Evaluation of incidence, predictive factors and treatment considerations for asymptomatic genitourinary granulomas after intravesical bacillus Calmette-Guérin therapy.

INTRODUCTION AND OBJECTIVES: Although the complications of intravesical BCG treatment are well described, asymptomatic genitourinary granulomas after BCG therapy have rarely been reported and management strategy for these conditions remains controversial. The objective of this study is to evaluate the incidence rate of asymptomatic genitourinary granuloma formation mimicking bladder cancer recurrence after intravesical bacillus Calmette-Guérin (BCG) therapy and to identify the diagnostic and treatment strategies according to patient conditions. PATIENTS AND METHODS: A retrospective review was conducted on 162 patients who underwent intravesical BCG therapy. For patients who developed granulomas, we evaluated the time interval between BCG instillation and the development of granuloma, the presence of acid-fast bacteria on pathology specimens, culture/polymerase chain reaction results, management strategies for the lesions, and clinical outcomes. RESULTS: Asymptomatic genitourinary masses developed in 14 patients, of whom 5 underwent histological examinations and all were confirmed to have granulomatous inflammation. The affected organs included the kidney, bladder, prostate, and penis. While four of the five patients did not receive treatment for their granulomas, one patient was administered antituberculous medication to prevent worsening of the lesion during the perioperative period of the scheduled cystoprostatectomy. None of the patients experienced worsening or recurrence of granulomatous lesions. Patients who developed asymptomatic masses (n = 14) were significantly younger than those who did not (p = 0.0076) and multivariate analysis also showed that younger age was independently associated with the development of clinically suspicious lesions (p = 0.032); however, none of the parameters were associated with histologically confirmed granuloma formation. CONCLUSIONS: Genitourinary granulomas mimicking recurrence of carcinoma may develop in nearly 10% of patients after intravesical BCG therapy. Most patients can be managed without potentially toxic antituberculosis therapy.

Pseudomonas Blight Caused by Pseudomonas syringae on Raspberry in California.

In February 2013 in coastal California (Santa Cruz County), plantings of red raspberry (Rubus idaeus var. strigosus) exhibited symptoms of a previously undocumented disease. Initial symptoms were small (less than 5 mm wide), angular, water-soaked lesions on leaf and petiole tissues of recently emerged foliage. Lesions were observable from both adaxial and abaxial leaf surfaces. As disease progressed, lesions enlarged and coalesced, resulting in significant dark brown to black blighting of the foliage. The foliage of severely affected plants was stunted and wilted. The disease affected 5 ha and incidence was approximately 30%. Cream-colored bacterial colonies were isolated from surface disinfested symptomatic tissue that was macerated and streaked onto King's medium B (KMB) and sucrose peptone agar (SPA). Fungi were not recovered from any tissue that was surface disinfested and placed into acidified potato dextrose agar. Four representative strains were fluorescent on KMB and gram-negative based on lysis by KOH. Strains were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices but induced a hypersensitive response in tobacco (Nicotiana tabacum L. cv. Samsun); strains thus belonged to Lelliot's LOPAT group 1, P. syringae (3). All four strains had identical DNA fragment-banding patterns generated by repetitive extragenic palindromic sequence (rep)-PCR using the BOXA1R primer (4). The pattern generated was different than all P. syringae pathovars in genomospecies 1 including P. syringae pv. syringae. According to multilocus sequence analysis conducted by previously described methods, the strains are most closely related to P. syringae pv. aceris and P. syringae pv. solidagae in genomospecies 1 (1). Potted raspberry plants were used to test four strains for pathogenicity. Inoculum was prepared by growing the bacteria on SPA for 48 h and suspending the bacteria in sterile distilled water (SDW) for a final concentration of approximately 107 CFU/ml. Suspensions were sprayed until runoff onto three replicate plants per strain. Control plants were sprayed with SDW until runoff. Plants were enclosed in plastic bags for 24 h and then maintained in a greenhouse (23 to 25°C). After 7 to 8 days, water soaked lesions developed on all inoculated plants; lesions later turned dark brown and appeared similar to symptoms observed in the field. Plants treated with water developed no symptoms. Bacteria re-isolated onto KMB from symptomatic tissues were fluorescent and appeared identical to the bacteria used to inoculate the plants; two selected re-isolated strains were identical to the original strains according to rep-PCR, fluorescence, and LOPAT reactions. The experiment was repeated and disease development and recovery of fluorescent strains on KMB was identical to the first experiment. To our knowledge, this is the first report of Pseudomonas blight of raspberry, caused by P. syringae, in California. Affected plants initially were stunted in growth but later in the summer exhibited no lasting effects from the disease. Pseudomonas blight has been reported in the Pacific Northwest region of the United States, the British Columbia region of Canada, and Serbia (2). References: (1) C. T. Bull et al. Phytopathology 101:847, 2011. (2) Z. Ivanovic et al. Eur. J. Plant Pathol. 134:191, 2012. (3) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966. (4) A. S. A. Marques, et al. Genet. Mol. Biol. 31:106. 2008.

First Report of Turnip mosaic virus in Tomatillo (Physalis philadelphica) in California.

