An Improved Evans Blue Staining Method for Consistent, Accurate Assessment of Plasmodiophora brassicae Resting Spore Viability.

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

Characterization of a Gene Identified in Pathotype 5 of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is an important disease of crucifers worldwide. Isolates of the pathogen can be classified into pathotypes according to their pathogenicity on differential hosts. In this study, the presence or absence of all database-available nonhousekeeping P. brassicae genes (118 in total) were assessed by polymerase chain reaction (PCR) analysis in isolates belonging to five P. brassicae pathotypes (2, 3, 5, 6, and 8 according to Williams' differential set). One gene, designated Cr811, was present exclusively in the isolate of pathotype 5. This was further confirmed by dot blot hybridization and by PCR using alternative DNA preparations and primers. Reverse transcription quantitative PCR analysis indicated that in planta expression of Cr811 was up-regulated during canola infection, especially in the stage of secondary plasmodia. Primers specific to Cr811 could distinguish a field isolate of P. brassicae belonging to pathotype 5 from two other field isolates representing pathotypes 3 and 8. These findings suggest that Cr811 is a gene that is potentially involved in clubroot pathogenesis and that it also might serve as a molecular marker for differentiation of pathotype 5 from other pathotypes.

First Report of Fusarium cuneirostrum Causing Root Rot Disease in Dry Bean (Phaseolus vulgaris) in Canada.

Root rot is a major disease of dry bean and can cause significant yield reductions due to weakened root systems and poor plant stands. An in-depth study on root rot pathogen identification was conducted in 2011 in three commercial dry bean fields from the major production areas in Manitoba. Ten plants, sampled at each of four random sites within each field, were rated for disease severity. Twenty roots were processed for pathogen isolation and identification in the laboratory. Roots were cut into eight sections (~1 cm) and surface-sterilized in a laminar flow bench. Four root sections were placed on potato dextrose agar plates amended with 0.02% streptomycin sulfate (PDA-Strep) and four root sections were placed on peptone-pentachloronitrobenzene agar amended with 0.1% streptomycin sulfate and 0.012% neomycin sulfate. Afterward, 960 monosporic cultures were obtained representing 320 single spore isolates of potential root rot pathogens per commercial field. Common monosporic cultures from each field were subcultured on PDA-Strep and Spezieller Nährstoffarmer Agar (SNA) media. Based on morphological characteristics, 74 isolates were identified as Fusarium cuneirostrum (1). Colonies grew slowly on PDA-Strep with undulated margins, radial cream-grey mycelia, and conidia pustules with a cream-greyish pigmentation. Sporodochial conidia were falcate, mostly 5-septate, with a wedge shape and slightly protruding basal foot cell (56.3 to 71.8 × 4.6 to 6.2 µm on average). Species identity was confirmed for two isolates by sequencing the translation elongation factor 1 alpha (EF1-α) gene (2), the internal transcribed spacer (ITS) region (4), and the ribosomal intergenic spacer (IGS) (3) (GenBank Accession Nos. KF530848, KF530849, and KF025648 to 51). Sequence homology was compared using BLAST analysis and the FUSARIUM-ID database. The F. cuneirostrum isolates were deposited at the Canadian Collection of Fungal Cultures (DAOM 242540 and 242541). Pathogenicity screenings of two isolates was performed using sterilized seed of navy bean cv. Envoy. Seeds were germinated on moist filter paper for 3 days at 25°C and then inoculated by immersion in a prepared conidial suspension (2.5 × 105 conidia/ml) for 5 min. Seeds of the controls were immersed in sterile water. After inoculation, the germinated seeds were planted in 10-cm diameter pots, filled with sterile soilless mix (Sunshine #3). In the greenhouse, the experiment was arranged as a completely randomized design with three replicates with four germinated seeds per isolate, and was repeated twice. Disease assessment was performed 14 days after inoculation. Infected plants displayed dark brown lesions on the hypocotyl and primary root with a disease severity of 4 scored on a 0 to 5 scale. Fusarium cuneirostrum was re-isolated from roots of symptomatic plants. To our knowledge, this is the first report of F. cuneirostrum causing root rot of dry bean in Canada. It has been previously isolated from mung bean (Vigna radiata) in Ontario (1). References: (1) T. Aoki et al. Mycoscience. 46:162, 2005. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (4) H. Wang et al. J. Clin. Microbiol. 49:1890, 2011. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance.

