V8 Juice Research Articles

First Report of Phytophthora cactorum Causing Fruit Rot on Avocado in Spain.

The area of avocado (Persea americana Mill.) orchards in southern Spain has increased recently and is currently at 8,063 ha. Avocado production in this part of Spain was 72,581 t during 2003. During February 2004, apical necrosis was observed on avocado fruits (cv. Hass) in one orchard in Vélez-Málaga, Málaga Province, southern Spain. Dark brown lesions and necrotic flecking of the flesh also were observed on fruits. Isolations from the skin of the fruit previously washed with tap water and disinfested with 20% sodium hypochlorite on potato dextrose agar (PDA) consistently resulted in mycelial colonies. Sporangia produced on V8 juice by successive washing of mycelia with saline solution (1) measured 31 to 37.2 (33.3) × 21.7 to 28.8 (24.2) μm in size. The pathogen was identified as Phytophthora cactorum on the basis of morphological structures (mycelia, sporangia, chlamydospores, and oospores) formed when grown on V8 juice and PDA (2). To confirm pathogenicity, a mycelial suspension was obtained by blending mycelia grown for 1 week on PDA in 200 ml of sterile water. Three healthy avocado fruits were inoculated with the suspension by injection; three other fruits were inoculated by placing a drop of suspension on the unbroken skin of the fruit. The same number of fruit was inoculated as controls using sterile water instead of mycelial suspension. The inoculated fruits were incubated for 5 days in a moist chamber at 24°C in darkness. Spots appeared on all fruits for both inoculation methods, and the pathogen was isolated and identified as P. cactorum. No symptoms appeared on the control fruits. To our knowledge, this is the first report of P. cactorum causing fruit rot on avocado in Spain.

First Report of Leaf Spot of Spinach Caused by Stemphylium botryosum in Arizona.

During the winter (December through February) of 2003-2004, and again during 2004-2005, spinach (Spinacia oleracea) crops in the Yuma region of Arizona developed a foliar disease that previously had not been diagnosed in this geographic area. The problem was found on only a few acres and severity was low. The first symptoms consisted of round to oval leaf spots that were gray to olive green and visible from both adaxial and abaxial leaf surfaces. The spots were 3 to 6 mm in diameter but expanded up to as much as 10 mm. As disease progressed, leaf spots became tan and dry and papery in texture. Fungal growth was not observed on the spots. Isolations from the edges of surface-sterilized lesions onto V8 juice agar consistently resulted in fungal colonies. The fungus was identified as Stemphylium botryosum based on the following morphological characteristics of isolates incubated under fluorescent lights: dark green-to-brown mycelial growth, unbranched conidiophores with distinctly swollen apical cells that had dark bands, and dictyoconidia. The conidia were brown, ellipsoidal to ovoid, verrucose, borne singly, and measured 17 to 28 × 13 to 19 μm. To test pathogenicity, inoculum of each of five isolates (approximately 1 × 105 conidia/ml) was sprayed separately onto 20 to 25 plants each of spinach cvs. Whitney, Rushmore, Lion, Springfield, Nordic IV, and Unipak 144. Inoculated plants were incubated in a humidity chamber for 48 h and then maintained in a greenhouse (24 to 26°C). After 10 to 14 days, leaf spots resembling those seen in the field developed on all inoculated plants, and S. botryosum was reisolated from the spots. Control plants were similarly inoculated with water but did not develop symptoms. To our knowledge, this is the first report of leaf spot of spinach caused by S. botryosum in Arizona. The possibility of seedborne S. botryosum (3) may account for the development of this disease in winter spinach crops in this arid region. Leaf spot could be damaging to spinach grown in this region if rainfall is higher than normal, such as in 2004-2005. This disease has been reported in production spinach crops in California, Delaware, Florida, and Maryland (2,4) and in spinach seed crops in Washington (1).

Root and Basal Stem Rot of Scotch Broom Caused by Phytophthora citricola and P. drechsleri in Italy

Scotch broom (Cytisus scoparius (L.) Link, Fabaceae), an evergreen shrub native to Europe, is cultivated as a garden plant. In 2003 and 2004, potted plants with symptoms of leaf chlorosis, defoliation, and eventual wilt and associated with root and collar rot were observed in ornamental nurseries in Sicily. As much as 10% of plants were affected in a single nursery. Two species of Phytophthora were consistently isolated alone or together from the same pot with the selective medium of Masago et al. (2). Pure cultures were obtained by single-hypha transfers and the species were identified as P. citricola Sawada (approximately 40% of isolations) and P. drechsleri Tucker (60% of isolations) on the basis of morphological, cultural characters, and electrophoretic phenotype. The isolates of P. drechsleri grew between 10 and 37°C (optimum 27°C) on potato dextrose agar (PDA). The sporangia produced on V8 juice agar (V8A) were ellipsoid to obpyriform, nonpapillate, persistent with internal proliferation, and often forming in a sympodium. Sizes varied, 30 to 60 × 20 to 40 μm (length/width ratio between 1.4 and 2.2). The hyphal swellings were produced in aqueous culture. All isolates were A1 mating type and formed plerotic oospores (mean diameter (ф) 25 μm) with amphigynous antheridia when paired with the A2 reference isolates of P. cryptogea on V8A plus β-sitosterol. The aryl-esterase and malate dehydrogenase isozymes of scotch broom isolates on polyacrylamide slab gels (1) were identical to those of the authentic isolate CBS 292.35 of P. drechsleri and differed from reference cultures of other nonpapillate species. The cardinal temperatures of P. citricola isolates on PDA ranged from 2 to 30°C (optimum 25°C). In liquid culture, the isolates produced irregular-shaped, obovoid to obpyriform sporangia 20 to 70 × 21 to 44 μm that were noncaducous, semipapillate or with inconspicuous papilla, often with two apices. The isolates were homothallic and produced oospores (mean ф 22 μm) with paragynous antheridia. The electrophoretic phenotype of these isolates was identical to the phenotype of P. citricola reference isolates and very different from that of the reference isolates of other semipapillate species. The pathogenicity tests of the representative isolates of P. drechsleri (IMI 391710) and P. citricola (IMI 391715) were carried out in a screenhouse. Twenty 3-month-old scotch broom seedlings were transplanted into pots (12 cm ф) filled with soil infested with the inoculum produced on a mixture of vermiculite and autoclaved oat seeds. The plants were maintained at 20 to 28°C and watered to field capacity once a week. After 30 to 40 days, all inoculated plants showed symptoms of wilting and root rot. The 20 control plants transplanted into pots containing noninfested soil remained healthy. P. citricola and P. drechsleri were reisolated from infected tissues. To our knowledge, this is the first report of P. citricola and P. drechsleri on scotch broom. A root rot of scotch broom caused by P. megasperma has been reported in central Italy (3).