Tomatillo is an important vegetable in Mexican cuisine. It is of Mesoamerica origin and now is grown widely in the Western Hemisphere. In 2011, 2% of commercially grown tomatillo plants in San Benito County, California exhibited severe stunting with foliage showing mosaic symptoms and leaf distortion. The fruits on infected plants were mottled and unmarketable. Flexuous filamentous-shaped virus particles of 800 to 850 nm long and 11 to 12 nm wide were observed from sap of the symptomatic plants with a transmission electron microscope. Sap from the diseased tomatillo plants reacted positively in an immunostrip assay for potyvirus (Agdia Inc., Elkhart, IN), indicating a potyvirus was associated with the disease. The causal agent was mechanically transmitted from the diseased field plants to six virus-free greenhouse tomatillo plants and all inoculated plants induced identical symptoms. The causal agent was also transmitted to Chenopodium quinoa and C. murale (chlorotic local lesions) and Nicotiana clevelandii, N. tabacum, and Physalis wrightii (systemic symptoms). The disease was also transmitted to tomatillo plants by the green peach aphid (Myzus persicae) in a nonpersistent manner (1-min acquisition access period and 1-min transmission access period with no latent period). To further identify the causal agent, total nucleic acids were extracted by a cetyltrimethylammoniumbromide (CTAB) method (2) and tested by reverse transcription-PCR using potyvirus degenerate primers CIFor and CIRev (1). An amplicon of approximately 700 bp from the diseased tomatillo was cloned and sequenced. Analysis of the 631-bp partial CI sequence (GenBank Accession No. JN601884) showed that the virus had 93.6% nucleotide identity and 100% amino acid identity with cognate regions of Turnip mosaic virus (TuMV) (GenBank Accession No. D10927). Our results indicated that the disease was caused by TuMV. To our knowledge, this is the first report of TuMV in tomatillo. Since TuMV has a wide host range and is readily transmitted by green peach aphids, TuMV could be a new threat to tomatillo production in California. References: (1) C. Ha et al. Arch. Virol. 153:25, 2008. (2) R. Li et al. J. Virol. Methods 154:48, 2008.

First Report of Bacterial Streak of Fennel (Foeniculum vulgare) in California Caused by Pseudomonas syringae pv. apii.

A previously undescribed disease appeared on commercially grown fennel (Foeniculum vulgare) in Salinas (Monterey County), CA in March 2010. Initial symptoms consisted of small, dark brown-to-black lesions on leaves and stems. As disease progressed, lesions expanded in a linear fashion and eventually spread down the stems and into the bulbs. Once the disease reached the fennel bulbs, the plants were unmarketable. Eighteen, gram-negative, blue-fluorescing bacterial isolates were obtained on sucrose peptone agar from individual plants of two outbreaks from different fields in Salinas. The isolates were dome shaped on sucrose-amended media and produced necrotic lesions when injected into tobacco. Isolates were oxidase negative and did not rot potatoes or hydrolyze arginine. These reactions corresponded to Lelliot's group 1, which includes Pseudomonas syringae (2). The fennel isolates were identical to recently characterized isolates of P. syringae pv. apii from parsley grown in coastal California (1) and similar to the pathotype strain of P. syringae pv. apii according to DNA fragment banding patterns of amplicons generated from repetitive extragenic palindromic sequence (rep)-PCR using the BOXA1R primer. The isolates were distinct from the pathotype strain P. syringae pv. coriandricola. Like P. syringae pv. apii, the fennel isolates did not nucleate ice but did hydrolyze gelatine. Six isolates were tested for pathogenicity in each of two independent experiments with a total of six replications per isolate. Healthy, potted fennel plant stems of a proprietary cultivar were pierced once with insect display pins that had been dipped into bacterial colonies grown for 48 h on nutrient agar. Control stems were inoculated with sterile pins. Plants were incubated for 48 h at 100% relative humidity and then held in a greenhouse. After 4 to 6 days, inoculated fennel plants developed symptoms similar to those originally observed in the field. Bacteria isolated from surface-disinfested symptomatic tissue were fluorescent and identical to strains used to inoculate plants as evaluated by rep-PCR, thus fulfilling Koch's postulates. Control plants remained symptomless. To our knowledge, this is the first report of this pathogen causing a disease on fennel; the disease has been named bacterial streak. The disease occurred in three fields in 2010 and incidence was <1% in each case. Similar symptoms were also observed on fennel in the 2011 season in at least two fields. Previously, the host range of P. syringae pv. apii was reported to be restricted to celery. This research expands the natural host range of P. syringae pv. apii; thus, care should be taken in choosing Apiaceae crops for plantings subsequent to the occurrence of this pathogen. References: (1) C. T. Bull et al. Phytopathology 101:847, 2011. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

Induction of DNA DSB and its rejoining in clamped and non-clamped tumours after exposure to carbon ion beams in comparison to X rays.

We studied double-strand breaks (DSB) induction and rejoining in clamped and non-clamped transplanted tumours in mice leg after exposure to 80 keV µm(-1) carbon ions and X rays. The yields of DSB in the tumours were analysed by a static-field gel electrophoresis. The OER of DSB after X rays was 1.68±0.31, and this value was not changed after 1 h rejoining time (1.40±0.26). These damages in oxygenated conditions were rejoined 60-70% within 1 h in situ. No difference was found between the exposure to X rays and carbon ions for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after exposure to carbon ions were similar to those after X rays, and the calculated relative biological effectivenesses of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not on the types of radiation quality.

Transforming growth factor-ß decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells.

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-ß (TGF-ß) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-ß repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-ß, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-ß negatively contributes to maintain the CICs within the cancer.

First Report of Bacterial Blight of Cabbage (Brassica oleracea var. capitata) Caused by Pseudomonas cannabina pv. alisalensis in California.