This study investigated how the timing of application of the biofungicide Serenade (Bacillus subtilis QST713) or it components (product filtrate and bacterial cell suspension) influenced infection of canola by Plasmodiophora brassicae under controlled conditions. The biofungicide and its components were applied as a soil drench at 5% concentration (vol/vol or equivalent CFU) to a planting mix infested with P. brassicae at seeding or at transplanting 7 or 14 days after seeding (DAS) to target primary and secondary zoospores of P. brassicae. Quantitative polymerase chain reaction (qPCR) was used to assess root colonization by B. subtilis as well as P. brassicae. The biofungicide was consistently more effective than the individual components in reducing infection by P. brassicae. Two applications were more effective than one, with the biofungicide suppressing infection completely and the individual components reducing clubroot severity by 62 to 83%. The biofungicide also reduced genomic DNA of P. brassicae in canola roots by 26 to 99% at 7 and 14 DAS, and the qPCR results were strongly correlated with root hair infection (%) assessed at the same time (r = 0.84 to 0.95). qPCR was also used to quantify the transcript activity of nine host-defense-related genes in inoculated plants treated with Serenade at 14 DAS for potential induced resistance. Genes encoding the jasmonic acid (BnOPR2), ethylene (BnACO), and phenylpropanoid (BnOPCL and BnCCR) pathways were upregulated by 2.2- to 23-fold in plants treated with the biofungicide relative to control plants. This induced defense response was translocated to the foliage (determined based on the inhibition of infection by Leptosphaeria maculans). It is possible that antibiosis and induced resistance are involved in clubroot suppression by Serenade. Activity against the infection from both primary and secondary zoospores of P. brassicae may be required for maximum efficacy against clubroot.

Isolation and Variation in Virulence of Single-Spore Isolates of Plasmodiophora brassicae from Canada.

Clubroot of crucifers, caused by Plasmodiophora brassicae, is emerging as an important disease of canola (Brassica napus) in Alberta, Canada. Populations of the pathogen often consist of a mixture of different pathotypes. Therefore, a simple and efficient method to isolate single resting spores of P. brassicae was developed, based on serial dilution of spore suspensions. The virulence of 24 single-spore isolates, representing five populations of the pathogen from Alberta, Ontario, and British Columbia, was characterized on the differentials of Williams and Somé et al. Symptoms were rated 6 weeks after inoculation and Fisher's least significant difference (P < 0.05) was used to differentiate resistant from susceptible host reactions. The pathotype composition of P. brassicae in Canada appeared more diverse when single-spore isolates were examined rather than populations of the pathogen. In Alberta, at least three and possibly four pathotypes were identified among the 14 isolates tested, whereas a maximum of only two pathotypes had been reported previously when populations of the pathogen were examined. Pathotype 3 or P2, as classified on the differentials of Williams and Somé et al., respectively, was found to be predominant in the province. The occurrence of other pathotypes at lower frequencies suggests that caution should be used in any breeding strategy, because rare pathotypes of P. brassicae may quickly become predominant if susceptible host genotypes are continuously grown.

A Specific and Sensitive Method for the Detection of Colletotrichum lindemuthianum in Dry Bean Tissue.

To facilitate early diagnosis and improve control of bean anthracnose, a rapid, specific, and sensitive polymerase chain reaction (PCR)-based method was developed to detect the causal agent, Colletotrichum lindemuthianum, in bean (Phaseolus vulgaris) seed. Based on sequence data of the rDNA region consisting of the 5.8S gene and internal transcribed spacers (ITS) 1 and 2 of four C. lindemuthianum races and 17 Colletotrichum species downloaded from GenBank, five forward primers were designed and evaluated for their specificity. Among them, one forward primer was selected for use in combination with ITS4 to specifically detect C. lindemuthianum. A 461-bp specific band was amplified from the genomic DNA template of 16 representative isolates of C. lindemuthianum, but not from 58 representative isolates of 17 other Colletotrichum species or 10 bean pathogens. Moreover, to enhance the sensitivity of detection, nested PCR was applied, which allowed the detection of as little as 10 fg of C. lindemuthianum genomic DNA and 1% infected seed powder, which was mixed with 99% healthy seed powder. The diagnostic analysis can be completed within 24 h, compared with about 2 weeks required for culturing. Furthermore, this method can be performed and interpreted by personnel with no specialized taxonomic expertise.

First Report of Rhizoctonia solani AG-4 and AG-2-2 on Lupinus angustifolius in Canada.