Detection of a Phytophthora sp. Causing Asparagus Spear and Root Rot in Michigan.

In the spring of 2004, a Phytophthora sp. was isolated from asparagus (Asparagus officinalis) spears, roots, and dormant crowns from several fields in Oceana and Ingham counties in Michigan. Symptomatic spears were often curved, had water-soaked lesions slightly above or below the soil line or were shriveled at the site of infection or both. Infected storage roots had water-soaked lesions but were not soft at the lesion site. Infected crowns had fewer roots than healthy crowns. In the laboratory, plant tissues were rinsed in tap water and blotted dry. Sections from the edge of lesions were placed aseptically onto BARP (25 ppm of benomyl, 100 ppm of ampicillin, 30 ppm of rifampicin, and 100 ppm of pentachloroni-trobenzene) amended unclarified V8 juice agar and incubated at 25°C for up to 7 days. Phytophthora sp. isolates recovered from the infected material produced ovoid, nonpapillate, noncaducous sporangia and amphigy-nous oospores on isolation media. Single-sporangium cultures made for each isolate were stored long term in sterile 2-ml microcentrifuge tubes containing two 7-mm mycelial plugs, two sterile hemp seeds, and 1 ml of sterile distilled water. Sporangia produced on dilute V8 juice agar averaged 45 μm long × 26 μm wide and oospores were 25 to 30 μm in diameter. Chlamydospores were not observed. Five detached 'Jersey Knight' spears were inoculated with a 7-mm mycelial plug from the edge of actively growing 5-day-old cultures and incubated at 23 to 25°C for 5 to 7 days in a moist chamber. After 3 days, water-soaked lesions and shriveling and curving of the spears were visible on all inoculated spears. The pathogen was always reisolated from the lesion edge. No symptoms were observed when spears were inoculated with sterile V8 juice agar plugs. DNA was extracted from representative isolates, and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified with ITS6 and ITS4 primers and sequenced. A BLAST search of the NCBI database with the ITS sequence revealed Phytophthora sp. UQ2141, Accession No. AF266795, as the closest match with 99% sequence similarity. These results, coupled with the morphological characteristics of the isolates, indicate that the Phytophthora sp. isolated from asparagus in Michigan is among the constituents of Phytophthora spp. included in the P. megasperma clade 6 (2), whose taxa are currently being reevaluated. Although a Phytophthora sp. has been described previously on asparagus (1,3), this is the first report, to our knowledge, of a Phytophthora sp. on asparagus in Michigan. The occurrence of excessive rainfall in the spring of 2004 is likely responsible for widespread disease and considerable yield losses in production fields.

First Report of Phytophthora cryptogea Root and Crown Rot on Peach Trees in Chile.