In 2008, field-grown cabbage (Brassica oleracea var. capitata L., cv. Grenader) from Monterey County, California showed symptoms on the wrapper leaves of immature plants that had formed heads. Initial symptoms consisted of small, brown, water-soaked flecks surrounded by chlorotic haloes. These flecks later coalesced into large, irregularly shaped, brown-black, necrotic lesions with chlorotic haloes visible on both adaxial and abaxial surfaces of the leaf. This disease resulted in lower quality and reduced marketability of the cabbage. Five gram-negative, blue-green fluorescing bacteria were isolated from separate lesions on different plants on King's medium B agar. The isolates were positive for levan formation and negative for oxidase and arginine dihydrolase. The isolates did not cause soft rot on potato slices but did induce a hypersensitive reaction in tobacco (Nicotiana tabacum L. cv. Samsun). These data indicated that the bacteria belonged to Lelliot's LOPAT group 1 (2). Repetitive extragenic palindromic sequence (rep)-PCR using the BOXA1R primer resulted in identical DNA fragment banding patterns for the cabbage isolates and the pathotype of Pseudomonas cannabina pv. alisalensis (formerly P. syringae pv. alisalensis). Additionally, both P. cannabina pv. alisalensis and the five cabbage isolates were sensitive to bacteriophage PBS1 while the pathotype strain of P. syringae pv. maculicola was not (1). Pathogenicity of the five cabbage isolates was evaluated in two independent experiments. Inoculum was prepared by growing the bacteria on nutrient agar for 48 h (27°C), suspending the bacteria in 0.01 M phosphate buffer (pH 7.0), adjusting each suspension to 0.6 OD at 600 nm (approximately 108 CFU/ml), and adding three to five drops of Tween 20. In each experiment, two cabbage, broccoli raab (Brassica rapa subsp. rapa cv. Sorrento), and oat (Avena sativa cv. Montezuma) plants were inoculated for each isolate by spraying until runoff. Positive control plants were inoculated with the pathotype strain of P. cannabina pv. alisalensis and negative control plants were treated with sterile 0.01 M phosphate buffer. The plants were placed in a mist chamber for 48 to 72 h and then in a greenhouse (20 to 25°C). After 7 to 10 days, foliar symptoms similar to symptoms observed on the original diseased cabbage plants developed on all inoculated plants including the positive control plants inoculated with P. cannabina pv. alisalensis. Additionally severe symptoms on broccoli raab and minor symptoms on oats developed on plants inoculated with cabbage strains or P. cannabina pv. alisalensis. For each experiment, bacteria reisolated from symptomatic tissue were identical to the bacteria used to inoculate the plants and to P. cannabina pv. alsialensis for rep-PCR DNA fragment banding pattern and sensitivity to phage PBS1. There were no symptoms on any of the cabbage and oat negative controls. Additionally, there were no symptoms on any broccoli raab negative controls in the first experiment; however, in the second experiment, a small (<1 mm) lesion was detected on one leaf of one plant. To our knowledge, this is the first report of P. cannabina pv. alisalensis causing bacterial blight of cabbage in California. This disease may have significant impact because of the large acreage of cabbage grown in California (approximately 5,666 ha annually). References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

Impact of superselective transarterial infusion therapy of high-dose cisplatin on maxillary cancer with orbital invasion.

BACKGROUND AND PURPOSE: We have been performing the superselective transarterial infusion of high-dose cisplatin for advanced maxillary cancer since 1998 and the local control rate, disease free survival rate, and organ preservation have improved markedly compared with our former therapy. This study evaluates the effectiveness of superselective transarterial infusion therapy by using high-dose cisplatin on maxillary cancer with orbital invasion. MATERIALS AND METHODS: We treated 23 patients with maxillary cancer by using superselective transarterial infusion therapy with high-dose cisplatin and concomitant radiation therapy for 10 years. Of all patients, 15 showed orbital invasion, with 11 of these tumors fed by both internal maxillary and ophthalmic arteries. In all patients, we performed superselective transarterial infusion therapy via the internal maxillary artery and/or the other feeding branches from the external carotid artery. After the operation, we determined whether a pCR had occurred by checking for the presence of viable cells. In addition, we calculated the overall survival rate, preservation rate of the eyeball, and disease-free survival rate. RESULTS: For all 23 patients, pCR and overall survival rates were 95.7% and 78.4%, respectively. To date, 2 of these patients died of lung metastasis without local recurrence. For the 15 patients with orbital invasion, the respective pCR and disease-free survival rates were 93.3% and 87.5%. Eyeballs were preserved in all patients, and local recurrence occurred in only 1 patient, at the inferior wall of the maxillary sinus (not in the orbit). CONCLUSIONS: Superselective transarterial infusion therapy with high-dose cisplatin remarkably improved the local control rate and disease-free survival rate of maxillary cancer. Even in patients with orbital invasion, a high local control rate was achieved, with preservation of the eyeball, through infusion only into branches of the external carotid artery.

First Report of Bacterial Blight of Brussels sprouts (Brassica oleracea var. gemmifera) Caused by Pseudomonas cannabina pv. alisalensis in California.

