatic seedlings were rinsed with distilled water. Tissue samples were excised and embedded in water agar amended with ampicillin (100 mg/liter) and incubated at 25°C. In addition to the isolation of Phytophthora cactorum, a known pathogen of ginseng, P. citricola, (five isolates) also was identified from single-zoospore cultures based on morphology (2). One-week-old, dilute V8 agar cultures were used to obtain single zoospores. Cultures were flooded with 20 ml of sterilized distilled water chilled to 10°C and incubated at 25°C for 25 min to induce zoospore release. Zoospore suspensions were spread onto water agar plates, and after 24 h at 25°C, single germinating zoospores were selected at random and transferred to benomyl, ampicillin, rifampicin, and pentachloronitrobenzene (BARP)-amended V8 agar plates. Sequence analysis of the internal transcribed spacer (ITS) region 1 and 2 of the rDNA was also used to distinguish P. citricola from P. cactorum. A representative sequence for the isolates of P. citricola (NCBI Accession No. FJ217388) matched (100% similarity) a P. citricola isolate deposited in GenBank (Accession No. DQ486661). To screen P. citricola for in vitro response to mefenoxam, agar plugs (7-mm diameter) from 1-week-old V8 agar cultures incubated at 25°C under fluorescent lighting were placed in the center of each of two V8 agar plates amended with 0 and 100 ppm of mefenoxam (Ridomil Gold EC, 48% a.i., suspended in sterile distilled water and added to V8 agar cooled to 49°C). The plates were incubated at 25°C for 3 days under fluorescent lighting. Isolates were assigned a mefenoxam sensitivity rating based on the percentage of radial mycelial growth on the amended V8 agar when compared with the unamended control. Each of the five isolates was scored as mefenoxam resistant with growth on 100-ppm plates >30% of the controls. Koch's postulates were conducted for the isolates of P. citricola recovered from ginseng seedlings to confirm pathogenicity. Previously, P. citricola was reported as nonpathogenic to ginseng (1). Three-week-old, healthy ginseng seedlings were planted into 89- × 64-mm pots filled with autoclaved medium-particle vermiculite and maintained in the greenhouse under 63% shade cloth with temperatures between 18 and 26°C. Pots were arranged in a completely randomized block design with eight seedlings per isolate as replicates and watered as needed. A 2-ml inoculum suspension (approximately 104 zoospores) was injected into the potting medium at the stem base of each seedling. All of the isolates were pathogenic to ginseng seedlings with 60% of inoculated seedlings per isolate exhibiting wilting, damping off, and blackened stems within 3 weeks after inoculation. P. citricola was reisolated from all inoculated plants. To our knowledge, this is the first report of P. citricola pathogenic on ginseng.

First Report of Stalk Rot Caused by Phytophthora tentaculata on Aucklandia lappa in China.

Phytophthora tentaculata causes root and stalk rot of Chrysanthemum spp., Delphinium ajacis, and Verbena spp. in nurseries in the Netherlands and Germany (2). In later years, P. tentaculata was isolated from Verbena hybrids (3) and lavender cotton (Santolina chamaecyparissus) in Spain (1). In August 2007, stalk rot symptoms were observed on Aucklandia lappa (Asteraceae), an economically important Chinese medicinal plant, in some fields in Yunnan Province of China. Small groups of infected plants were randomly distributed throughout the fields. Plants showing stalk rot and wilting died rapidly. Diseased tissues were cut into 10-mm pieces and plated onto Phytophthora selective medium, P5ARP (2), to obtain the pure cultures. Seven isolates were obtained, and five isolates were grown on solidified LBA (60 g of lima bean powder and 15 g of agar per 10,000 ml of distilled water) and 10% V8 juice liquid medium for examination of morphological and physiological characteristics (4). The colony surface texture was uniform and formed sparse, loosely branched mycelium on LBA medium. Radial growth rate was 2 to 3 mm per day at 24°C on LBA. In water, relatively small swellings were formed at hyphal branches. Sporangia were spherical or ovoid to obpyriform and some were distorted and papillate with a narrow exit pore. Approximately 10% of the sporangia were caducous with a short pedicel. Sporangial dimensions were 28 to 47 (35) × 21 to 36 (29) μm, length/breadth ratio 1.2. Chlamydospores formed on LBA after 1 week and were terminal, spherical, thin walled, and 21 to 31 (27) μm in diameter. The isolates were homothallic. Oogonia abundantly formed on LBA and were 25 to 36 (31) μm in diameter. One or two paragynous antheridia (15 × 10 μm) were attached to the oogonia. Oospores were spherical, hyaline, aplerotic, and 20 to 32 (25) μm in diameter. The minimum temperature for mycelium growth was 8°C and maximum temperature was 34°C. The internal transcribed spacer (ITS) region was amplified and sequenced and agreed 100% with sequences of four P. tentaculata isolates deposited in GenBank (Accession Nos. AJ854302, AY881001, DQ335634, and AF266775). Pathogenicity was assessed by flooding three potted A. lappa plants with a 104 ml-1 zoospore suspension and incubating at 20 to 22°C. As controls, two potted A. lappa plants were flooded with deionized water. All three inoculated A. lappa plants exhibited stalk rot after 15 days, from which the pathogen was reisolated using selective medium, P5ARP. Controls remained healthy 15 days after inoculation, To our knowledge, this is the first report of P. tetaculata H. Kroeber & R. Marwitz infection of A. lappa in China. We speculate that the pathogen might have been introduced from other countries on seeds.

First Report of Crown and Root Rot Caused by Phytophthora capsici on Hydroponically Grown Cucumbers in Norway.

In December 2004, symptoms of root and crown rot were observed on cucumbers (Cucumis sativus L.) in a greenhouse in Norway. Cucumbers were the only crop of the greenhouse that used rockwool as a growing substrate in a hydroponical system. The first symptoms were detected in propagation material. One week after planting, symptoms of root and crown rot were observed and approximately 10% of the plants died. Later, losses of 50% in some greenhouses were observed. A yield reduction as much as 65% was estimated in the winter period (January and February). The two main cucumber cultivars planted were Armada and Lopez. In February 2005, Phytophthora capsici (Leonian) (1) was isolated on potato dextrose agar from a sample of cv. Lopez. The isolate produced deciduous, papillate sporangia (occasionally with two or three papilla) and pedicels that were sometimes longer than the sporangia. Sequencing of amplicons of the internal transcribed spacer region (ITS) rDNA and of the mitochondrial cytochrome c oxidase subunit 1 (Cox1) gene (2) confirmed the identification. Three isolates collected through 2005 from the same greenhouse were crossed with tester strains of P. cryptogea. Formation of oogonia and amphigynous antheridia was always observed in crosses with mating type A2; thus, all isolates were the A1 mating type. All three isolates grew well at 35°C and did not produce chlamydospores. A pathogenicity test was performed with one isolate of P. capsici. Four plants of cucumber cvs. Indira and Jessica were grown in a growth chamber at 24°C. Plants at the two-leaf stage were drenched with 20 ml of a zoospore suspension of 106 zoospores per ml per plant. After 18 days, all plants of both cultivars developed symptoms of crown rot or wilted and died. P. capsici was reisolated from inoculated plants of both cultivars. The pathogenicity test was repeated in the same way, but in a greenhouse with temperatures that ranged between 18 and 29°C. In addition, four plants of both cultivars at the four-leaf stage were inoculated with a suspension of 105 zoospores per ml. After 1 week, all plants developed crown rot or were irreversibly wilted, independently of the plant age or the zoospore concentration. To our knowledge, this is the first report of P. capsici in Norway.