Phytophthora cryptogea Pethybridge and Lafferty was consistently isolated from the Nemaguard rootstock (Prunus persica × P. davidiana) of peach trees in Chile. Symptoms included reddish necrotic tissues at the base of the trunk often extending to the main roots, root rot, gummosis, foliar chlorosis, lack of vigor, and dieback, with severely infected trees dying. Isolations with corn meal agar (Difco, Detroit, MI) amended with antibiotics and fungicides (ACMA) (2) and pure cultures were obtained by hyphal tip transfers to ACMA. Ten isolates from different locations were identified by morphology and growth at cardinal temperatures (1). These isolates produced hyphal swellings and sporangia by using carrot juice broth (2) for 48 h followed by the addition of 1% (w/v) nonsterilized soil extract. Sporangia were nonpapillate, internally proliferating, noncaducous, ovoid to obpyriform, and with mean dimensions of 36 × 24 μm. All isolates were typed as A1 using a P. cinnamomi A2 culture as the test isolate and produced oospores on clarified V8 juice agar amended with thiamine, tryptophan, and β-sitosterol (2) after 15 to 30 days at 20°C in the dark. Mycelia grew between 5 and 30°C, with optimal growth at 20°C and no growth at 35°C. The ITS sequence analysis performed by IMI (CABI Bioscience, Wallingford, UK) indicated a close match with P. cryptogea, P. drechsleri, and P. erythroseptica, however, peach isolates were differentiated from P. drechsleri and P. erythroseptica by the absence of mycelial growth at 35°C and the heterothallic production of oospores, respectively. Four P. cryptogea isolates were pathogenic when inoculated onto 1-year-old twigs detached from a Nemaguard tree, causing reddish necrotic lesions varying from 7 to 29 mm long after 15 days of incubation at 20°C. Two-year-old P. persica var. nectarina cv. Ruby Diamond budded on Nemaguard and cultivated in 100-liter containers in open field conditions were inoculated with 500-ml of a mycelial suspension (4.5 × 106 propagules per ml) per plant added to 15-cm-deep holes around the trunk. Plants were immediately irrigated and maintained at field capacity during the experiment. An equal number of noninoculated trees treated similarly were left as controls. During the spring, plants developed a general chlorosis and declined within 90 days postinoculation. Tests were repeated with similar results. In other preliminary tests, plants developed cankers characterized by gumming and reddish necrotic lesions underneath the bark following trunk inoculations with mycelium in agar of the same isolates obtained on ACMA. These tests were also repeated. The pathogen was consistently reisolated from symptomatic tissue. To our knowledge, this is the first report of P. cryptogea as a pathogen on peach trees in Chile.

Leaf Blight of Fatsia japonica caused by Phytophthora cactorum

A leaf blight caused by a species of Phytophthora was found on fatsia plants (Fatsia japonica Decne et Planch.) growing in an apartment garden in Daegu, Korea in late April to May, 2003. The species of Phytophthora isolated from the diseased plants produced sporangia and sex organs on V8 juice agar medium. Sporangia were papillate, ovoid to subspherical, and caducous with a pedicel. The dimensions of the sporangia were <TEX>$31.2-46.8\times23.4-33.2{\mu}m$</TEX> in range, <TEX>$39.6\pm4.1\times28.3\pm2.8{\mu}m$</TEX> in <TEX>$average{\pm}standard$</TEX> deviation, I/b ratio approximately 1.40, with papillae about <TEX>$3.6{\mu}m$</TEX> high, and pedicels <TEX>$0.9­5.8{\mu}m$</TEX> long. Oogonia were spherical, <TEX>$25.0-32.5{\mu}m$</TEX> in range with an average of <TEX>$28.2\pm2.3{\mu}m$</TEX> in diameter. Antheridia were predominantly paragynous, globose to ovoid, <TEX>$8.8-13.8\times7.5-10.0{\mu}m$</TEX> with an average of <TEX>$10.9\pm1.2 \times9.2\pm1.1{\mu}m$</TEX>. Oospores in the oogonia were aplerotic or plerotic, and <TEX>$20.0-25.0{\mu}m$</TEX> in diameter with an average of <TEX>$23.5\pm1.5{\mu}m$</TEX>. Pathogenicity of the isolate was confirmed on detached leaves of fatsia. The disease was observed only in April and May of 2003 when the weather was unusually wet. It then diminished with increase of temperature in the year and did not appear again on the same plants in 2004. Thus, the fungus appeared to be a relatively weak pathogen of fatsia.

First Report of Brown Stripe of Saltgrass Caused by Bipolaris heveae in Colorado.

Inland saltgrass (Distichlis spicata var. stricta (L.) Greene) is indigenous to western North America and Australia and is a dioecious, rhizomatous, perennial, warm-season grass. It is commonly found in areas where salinity, alkalinity, and drought have eliminated many other types of vegetation (1). It has potential for revegetation of mine spoils or use along roadsides (2). During September 2004, multiple lenticular, brown lesions were observed on leaves of saltgrass accession no. 1023 at the Horticulture Field Research Center, Colorado State University, Fort Collins. Segments of symptomatic leaf tissue were surface sterilized in 0.5% sodium hypochlorite and placed on one-quarter-strength potato dextrose agar and incubated at 25°C in the dark. Dark green fungal colonies with aerial mycelium consistently grew on the medium. Slightly curved, ellipsoidal, pale-to-golden brown, smooth conidia 46 to 80 μm long and 13 to 17 μm wide (average 64.5 × 14.7 μm) with 6 to 9 septations formed after 7 days in cultures grown on V8 juice agar. The morphology and bipolar germination of conidia was consistent with the genus Bipolaris, however, conidia were often shorter than previously reported (3). The rDNA internal transcribed spacer (ITS) regions of one isolate were amplified using polymerase chain reaction (PCR) with universal fungal rDNA primers ITS1 and ITS4. PCR products were sequenced (555 bp) and exhibited 99% nucleotide identity to Bipolaris heveae isolates collected from zoysiagrass and bermudagrass in Japan (3) and rubber in Nigeria (4). To confirm pathogenicity, a suspension of 104 conidia per ml of water containing 0.1% Tween 20 was sprayed on saltgrass leaves to runoff. Plants were covered with transparent plastic bags and incubated at 25°C in the dark. After 72 h, the bags were removed and plants were placed in the greenhouse. Brown stripe symptoms were observed on all plants after 7 days, and B. heveae was consistently isolated from symptomatic tissue. To our knowledge, this is the first report of brown stripe on inland saltgrass caused by B. heveae.