Greenhouse-grown Brussels sprouts (Brassica oleracea L. var. gemmifera) transplants from Monterey County, California showed symptoms in 2006 of a previously undescribed disease. Initial symptoms consisted of small (1 to 2 mm in diameter), angular, water-soaked flecks, some of which were surrounded by chlorotic haloes. These flecks coalesced into large, irregularly shaped, gray brown lesions as large as 10 mm. Lesions were visible from both adaxial and abaxial leaf surfaces and generally retained chlorotic borders. This disease resulted in decreased quality and reduced marketability of the transplants. Gram-negative, blue-green fluorescing bacteria were consistently isolated from lesions on King's medium B agar. Ten isolates were selected and used in further studies. Isolates were levan positive, oxidase negative, and arginine dihydrolase negative. Isolates did not rot potato slices but induced a hypersensitive reaction in tobacco (Nicotiana tabacum L. cv. Samsun). These data indicated that the bacteria belonged to Lelliot's LOPAT group 1 (2). Repetitive extragenic palindromic sequence (REP)-PCR using the BOXA1R primer resulted in identical DNA fragment banding patterns for the Brussels sprouts isolates and the pathotype of Pseudomonas cannabina pv. alisalensis (formerly P. syringae pv. alisalensis). Like P. cannabina pv. alisalensis, the isolates from Brussels sprouts were sensitive to bacteriophage PBS1 (1). All 10 isolates were used in two independent pathogenicity experiments. Inoculum for pathogenicity studies was prepared by growing the bacteria on nutrient agar for 48 h (27°C), suspending the bacteria in 0.01 M phosphate buffer (pH 7.0), and adjusting each suspension to 0.6 OD at 600 nm (approximately 108 CFU/ml). In each experiment, six Brussels sprouts plants were inoculated for each isolate by spraying until runoff or by swabbing a suspension of the appropriate bacterial isolate to which Carborundum had been added. Additionally, four Brussels sprouts isolates were used to spray inoculate six broccoli raab (Brassica rapa subsp. rapa) plants. Positive control plants were inoculated with the pathotype of P. cannabina pv. alisalensis, and the negative control plants were inoculated with sterile buffer or sterile buffer with Carborundum. Inoculated plants were placed in a mist chamber for 48 h and then in a greenhouse (20 to 25°C). After 5 to 7 days, foliar symptoms similar to symptoms observed on the original diseased Brussels sprouts plants developed on all inoculated plants, including the positive control plants inoculated with P. cannabina pv. alisalensis. Negative control plants remained symptomless. In each experiment, bacteria reisolated from symptomatic tissue were identical to the bacteria used to inoculate the plants and to P. cannabina pv. alisalensis for LOPAT reactions, REP-PCR DNA fragment banding pattern, and sensitivity to phage PBS1. To our knowledge, this is the first report of P. cannabina pv. alisalensis causing bacterial blight of Brussels sprouts. References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

First Report of Tobacco rattle virus in Spinach in California.

In 2009 in coastal California (Santa Barbara County), commercially grown spinach (Spinacia oleracea) in two nearby fields exhibited symptoms of a previously unrecognized virus-like disease. Symptoms consisted of general chlorosis and bright yellow blotches and spots. Necrotic spots were also associated with the disease. In affected fields, disease occurred in limited, irregularly shaped patches that ranged from one to several meters in diameter. Symptomatic plants were unmarketable and these small patches of spinach were not harvested. With a transmission electron microscope, rigid, rod-shaped particles with a clear central canal were observed from plant sap of the symptomatic spinach. Analysis by a double-antibody sandwich-ELISA assay (Agdia Inc., Elkhart, IN) for Tobacco rattle virus (TRV) showed that the symptomatic plants were positive. Symptomatic spinach from the field was used for mechanical transmission to Chenopodium quinoa, C. murale, C. capitatum, spinach, and sugar beet (Beta vulgaris). All inoculated plants showed chlorotic local lesions and sugar beet showed chlorotic local lesions with rings. To further confirm the presence of TRV, reverse transcription (RT)-PCR was conducted. Total RNA was extracted from the mechanically inoculated symptomatic spinach plants using an RNeasy Plant Kit (Qiagen Inc., Valencia, CA) and used as a template in RT-PCR with forward (5'-TACATCACATCTGCCTGC-3') and reverse (5'-CTTCATTCACACAACCCTTG-3') primers specific to the movement protein gene from the spinach isolate of TRV (GenBank Accession No. AJ007294). Amplicons of the expected size (approximately 562 bp) were obtained. The RT-PCR products were sequenced (GenBank Accession No. GU002156) and compared with TRV sequences in GenBank to confirm the identity of the products. Sequences obtained had 96% nucleotide identity and 97% amino acid identity with TRV sequences available under the GenBank Accession Nos. FJ357571 and AJ007294. On the basis of the data from electron microscopy and serological and molecular analyses, the virus was identified as TRV. Soil samples collected from one of the fields were assayed for nematodes; however, Paratrichodorus or Trichodorus species were not recovered. To our knowledge, this is the first report of TRV in spinach in California. TRV has also been reported in spinach in England (1) and Germany (2). References: (1) A. Kurppa et al. Ann. Appl. Biol. 98:243, 1981. (2) K. Schmidt and R. Koenig. Arch. Virol. 144:503, 1999.

First Report of Blossom Blight of Strawberry (Fragaria × ananassa) Caused by Pseudomonas marginalis.