Effect of media and temperature on sporulation of Septoria apiicola, and of inoculum density on septoria blight severity in celery

The number of conidia produced by Septoria apiicola was quantified on seven media incubated at 20°C. Conidia were produced abundantly on celery agar (CA) and celery potato dextrose agar (CPDA) after 12 days of incubation. In general, media containing celery decoction, including cleared V8 juice agar (CVA), produced more conidia than those without celery decoction. The number of conidia and the colony area produced per plate on CA increased with increase in temperature up to an optimum temperature and then decreased markedly. A second-order linear regression equation explained 99% of the variation in the number of conidia produced on CA plates incubated at temperatures ranging from 15 to 30°C, with the predicted optimum temperature of 22.4°C. Ninety eight percent of the variations in the colony area were explained by a linearized BETE-equation with the predicted optimal temperature of 22.7°C. Blight severity in celery (Apium graveolens var. dulce ) increased with increase in inoculum density. For inoculum density ranging from 5-140 conidia/cm2 of leaf surface, 92% of the variations in the number of lesions per leaf, pooled from two experiments, were explained by a second-order linear regression equation. The model was less reliable for inoculum densities less than 17 conidia/cm2 of leaf surface.

First Report of Macrophomina phaseolina Causing a Crown Rot of Strawberry in Florida.

Strawberry (Fragaria × ananassa Duchesne) is produced as an annual winter crop in raised, plastic-mulched beds on 2,800 ha in west central Florida. In December 2001, a grower submitted collapsed and dying strawberry plants from a commercial field to the University of Florida in Dover. The cut crowns of affected plants revealed dark brown necrotic areas on the margins and along the woody vascular ring. Macrophomina phaseolina was isolated from pieces of infected tissue cut aseptically from the crowns and placed on a medium containing 12 g of Difco potato dextrose broth, 17 g of Bacto agar, 250 mg of ampicillin, and 100 mg of streptomycin sulfate per liter of water. The fungus produced numerous, dark, oblong sclerotia in the isolation medium after 4 to 5 days incubation at 24°C under constant fluorescent lighting. In 10-day-old cultures, sclerotia ranged in size from 55 to 190 μm long by 50 to 135 μm wide (average 105 × 74 μm). Ostiolate pycnidia bearing relatively large, broadly ellipsoidal, hyaline conidia occasionally developed on the host tissue after 8 to 10 days of incubation (2). During the 2003-2004 season, M. phaseolina was isolated from dying strawberry plants taken from the original field and two additional farms. Affected plants were often found along field margins or other areas inadequately fumigated with methyl bromide. Two single-spore isolates from different fields were tested for pathogenicity on nursery runner plants (cv. Strawberry Festival) grown for 4 weeks in the greenhouse on artificial potting soil. The fungal isolates were grown on corn meal agar at 24°C for 4 days and allowed to colonize sterile wooden toothpicks placed on the medium for an additional 5 days. Prior to use, the toothpicks were sterilized by autoclaving twice in deionized water and a third time in V8 juice. Six plants were inoculated with each isolate by inserting a colonized toothpick into each crown. Sterile, V8-infused toothpicks were inserted into the crowns of corresponding control plants. The plants were incubated in a greenhouse in a randomized complete block design with two replicates of three plants each. After 3 days, 33 to 100% of the inoculated plants developed wilting in one or more leaves. All inoculated plants collapsed or died within 2 weeks of inoculation, while the control plants remained healthy during the observation period. The pathogen was readily reisolated from inoculated plants. Charcoal rot disease caused by M. phaseolina has been reported on strawberry in France, India, and Illinois (2,3). To our knowledge, this is the first report from Florida. M. phaseolina may be an emerging threat as the Florida strawberry industry transitions from methyl bromide to other fumigants in 2005.

Impact of Label Information on Consumer Assessment of Soy‐enhanced Tomato Juice

ABSTRACT: The impact of label information on the liking and closeness to ideal of tomato juice beverages was examined by having 100 judges assess 3 tomato juice beverages (Campbell's tomato juice, V8 juice, and an experimental tomato juice enhanced with soy) either with or without labeling information. Judges rated overall liking of each product and then rated appropriateness of various attributes (saltiness, tomato flavor, thickness, texture, red color, orange color, and brown color) on 5‐point just‐right scales. Only half of the judges were presented with product‐related label information during evaluation. Overall liking scores were analyzed using repeated‐measures ANOVA, whereas different attribute ratings were analyzed using Thurstonian Ideal Point modeling and Chi‐square. Product‐associated label information did not significantly alter overall liking ratings (P &gt; 0.05), although a significant difference in liking was found between products (P &lt; 0.05). Similarly, the label information did not impact comparison of product attribute levels to ideal attribute levels. Both V8 and Campbell's were significantly different from the ideal for 3 of the attributes (P &lt; 0.05). For the soy‐enhanced tomato juice, all 7 attributes were significantly different from the ideal (P &lt; 0.05). In this instance, labeling information had no notable impact on assessments.

First Report of Spot Type of Barley Net Blotch Caused by Pyrenophora teres f. sp. maculata in Uruguay.

In September 2003, leaves exhibiting spot-type lesions similar to those produced by Cochliobolus sativus Drechs. ex Dastur were widely observed in six commercial barley crops of cvs. Norteña Daymán, Norteña Carumbé, and MUSA 936 in Soriano and Río Negro provinces, the main barley production region in western Uruguay. Spot lesions were tan to dark brown, circular to elliptical, and 3 to 10 mm in diameter. Larger lesions were surrounded by a chlorotic margin of varying width. Affected leaf pieces (10 to 15) from each field were placed in a moist chamber for 2 days to promote sporulation. A fungus identified morphologically as Pyrenophora teres (Died.) Drechs. (1) was consistently isolated from infected leaves. However, symptoms did not correspond to the net-type lesions of net blotch commonly produced by P. teres f. sp. teres in Uruguay. Three monoconidial cultures were obtained by transferring single conidia to potato dextrose agar and then to 10% V8 juice agar and incubated at 20 to 22°C with a 12-h photoperiod for 10 days. Adding sterile water to each plate and gently rubbing the surface with a microscope slide prepared inoculum for pathogenicity tests. Conidia concentration was adjusted to 1 × 104 conidia per ml. Sixty-eight barley genotypes from Uruguay, ICARDA/CIMMYT, and North Dakota were grown in the greenhouse for 2 weeks at 20 to 22°C with a 14-h photoperiod. For each monoconidial isolate, three seedlings of each genotype were inoculated at the three-leaf stage 15 to 16 days after seeding with 0.4 ml of the inoculum suspension with an airbrush inoculator. A drop of Tween 20 was added per 40 ml of inoculum suspension. One set of each genotype was inoculated with sterile water as a control. Seedlings were placed in a dew chamber at 20°C and 100% relative humidity in the dark for 24 h and then returned to prior conditions. The first lesions developed after 7 to 9 days. Leaves two and three of the plants were visually rated for disease (3) 13 days after inoculation. Control plants were disease free. The most susceptible reactions were observed on cvs. Norteña Daymán, MUSA 936, and line CLE 230 (Uruguay). Symptoms were similar in shape and size to those observed in the fields. The most resistant infection types were observed on several Uruguayan and North Dakota advanced lines. The fungus was consistently reisolated from inoculated plants. On the basis of morphology and symptoms produced, the pathogen was identified (2) as P. teres. f. sp. maculata Smedeg. To our knowledge, this is the first report of this fungus causing spot-like symptoms of net blotch in Uruguay.

Leaf Blight of Ailanthus altissiman Caused by Phytophthora boehmeriae

A leaf blight disease was found on Ailanthus altissiman growing in the Manchon Mountain Park in Daegu city. When isolated, the causal fungus readily formed sex organs, being homothallic. Oogonia were spherical, 19.5-42.9 <TEX>${\mu}{\textrm}{m}$</TEX> in diameter with an average of 29.4<TEX>$\pm$</TEX>4.2 <TEX>${\mu}{\textrm}{m}$</TEX>. Antheridia were amphigynous, round to ovoid, and measured 11.3-15.0 <TEX>${\mu}{\textrm}{m}$</TEX> long and 12.5-14.5 <TEX>${\mu}{\textrm}{m}$</TEX> wide. Oospores in the oogonia were spherical, 26.1-29.0 <TEX>${\mu}{\textrm}{m}$</TEX> in diameter. Sporangia that formed in water were spherical to sub-spherical with a conspicuous papilla and measured 19.5-56.6 <TEX>${\times}$</TEX> 15.6-44.9 <TEX>${\mu}{\textrm}{m}$</TEX> with an average of 44.0<TEX>$\pm$</TEX>8.7<TEX>${\times}$</TEX>32.7<TEX>$\pm$</TEX>6.3 <TEX>${\mu}{\textrm}{m}$</TEX>. The mean length/breadth (I/b) ratio was 1.35. Papillae were 3.9-11.7 <TEX>${\mu}{\textrm}{m}$</TEX>l high and 3.9-9.8 <TEX>${\mu}{\textrm}{m}$</TEX> wide. Sporangia formed slowly on V8 juice agar medium when cultured under fluorescent light at 12-hour alternation. The sporangia that formed on the agar medium were more spherical and measured 26.5-39.0<TEX>${\times}$</TEX>23.4-35.1 <TEX>${\mu}{\textrm}{m}$</TEX> with an average of 33.6<TEX>$\pm$</TEX>3.4<TEX>${\times}$</TEX>28.2<TEX>$\pm$</TEX>3.2 <TEX>${\mu}{\textrm}{m}$</TEX> and I/b ratio of 1.19. Disease symptom was repro-duced by artificial inoculation of the healthy plants with the isolate. The causal organism was identified as Phyto-phthora boehmeriae on the basis of its morphological characteristics.

First Report of Resistance to Mefenoxam in Phytophthora cactorum in the United States and Elsewhere.