In 2003, a new disease was observed on commercial strawberries (Fragaria × ananassa Duch.) grown in multiple fields in Watsonville, CA. Initial symptoms consisted of brown lesions on the undersides of the sepals of strawberry flowers. The lesions coalesced and spread to upper sepal surfaces and anther bases. No leaf symptoms were observed. Fields affected with this disease appeared to have a greater number of deformed fruit, though incidence data were not collected. A gram-negative, blue-green fluorescent pseudomonad was isolated from lesions on King's medium B agar from both sepals and anthers from 23 of 24 samples from three different fields. All isolates were levan, oxidase, and arginine dihydrolase positive. The strains did rot potato slices but did not induce a hypersensitive reaction in tobacco (Nicotiana tabacum L. cv. Sansun), indicating that the bacteria belonged to Lelliot's LOPAT group IVa, P. marginalis (3). Isolates from strawberry were compared with pathotype strains of Pseudomonas marginalis pv. marginalis, P. marginalis pv. alfalfae, and P. marginalis pv. pastinaceae. The 16S rDNA sequence of type strain of P. marginalis (Z76663) was 97 to 99% similar to the four strawberry isolates sequenced (GQ845121). Identity was further supported by analysis of fatty acid methyl esters (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE). Polymerase chain reaction using BOX-A1R primers (repetitive sequence-based (rep)-PCR [1]) resulted in DNA fragment banding patterns that were identical among strawberry isolates. These banding patterns were different from the three distinct patterns of the P. marginalis pathotypes. Pathogenicity on strawberry (cv. Albion) was confirmed in three experiments using four strawberry isolates originally isolated from plants from three different fields and the P. marginalis pathotype strains. Inoculum was produced by growing bacteria in nutrient broth shake cultures for 48 h (24°C) and washing and suspending the cultures in 0.01 M phosphate buffer (pH 7.0). Three to five attached strawberry flowers on separate plants were dipped in the bacterial suspensions (106 CFU/ml) or sterile buffer for 1 min. To maintain high humidity, flower buds were enclosed in plastic bags for 36 to 48 h and then incubated in the greenhouse (24 to 26°C). After 7 days, approximately half of the flowers inoculated with the strawberry isolates had symptoms on sepals that were identical to symptoms seen in the field. Additionally, reisolates obtained from the symptomatic, inoculated flowers were identical to those used to inoculate the plants as confirmed by LOPAT reactions and rep-PCR, thus completing Koch's postulates. Flowers dipped in phosphate buffer or the P. marginalis pathotype strains did not develop symptoms and no bacteria were reisolated. To our knowledge, this is the first report of blossom blight of strawberry caused by P. marginalis and the first report of P. marginalis on strawberry in California. P. marginalis causes leaf bud rot of strawberry in Japan (2). Further research is needed to determine if the strawberry isolates represent a new or previously described pathovar of P. marginalis. References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) T. Kijima et al. Bull. Tochigi. Agric. Exp. Stn. 36:59, 1989. (3) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

First Report of Apium virus Y on Cilantro, Celery, and Parsley in California.

Recently, Apium virus Y (ApVY) was detected in field-grown cilantro (Coriandrum sativum), celery (Apium graveolens), and parsley (Petroselinum crispum) in California. In 2003, cilantro plants growing in three different fields in California (Monterey, San Joaquin, and San Luis Obispo counties) expressed symptoms of mosaic, vein clearing, and stunting. When plant sap was examined by transmission electron microscopy, flexuous, rod-shaped virus particles were observed. Total RNA was extracted from the symptomatic cilantro plants and used as a template in reverse transcription (RT)-PCR using universal potyvirus primers according to Chen et al. (1). The RT-PCR product was cloned into pGEM-T (Promega, Madison, WI) and the insert of 1,713 bp was sequenced (GenBank Accession No. EU515125). Nucleotide sequences from clones derived from three different infected cilantro plants were 89 to 97% identical to ApVY sequences encoding partial sequence of polyprotein in GenBank (Accession Nos. AY049716, EU127499, AF207594, AF203529, and EU255632). In 2007, celery plants showing necrotic line patterns and necrotic lesions on lower leaves and petioles were observed in several fields in two coastal counties in California (Monterey and Santa Clara counties). Flexuous, rod-shaped virus particles were also observed in the sap of those plants. ELISA for Cucumber mosaic virus and RT-PCR for Celery mosaic virus were negative. ApVY specific primers were designed on the basis of a consensus sequence of ApVY identified from cilantro in 2003; reverse primer 5'-GGCTCTTGCTATAGACAAATAGT-3' and forward primer 5'-GAAGACCAAGCCAATGTGTGTA-3'. The sequence of RT-PCR products (GenBank Accession No. EU515126) amplified from infected celery had 90 to 98% nucleotide identity to ApVY. When the deduced amino acid sequences of NIb and CP regions from both cilantro and celery were used for comparison, they showed 95 to 99% identity with the known ApVY GenBank sequences mentioned above. More than 10 asymptomatic parsley plants growing in fields adjacent to the infected celery were also tested for ApVY and found to be infected. ApVY was previously identified in three Apiaceae weeds in Australia (2) and in celery in New Zealand (3). To our knowledge, this is the first report of ApVY on cilantro, celery, and parsley in California. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) J. Moran et al. Arch. Virol. 147:1855, 2002. (3) J. Tang et al. Plant Dis. 91:1682, 2007.

First Report of Impatiens necrotic spot virus Infecting Lettuce in California.

Impatiens necrotic spot virus (INSV; family Bunyaviridae, genus Tospovirus) is an important pathogen of ornamental plants in North America and Europe, particularly in the greenhouse industry (2,3). However, INSV is now emerging as a pathogen of vegetable crops. During the 2006 and 2007 growing seasons, lettuce (Lactuca sativa) in Monterey County, CA showed necrotic spotting, leaf chlorosis, and plant stunting typical of symptoms induced by Tomato spotted wilt virus (TSWV). Significant and damaging outbreaks of these disease symptoms were found in numerous romaine, greenleaf, redleaf, butterhead, and iceberg lettuce fields in Monterey and San Benito counties. Samples from symptomatic plants from 21 of 27 fields in Monterey County were negative when tested with TSWV immunostrips (Agdia, Elkhart, IN); however, tests of the TSWV-negative samples with INSV immunostrips were positive. In most fields where INSV was detected, disease development was limited to the edges of fields and disease incidence was <5%; however, some fields had incidences >50% and crop loss was experienced. The virus causing the tospovirus symptoms in the TSWV-negative lettuce was sap transmitted to Nicotiana benthamiana and lettuce, where it induced chlorosis and necrosis. Symptoms in N. benthamiana were consistent with INSV infection, and those in lettuce were similar to symptoms observed in the field. Immunostrip tests confirmed that symptomatic N. benthamiana and lettuce plants were infected with INSV. To further confirm the identity of this virus, reverse transcription (RT)-PCR analysis was conducted with an INSV primer pair that directs the amplification of a ~1.3-kb fragment from the small RNA of INSV (4). The 1.3-kb fragment was amplified from RNA from symptomatic lettuce plants that were INSV positive with immunostrips, and not from asymptomatic lettuce. A total of 38 of 54 samples showing tospovirus-like symptoms were confirmed to be infected with INSV by RT-PCR. Sequences of two representative 1.3-kb DNA fragments were 98 to 99% identical with sequences of INSV isolates from Japan, Italy, and The Netherlands (GenBank Accession Nos. AB109100, DQ425096, and X66972). Taken together with the previous identification of the INSV vector, the western flower thrips (Frankliniella occidentalis), in central California lettuce (1), these results confirm that INSV induced tospovirus symptoms in lettuce fields in Monterey County in 2007. To our knowledge, this is the first report of the occurrence of INSV infecting lettuce in California. References: (1) W. E. Chaney. Annu. Rep. California Lettuce Res. Board. 2006. (2) M. Daughtrey et al. Plant Dis. 81:1220, 1997. (3) M. D. Law and J. W. Moyer. J. Gen. Virol. 71:933, 1990. (4) R. A. Naidu et al. Online publication. doi: 10.1094/PHP-2005-0727-01-HN. Plant Health Progress, 2005.