Phytophthora cactorum causes crown rot of strawberry (Fragaria × ananassa) (2), a disease that has been particularly severe during the last 5 years in the southeastern United States. In the fall of 2001, strawberry plants (cv. Camarosa) in a field in Lexington County, South Carolina exhibited typical crown rot symptoms (2) 1 to 2 weeks after transplanting, even though plants had been drenched with mefenoxam (Ridomil Gold; Syngenta Crop Protection, Greensboro, NC) immediately after transplanting. Initially, we observed leaves that had marginal necrosis, were smaller than normal, and were discolored. Soon after, diseased plants appeared stunted and unthrifty compared with other plants in the field, and some of these plants eventually wilted and died. Severely affected plants had necrotic roots and decayed crowns. Ten symptomatic plants were collected for isolation. In the laboratory, root and crown tissues were rinsed in running tap water and blotted dry, small pieces of necrotic tissue were placed aseptically on PAR-V8 selective medium (1), and isolation plates were placed at 20°C in the dark for up to 7 days. P. cactorum was recovered from six plants. Isolates produced characteristic asexual and sexual structures directly on the isolation plates (i.e., papillate sporangia on sympodial sporangiophores and oospores with paragynous antheridia) (2). A single hypha of an isolate from each plant was transferred to fresh PAR-V8, and pure cultures were stored on cornmeal agar in glass vials at 15°C in the dark. All six isolates from the Lexington County field and nine other isolates of P. cactorum from strawberry (three from South Carolina, three from North Carolina, and three from Florida) were tested for sensitivity to mefenoxam on fungicide-amended medium. Mefenoxam was added to 10% clarified V8 juice agar (cV8A) after autoclaving so the concentration in the medium was 100 ppm. Agar plugs from active colonies were transferred to mefenoxam-amended and nonamended cV8A (three replicates per treatment), plates were placed at 25°C in the dark for 3 days, and linear mycelium growth was measured. All six isolates from Lexington County were highly resistant to mefenoxam with mycelium growth relatively unrestricted on mefenoxam-amended medium (73 to 89% of that on nonamended medium). In comparison, the other nine isolates were sensitive to mefenoxam with mycelium growth severely restricted by 100 ppm of mefenoxam (0 to 7% of that on nonamended medium). To our knowledge, this is the first report of mefenoxam resistance in P. cactorum on strawberry or any other crop in the United States and elsewhere. Because mefenoxam is the primary fungicide used to manage Phytophthora crown rot in the southeastern United States, resistance may limit use of this fungicide in strawberry production.

Experience with the MicroSeq D2 large-subunit ribosomal DNA sequencing kit for identification of filamentous fungi encountered in the clinical laboratory.

Described herein is our experience with the MicroSeq D2 large-subunit rDNA sequencing kit for the identification of filamentous fungi encountered in the mycology laboratory at the Mayo Clinic. A total of 234 filamentous fungi recovered from clinical specimens were used in the evaluation. All were identified by using phenotypic characteristics as observed macroscopically and microscopically on any medium or a combination of media, which included Sabouraud's dextrose, inhibitory mold, cornmeal, Czapek-Dox, potato dextrose, and V8 juice agars; all isolates were sequenced using the MicroSeq D2 large-subunit rDNA sequencing kit. Of the of 234 isolates, 158 were correctly identified to the appropriate genus or genus and species by using nucleic acid sequencing. Sequences for 70 (29.9%) of the isolates (27 genera) were not included in the MicroSeq library. Of the 80 dematiaceous and 154 hyaline fungi sequenced, 65 and 51.2%, respectively, gave results concordant with those determined by phenotypic identification. Nucleic acid sequencing using the MicroSeq D2 large-subunit rDNA sequencing kit offers promise of being an accurate identification system; however, the associated library needs to include more of the clinically important genera and species.

First Report of Stem and Root Rot of Tomato Caused by Phytophthora capsici in South Africa.

Tomato plants, grown in open hydroponic systems under shadecloth and plastic near Barberton and Pretoria in South Africa and Srelebi Phikwe in Botswana, developed symptoms of wilting with brown-to-black cankers on the lower stems, blackening of the vascular tissues, and root rot. Pathogens isolated from affected tissues were identified as Phytophthora capsici Leonian (1) and Pythium aphanidermatum (Edson) Fitzp. (2).They occurred separately or together. Pythium aphanidermatum has previously been recorded on tomato in South Africa. P. capsici isolates were papillate, caducous, grew at >36°C, had tapered sporangial bases, and a maximum sporangial length of >70 μm. Koch's postulates were confirmed by inoculating 4-week-old tomato seedlings (cv. Floradade) grown at 22 to 30°C in a steam-pasteurized mixture of sawdust compost, pine bark, and vermiculite (3:2:1). Plugs from V8 juice agar cultures of P. capsici were placed on wounds made on the stems of 10 seedlings. Ten wounded uninoculated plants served as controls. Water-soaked lesions were visible on the stems of all inoculated plants after 2 days. Control plants remained healthy. After 4 days, lesions turned dark brown with affected plants wilted or dead. Reisolation yielded P. capsici. The experiment was repeated with similar results. To our knowledge, this is the first report of P. capsici on tomatoes in South Africa.

First report of cercospora leaf spot on Swiss chard caused by Cercospora beticola in Turkey