First Report of Bacterial Blight of Rutabaga (Brassica napus var. napobrassica) Caused by Pseudomonas syringae pv. alisalensis in California.

In 2005, commercial, organically grown rutabaga (Brassica napus var. napobrassica) in San Benito County, CA showed symptoms of a previously undescribed disease on approximately 30% of the plants. Initial symptoms consisted of small (1 to 2 mm in diameter), angular, water-soaked flecks that often were surrounded by chlorotic haloes. These flecks enlarged and coalesced into large, irregularly shaped, gray brown lesions that could be as long as 10 mm. Lesions were visible from both adaxial and abaxial leaf surfaces and generally retained the chlorotic borders. A blue-green fluorescing pseudomonad was consistently isolated from lesions on King's medium B. Eight isolates were characterized and were levan positive, oxidase negative, and arginine dihydrolase negative. Isolates did not rot potato slices but induced a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Samsun). These data indicated that the bacteria belonged to Lelliot's LOPAT group 1 (2). This was confirmed with data from fatty acid methyl ester analysis (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE) that showed that the isolates were highly similar (similarity = 0.922 or greater) to Pseudomonas syringae. Amplification of repetitive bacterial sequences (rep-PCR) using the BOXA1R primer and the polymerase chain reaction resulted in identical banding patterns for the rutabaga isolates and the P. syringae pv. alisalensis pathotype strain. Pathogenicity was demonstrated by growing inocula of six isolates in nutrient broth shake cultures for 48 h (24°C), adjusting the bacterial suspension to 106 CFU/ml, and misting the resulting suspensions onto rutabaga (cv. American Purple Top). Plants were enclosed in plastic bags for 24 h and then incubated in a greenhouse (24 to 26°C). Control plants were misted with sterile water and treated the same way. After 5 to 7 days, foliar symptoms similar to symptoms seen in the field developed on all inoculated plants, and reisolated bacteria were characterized and found to be P. syringae pv. alisalensis. Control plants remained symptomless. The results of two sets of pathogenicity tests were the same. To our knowledge, this is the first report of commercially grown rutabaga as a host of P. syringae pv. alisalensis and the first report of a B. napus host of this pathogen. This bacterial pathogen has previously been reported on commercial plantings of arugula (Eruca sativa), broccoli (Brassica oleracea var. botrytis), and broccoli raab (Brassica rapa var. rapa) in California and under experimental conditions it causes disease on additional hosts, including members of the Poaceae (1). References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

ELAC2, a putative prostate cancer susceptibility gene product, potentiates TGF-beta/Smad-induced growth arrest of prostate cells.

Transforming growth factor-beta (TGF-beta) elicits a potent growth inhibitory effect on many normal cells by binding to specific serine/threonine kinase receptors and activating specific Smad proteins, which regulate the expression of cell cycle genes, including the p21 cyclin-dependent kinase (CDK) inhibitor gene. Interestingly, cancer cells are often insensitive to the anti-mitogenic effects of TGF-beta for which the molecular mechanisms are not well understood. In this study, we found that the candidate prostate cancer susceptibility gene ELAC2 potentiates TGF-beta/Smad-induced transcriptional responses. ELAC2 associates with activated Smad2; the C-terminal MH2 domain of Smad2 interacts with the N-terminal region of ELAC2. Small interfering siRNA-mediated knock-down of ELAC2 in prostate cells suppressed TGF-beta-induced growth arrest. Moreover, ELAC2 was shown to specifically associate with the nuclear Smad2 partner, FAST-1 and to potentiate the interaction of activated Smad2 with transcription factor Sp1. Furthermore, activation of the p21 CDK inhibitor promoter by TGF-beta is potentiated by ELAC2. Taken together our data indicate an important transcriptional scaffold function for ELAC2 in TGF-beta/Smad signaling mediated growth arrest.

First Report of Bacterial Blight of Romanesco Cauliflower (Brassica oleracea var. botrytis) Caused by Pseudomonas syringae pv. alisalensis in California.

In 2005, a new disease was detected on commercial, organically grown romanesco (green) cauliflower (Brassica oleracea var. botrytis) grown in San Benito County, California. Initial symptoms consisted of small (1 to 2 mm in diameter), angular, water-soaked flecks. These flecks developed into tan-to-gray, angular lesions measuring as much as 5 mm in diameter. Lesions were usually surrounded by chlorotic borders. Coalescing lesions caused the leaf to turn papery in texture and have a blighted appearance. A blue-green fluorescing pseudomonad was consistently isolated from lesions on King's medium B. Strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Samsun). These data indicated that the bacteria belonged to Lelliot's LOPAT group 1 (2). This was confirmed with data from fatty acid methyl ester analysis (MIS-TSBA version 4.10, MIDI, Inc., Newark, DE), which showed that the strains were highly similar (similarity = 0.921 or greater) to Pseudomonas syringae. Amplification of repetitive bacterial sequences (rep-PCR) using the BOXA1R primer and the polymerase chain reaction resulted in identical banding patterns for the romanesco strains and the P. syringae pv. alisalensis pathotype strain. Pathogenicity was demonstrated by growing inoculum of six strains in nutrient broth shake cultures for 48 h (24°C), adjusting the bacterial suspension to 106 CFU/ml, and spraying the resulting suspension onto green cauliflower (cv. Romanesco Precoce). Plants were enclosed in plastic bags for 24 h and then incubated in a greenhouse (24 to 26°C). Control plants were misted with sterile water and treated the same way. After 5 days, foliar symptoms identical to symptoms seen in the field developed on all inoculated plants, and reisolated strains were characterized and found to be identical to P. syringae pv. alisalensis by the tests described above. Control plants remained symptomless. The results of two sets of pathogenicity tests were the same. To our knowledge, this is the first report of commercially grown romanesco green cauliflower as a host of P. syringae pv. alisalensis. The infested field had approximately 30% of the plants affected, with perhaps 10% sustaining some crop loss. This bacterial pathogen has previously been reported on commercial plantings of arugula (Eruca sativa), broccoli (Brassica oleracea var. botrytis), and broccoli raab (Brassica rapa var. rapa) and under experimental (greenhouse) conditions causes disease on additional hosts, including members of the Poaceae (1). References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

First Report of Bacterial Leaf Spot of Italian Dandelion (Cichorium intybus) Caused by a Pseudomonas syringae Pathovar in California.

Italian dandelion (Cichorium intybus) is a leafy, nonhead forming chicory plant that is eaten as a fresh vegetable in salads. During the late summer (August through October) of 2002, in the Salinas Valley (Monterey County) in California, a previously unreported disease was found in commercial Italian dandelion fields. Early symptoms were angular, vein delimited, dark, water-soaked leaf spots that measured 2 to 7 mm in diameter. As disease developed, spots retained angular edges but exhibited various irregular shapes. Spots commonly formed along the edges of the leaves; in some cases these spots developed into lesions that measured between 10 and 30 mm long. Spots were visible from adaxial and abaxial sides and were dull black in color. A cream-colored pseudomonad was consistently isolated from leaf spots that were macerated and streaked onto sucrose peptone agar. Fungi were not recovered from any of the spots. Recovered strains were blue-green fluorescent when streaked onto King's medium B agar. Bacterial strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum cv. Turk). These data indicated that the bacteria belonged to LOPAT group 1 of Pseudomonas syringae (1). Pathogenicity of six strains was tested by growing inoculum in nutrient broth shake cultures for 48 h, diluting to 106 CFU/ml, and spraying onto 12 6-week-old plants of Italian dandelion cv. Catalogna Special. Untreated control plants were sprayed with sterile nutrient broth. After 10 to 12 days in a greenhouse (24 to 26°C), leaf spots similar to those observed in the field developed on all inoculated plants. Strains were reisolated from the spots and identified as P. syringae. Control plants remained symptomless. These inoculation experiments were done twice and the results were the same. Amplification of repetitive bacterial sequences (repetitive sequence-based polymerase chain reaction [rep-PCR]) demonstrated that all Italian dandelion strains had the same rep-PCR fingerprint, which differed from fingerprints of P. syringae pv. tagetis and P. syringae pv. tabaci. Additionally, toxin specific primers did not amplify tagetitoxin or tabtoxin biosynthesis genes from Italian dandelion strains. To our knowledge, this is the first report of bacterial leaf spot of commercially grown Italian dandelion in California caused by a P. syringae pathovar. Because fields were irrigated with overhead sprinklers, the disease was severe in several fields and as much as 30% of those plantings were not harvested. Reference: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966.

Evidence from mucin core protein expression that some Paget's disease on areola can be of extramammary-like histogenesis and part of multisite disease.

Triple extramammary Paget's disease, which consists ordinarily of bilateral axillary and genital lesions, is uncommon. Triple extramammary Paget's disease involving other sites has never been reported, although solitary extramammary Paget's disease can occur at various sites around the body. Erythematous plaques on the areola, axilla and genitalia of a 91-year-old man were surgically removed under the clinical diagnosis of multiple extramammary Paget's disease. Histology revealed that all three lesions consisted of intraepidermal nests of Paget cells and other isolated Paget cells scattered in the epidermis. Although adnexal invasion was observed in the genital lesion, neither intraductal invasion nor underlying breast carcinoma was detected in the areolar lesion. Immunohistochemically, the Paget cells in all lesions expressed simple epithelial cytokeratins (CK8, 18 and 19), mucin (MUC)1 and MUC5AC, but neither CK20 nor MUC2. From the histological findings, the present case was interpreted as triple extramammary Paget's disease rather than synchronous mammary and extramammary Paget's disease. Furthermore, the mucin core protein expression pattern, which was identical to that observed in extramammary Paget's disease, supported the above interpretation.

Bacterial Blight on Arugula, a New Disease Caused by Pseudomonas syringae pv. Alisalensis in California.