In February and November 2002, a severe outbreak of a foliar disease was noted in the main producing Swiss chard ( Beta vulgaris var. cicla ) areas in the Hatay province of Turkey. In this region, disease was observed in 90–95% of fields inspected. Symptoms began as small circular, light brown spots with definite dark brown border on both sides of the leaf. The circular spots increased in size (up to 4–6 mm in diameter), developing into depressed lesions, which were brownish in colour and bordered by a purple margin. As lesions matured, the centre of the spots became whitish grey to black, due to the production of conidia and conidiophores. Microscopic examination of the whitish-grey lesions revealed the presence of tiny, black stromata within the leaf substomatal cavities. The stromata produced dense fascicles of conidiophores (up to 35 conidiophores per fascicle) that emerged through the stomata. Conidiophores were simple, one to three septate, pale brown at the base but subhyaline at the apex, straight (3·5–4·5 × 20– 65 μ m), unbranched and slightly attenuated towards to apex. Conidia, borne singly, were filiform, hyaline, pluriseptate (five to 18), nonconstricted at the septa, slightly curved, subtruncate at the base, obtuse to subacute at the apex and varying considerably in length (2·5–4 × 40– 125 μ m). The causal organism was identified as Cercospora beticola based on morphological characteristics (Kim & Shin, 1998). A single-spore isolate was cultured on V8 juice agar. Pathogenicity tests were conducted on 4week-old chard seedlings (cv. Zilk), by spraying the leaves with a conidial suspension (10 4 spores per mL). Inoculated plants were kept in a moist chamber (100% RH) at 25 ° C for 3 days, then moved to a growth chamber at 26 ° C with a 16-h photoperiod. Many small elongated lesions (1–2 mm wide) were observed on inoculated leaves 5 days after treatment. After 21 days, these developed into lesions similar to those produced on naturally infected leaves. The pathogen was reisolated from inoculated leaves. This is the first report of C. beticola on Swiss chard in Turkey. Previous reports list C. beticola on Swiss chard in Egypt (El-Kazzaz, 1977) and Korea (Kim & Shin, 1998). The fungus was also reported on several host plants such as cultivated and wild Beta spp., spinach and lambs quarter (Nyvall, 1989; Rossi et al ., 1995).

A New Phytophthora sp. Causing a Basal Canker on Beech in Italy.

In autumn 2001, bleeding cankers were observed on the basal portion of the trunk of a declining tree in a forest stand of European beech (Fagus sylvatica L.) in Latium (central Italy). A Phytophthora sp. was isolated consistently from infected trunk bark using whole apples as bait. Isolations were made from brown lesions that developed in the apple pulp around the inserted bark pieces. Pure cultures were obtained by using hyphal tip transfers. Colonies were stellate on V8 juice agar (V8A), uniform to slightly radiate on cornmeal agar, and cottony, without a distinct growth pattern on potato dextrose agar (PDA). On V8A, radial growth rates were 2.1, 4.8, and 4.5 mm/day at 10, 15, and 20°C, respectively. Colonies grew slowly at 5 and 25°C, but failed to grow at 30°C. On PDA, growth was 1.7 and 1.4 mm/day at 15 and 20°C, respectively. Catenulate hyphal swellings formed on solid and liquid media. Sporangia formed abundantly at 15°C, were ovoid to obpyriform, semipapillate, occasionally bipapillate, and had narrow exit pores (mean diameter = 5.4 μm). On V8A, pores were 40 to 50 μm in length and 25 to 40 μm in breadth. Isolates were homothallic with paragynous antheridia, oogonia were spherical with diameters from 32 to 35 μm, and oospores were plerotic with diameters from 20 to 30 μm. Electrophoretic banding patterns of mycelial proteins and isozymes (alkaline phospatase, esterase, glucose-6-phospate dehydrogenase, malate dehydrogenase, and superoxide dismutase) of beech isolates were distinct from those of reference isolates of semipapillate Phytophthora species, including P. citricola, P. hibernalis, P. ilicis (IMI 158964), P. psychrophila (CBS 803.95), and P. syringae from citrus fruits, whose identification had been confirmed on the basis of internal transcribed spacer (ITS)-restriction fragment length polymorphism (RFLP) patterns and sequences. Conversely, the electrophoretic phenotype and the ITS-RFLP pattern (and sequence) of the beech isolates were identical to those of a reference isolate (Ph24) from Quercus cerris, which was originally identified as P. syringae on the basis of morphological and cultural characters (1). However, the isolate Ph24 has been reexamined, and morphological and cultural characteristics as well as the ITS sequence would indicate that this isolate is a new species not yet formally described, for which the name P. pseudosyringae has been suggested (2). The pathogenicity of a beech isolate (IMI 390500) was compared to that of an Italian P. cambivora isolate from European chestnut by inoculating the stems of 16-month-old beech seedlings (10 replicates), which were placed at 18°C with a 12-h photoperiod. The beech isolate produced lesions averaging 2 cm long after 2 months, while those produced by the P. cambivora isolate averaged 3 cm. Control seedlings inoculated with sterile agar did not develop symptoms. The pathogen was reisolated from lesions to fulfil Koch's postulates. To our knowledge, this is the first report of this new Phytophthora sp. on beech in Italy. Conversely, the same species has been reported to be associated with decline of oak stands (1).

First Report of Foliar Infection of Starflower by Phytophthora ramorum.

In March 2002, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from pacific or western starflower (Trientalis latifolia Hook.), an herbaceous perennial of the Primulaceae family, at Castro Canyon in Big Sur, Monterey County, California. Affected leaves had numerous necrotic lesions >5 mm in diameter surrounded by a yellow halo, and the lesions coalesced with time. Isolates were identified as P. ramorum by the large chlamydospores, caduceus, semipapillate sporangia, and sequences of the internal transcribed spacer (ITS) region of the rDNA (1,2). The same symptoms were observed on starflower in a second location at the Soquel Demonstration Forest, Santa Cruz County. Although P. ramorum was not isolated from symptomatic leaves on the plants in Santa Cruz County, the ITS region of the pathogen was amplified and sequenced using P. ramorum-specific primers. Both sites were mixed forests of coast redwood (Sequoia sempervirens), bay laurel (Umbellularia californica), and tanoak (Lithocarpus densiflorus), which are confirmed hosts of P. ramorum. To test for pathogenicity to starflower, asymptomatic plants were carefully excavated from the two forest locations, replanted in 15-cm paper cups in the original forest soil, and the foliage was inoculated with zoospores of P. ramorum isolate Pr-52, an isolate used in previous inoculations. The zoospores were produced by placing agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar into 20 to 30 ml of sterile deionized water in petri dishes. After 2 days incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then to room temperature for 45 to 60 min. Three hundred μl of the zoospore suspension (approximately 2 × 104 zoospores/ml) was poured into 500-μl modified microcentrifuge tubes in which tips of leaves of starflower were submerged. Control leaves were dipped in sterile deionized water. Plants were placed in a humid-chamber consisting of moist paper towels placed on the tray and covered with a clear-plastic lid that was sprayed with sterile water. The chambers were maintained at 20 to 24°C in the laboratory. Two or three leaves were inoculated, and one leaf was left as the control on each of seven or eight plants in two separate trials. In both trials, water-soaked lesions were observed on the leaves 12 h after inoculation with P. ramorum. At 8 or 11 days after inoculation, necrotic lesions were present on all inoculated leaves starting from the leaf tips. Lesions averaged 29 mm (range 13 to 39 mm) and 45 mm (range 31 to 56 mm) in length in the respective trials. Some lesions covered entire leaves. P. ramorum was reisolated on Phytophthora-selective agar medium (1) from the lesions in both trials. Control leaves had no lesions, and P. ramorum was not reisolated. Infection of starflower and other understory species appears to occur under infested tree hosts such as bay laurel, which is known as a source of inoculum for P. ramorum. To our knowledge, this is the first report of an herbaceous host for P. ramorum and the first report of the disease on the Primulaceae. Previously, only woody hosts were known. Starflower is unlikely to play a major role in the natural spread of the disease, but the pathogen may be spread via movement of plants through the horticultural industry. Furthermore, Trientalis spp. in Europe where P. ramorum is present may also be potential hosts.

First Report of Phytophthora cinnamomi on High-Bush Blueberry in Italy.

Beginning in the summer of 1996, a disease of high-bush blueberry (Vaccinium corymbosum L.) appeared on cvs. Sparta and Berkeley in commercial plantings near Cuneo and in a nursery in Pinerolo (northwest Italy). In the field, the disease became evident 5 to 8 months after planting. Symptoms included stunting, pale green-to-reddish leaves, premature defoliation, and root and crown rot. The root system was reduced, with few feeder roots and a dark discoloration of the root cortex. Root, crown, and basal stem rot led to death of the plants. Pieces of the discolored organs were surface-sterilized in sodium hypochlorite (2%) for 5 min, rinsed in sterile water, dried on sterile filter paper, and plated on acidified malt extract agar (pH 4.6) (MEA) and a selective medium for oomycetes (2). Slow-growing colonies developed on MEA, whereas colonies characteristic of Phytophthora spp. grew on the selective medium for oomycetes. Six isolates from plants collected from the field and the nursery were inoculated as V8 juice culture homogenate on the roots of blueberry cv. Berkeley, azalea, and 1-year-old chestnut plants in pots. All six isolates reproduced the symptoms in blueberry and caused root and foot rot in the other hosts. The original fungus was reisolated from all inoculated hosts. The isolates were examined and compared with strain 0229 of P. cinnamomi Rands from the International Mycological Institute. They were identified as P. cinnamomi on the basis of morphological characteristics and total mycelial soluble protein patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In Italy, blueberry is a new host for this pathogen that was previously observed in rhododendron (1), and recently in chestnut (3) and other forest species. Because of the high risk of spreading the pathogen to areas where susceptible plants are grown, it is recommended that high-bush blueberry nursery stock be tested for the pathogen.

Induced Resistance as a Possible Means to Control Diseases of Strawberry Caused by Phytophthora spp.

Two putative elicitors of disease resistance (acibenzolar-S-methyl and chitosan) were tested for their effect on crown rot (Phytophthora cactorum) in strawberry. The effect of both compounds was enhanced when the time between treatment and inoculation was prolonged from 2 to 20 days. There were no significant differences between treatments when the concentration of acibenzolar-S-methyl was increased from 10 to 1,000 μg a.i./plant. The lowest tested concentrations of chitosan (10 and 50 μg a.i./plant) resulted in a lower disease score compared with the highest concentrations (250 or 1,000 μg a.i./plant). There were no differences in disease score between treatment with fosetyl-Al, acibenzolar-S-methyl, or chitosan when applied 5 or 15 days before inoculation. The effect of acibenzolar-S-methyl and chitosan also was tested against P. fragariae var. fragariae in alpine strawberry (Fragaria vesca var. alpina cv. Alexandria). Chitosan had no effect, whereas fosetyl-Al and all treatments with acibenzolar-S-methyl (50 or 250 μg a.i./plant; 5, 10, 20, or 40 days before inoculation) reduced the severity of the disease. There were no significant differences between acibenzolar-S-methyl and fosetyl-Al when applied at the same time. Acibenzolar-S-methyl and chitosan at concentrations of 0.5, 5, 50, and 500 μg a.i. ml-1 in V8 juice agar were tested for possible effects on P. cactorum and P. fragariae var. fragariae in vitro. Only chitosan at concentrations of 50 and 500 μg a.i. ml-1 had a growth-retarding effect on P. cactorum. Both acibenzolar-S-methyl and chitosan at a concentration of 500 μg a.i. ml-1 reduced the growth rate of P. fragariae var. fragariae.