Beginning in 1995, a leaf spot disease has occasionally developed on the leafy crucifer arugula (Eruca vesicaria subsp. sativa) that is grown in coastal California as a fresh market commodity used mostly in bagged salad mixes. Initially, symptoms consist of small (<2 mm in diameter), angular, water-soaked spots that are visible from both sides of the leaf. The spots later enlarge, remain angular in shape, and turn brown to tan. A purple margin sometimes occurs around the spots. An important diagnostic feature is that this disease closely resembles downy mildew infections that have not produced sporangia (3). A blue-green fluorescent pseudomonad was consistently isolated from both types of lesions on King's medium B. Strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum L. cv. Turk). These data indicated that the bacteria belonged to Lelliot's LOPAT group 1 (4). This was confirmed with data from fatty acid methyl ester analysis (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE), which indicated that the strains were highly similar (similarity > = 0.758) to Pseudomonas syringae. Amplification of repetitive bacterial sequence-based polymerase chain reaction (rep-PCR) was used to determine the relationship between the P. syringae strains isolated from arugula and two common crucifer pathogens, P. syringae pv. maculicola and P. syringae pv. alisalensis (1). Using the BOXA1R primer, banding patterns for the arugula strains and the P. syringae pv. alisalensis pathotype were similar, differing by only one band. In contrast, the banding patterns of the arugula strains differed significantly from those of P. syringae pv. maculicola. Additionally, the arugula isolates were sensitive to a bacteriophage originally isolated for its ability to lyse P. syringae pv. alisalensis (1). Previously, the pathogen from arugula was reported to be P. syringae pv. maculicola (2). It is the intent of this disease note to clarify this identification. We completed Koch's postulates by confirming pathogenicity on arugula (cv. Rocket Salad). The strains were grown as nutrient broth shake cultures for 48 h at 24°C, adjusted to 108 CFU/ml, and misted onto 2- to 3-week old plants. Control plants were misted with sterile nutrient broth. After 4 to 5 days in a greenhouse (24 to 26°C), large, angular leaf lesions developed on all inoculated arugula plants. Strains were reisolated from symptomatic tissue and identified as P. syringae pv. alisalensis. Control plants remained symptomless. Similar methods confirmed that the host range of the arugula isolates were identical to that of P. syringae pv. alisalensis. The arugula and P. syringae pv. alisalensis isolates caused disease on broccoli (Brassica oleracea var. botrytis cvs. Patriot and Titleist), broccoli raab (B. rapa subsp. rapa cv. Sorento), and oats (Avena sativa cv. Montezuma), while P. syringae pv. maculicola caused disease on broccoli only. Pathogenicity tests were conducted two times with identical results. This confirms that the bacterial blight that has been occurring on commercial plantings of arugula is caused by P. syringae pv. alisalensis. References: (1) N. A. Cintas et al.Plant Dis. 86:992, 2002. (2) S. T. Koike et al. Plant Dis. 80:464, 1996. (3) S. T. Koike. Plant Dis. 82:1063, 1998. (4) R. A. Lelliott, J. Appl. Bacteriol. 29:470, 1966.

First Report of Bacterial Leaf Spot of Swiss Chard Caused by Pseudomonas syringae pv. aptata in California.

From 1999 through 2003, a previously unreported disease was found on commercial Swiss chard (Beta vulgaris subsp. cicla) in the Salinas Valley, (Monterey County) California. Each year the disease occurred sporadically throughout the long growing season from April through September. Initial symptoms were water-soaked leaf spots that measured 2 to 3 mm in diameter. As disease developed, spots became circular to ellipsoid, 3 to 8 mm in diameter, and tan with distinct brown-to-black borders. Spots were visible from the adaxial and abaxial sides. Cream-colored bacterial colonies were consistently isolated from spots. Strains were fluorescent on King's medium B, levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum cv. Turk). The isolates, therefore, belong in LOPAT group 1 (1). Fatty acid methyl esters (FAME) analysis (MIS-TSBA version 4.10, MIDI Inc., Newark, DE) gave variable results that included Pseudomonas syringae, P. cichorii, and P. viridiflava with similarity indices ranging from 0.91 to 0.95. BOX-polymerase chain reaction (PCR) analysis gave identical banding patterns for the chard isolates and for known P. syringae pv. aptata strains, including the pathotype strain CFBP1617 (2). The bacteria were identified as P. syringae. Pathogenicity of 11 strains was tested by growing inoculum in nutrient broth shake cultures for 48 h, diluting to 10 × 6 CFU/ml, and spraying onto 5-week-old plants of Swiss chard cvs. Red, White, Silverado, and CXS2547. Untreated control plants were sprayed with sterile nutrient broth. After 7 to 10 days in a greenhouse (24 to 26°C), leaf spots similar to those observed in the field developed on all inoculated plants. Strains were reisolated from the spots and identified as P. syringae. Control plants remained symptomless. To investigate the host range of this pathogen, the same procedures were used to inoculate three strains onto other Chenopodiaceae plants: five cultivars of sugar beet (B. vulgaris), and one cultivar each of spinach (Spinacia oleracea) and Swiss chard. In addition, five chard strains and strain CFBP1617 were inoculated onto two cultivars of sunflower (Helianthus annuus), and one cultivar each of cantaloupe (Cucumis melo), sugar beet, spinach, and Swiss chard. All Swiss chard, cantaloupe, sunflower, and sugar beet plants developed leaf spots after 7 days. The pathogen was reisolated from spots and confirmed to be the same bacterium using BOX-PCR analysis. Spinach and untreated controls failed to show symptoms. All inoculation experiments were done at least twice and the results were the same. The phenotypic data, fatty acid and genetic analyses, and pathogenicity tests indicated that these strains are P. syringae pv. aptata. To our knowledge this is the first report of bacterial leaf spot of commercially grown Swiss chard in California caused by P. syringae pv. aptata. The disease was particularly damaging when it developed in Swiss chard fields planted for "baby leaf" fresh market products. Such crops are placed on 2-m wide beds, planted with high seed densities, and are sprinkler irrigated. This disease has been reported from Asia, Australia, Europe, and other U.S. states. References: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (2) J. L. W. Rademaker et al. Mol. Microbiol. Ecol. Man. 3.4.3:1-27, 1998.