Narrow-leaved lupine (Lupinus angustifolius L.) is grown as a grain legume crop in Australia and Europe where it is used as feedstock in the livestock and aquaculture industries. During July 2003, a stem rot disease was observed in narrow-leaved lupine (cv. Arabella) plants in a research plot at the Crop Diversification Centre North (CDCN), Edmonton, Alberta, Canada. The disease was also found on cv. Rose at the CDCN and cv. Arabella in experimental fields near Devon, Ellerslie, and Westlock, Alberta during the late spring of 2004. Diseased plants showed dark brown-to-black stems with sunken and constricted lesions at the soil level. Young leaves became shrunken and twisted and seedlings collapsed. Rhizoctonia solani was consistently isolated from lesions on taproots and basal stems of diseased plants. Colonies of cream-colored mycelia grew close to the surface of potato dextrose agar (PDA). Most sclerotia formed inside the medium. Agar disks (1 cm in diameter) of isolates LP-11Bb, LP-24C, and LP-25C were attached to the opposite sides of basal stems (180° apart) of 1-month-old lupine seedlings (cv. Arabella). Inoculated plants were incubated for 2 days in black plastic bags under a greenhouse bench at approximately 20°C. All isolates caused brown lesions on the lower stems (extending up to 7 cm above ground level), girdling, and root rot. Plants wilted within 7 to 10 days after inoculation, and aerial mycelia appeared on the basal stems. R. solani was reisolated from the infected crown tissues using PDA to complete Koch's postulates. The isolates were paired with AG tester strains of R. solani. Isolates LP-11Bb and LP-24C were identified as AG-4 while isolate LP-25C was identified as AG-2-2. In another trial, eight isolates of R. solani (unknown AG types) were tested for virulence on L. angustifolius cv. Arabella using the inoculation method described above. All isolates were pathogenic, and disease severity that was based on a 0 to 4 scale ranged from 2.7 to 3.2. The most virulent strain was LP-24C, which caused a 77% loss in fresh weight compared with the noninoculated control plants. R. solani AG-8 is associated with Rhizoctonia disease of lupine in Australia (1) and also causes bare patch disease of wheat. To our knowledge, this is the first report of R. solani on lupine in Canada. This disease could have a significant impact on the commercial production of lupine in Alberta. Reference: (1) M. W. Sweetingham et al. Pages 466-486 in: Advances in Lupin Disease Management in Australia. Proc. Int. Lupin Conf., 8th. G. D. Hill, ed. International Lupin Association, Canterbury, New Zealand, 1999.

Characterization of the metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas (Pisum sativum L.).

The metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas, was detected by thin layer chromatography (TLC) and was analyzed for its chemical and pathogenic characteristics. One blue dot was detected using 254nm UV light on TLC plate, and a spray of rho-anisaldehyde (110 degrees C, 30 min) also produced a blue dot. The solvent systems used for TLC analysis were ethyl acetate/water/acetone (5/2/5), chloroform/methanol/glacial acetic acid (19/10/2), toluene/ethyl acetate/90% formic acid (6/3/1), diethylether/methanol/water/90% formic acid (95/4/1/1), and bezene/methanol/acetic acid (24/2/1), with R(f) values (min-max) of 0.09-0.18, 0.88-0.95, 0.06-0.15, 0.39-0.47 and 0.05-0.12, respectively. The recovered metabolite from the TLC plate displayed UV absorption peaks at 212, 244, 250, 256 and 261 nm. The proposed formula of the main component of the metabolite was C16H12N3O6. The TLC-purified metabolite induced symptom of discoloration on detached pea leaves.

Stem Smut (Ustilago hypodytes) on Intermediate Wheatgrass in Canada.

Intermediate wheatgrass (Thinopyrum intermedium [Host] Barkworth & D.R. Dewey) (syn. Agropyron intermedium [Host] Beauv.) is becoming an important forage grass species in Alberta, Canada. Severe losses in seed yield due to stem smut (Ustilago hypodytes [Schlecht.] Fr.) were noted in a 70-acre field near Warner, AB, in 1999. The crop had been established in 1993 and harvested for seed each year. Smut symptoms (5% incidence) were noted initially in 1997. Incidence, determined by counting the number of symptomatic stems, increased to 10% in 1998 and 50% in 1999. The symptoms usually appeared in the first week of June. Brown sori developed on infected stems, especially between the uppermost node and the leaf below the flag leaf, and gradually became black during the period of seed filling, which is characteristic of stem smut (1). Teliospores were smooth, spherical to oval, light to dark brown, and 4.5 to 5.0 × 5.0 to 6.8 µm in dimension, which is also consistent with previous descriptions of U. hypodytes. Infected stems occasionally flowered, but did not set seed, so seed yield losses were proportional to disease incidence. Plants infected with stem smut were often stunted. Tissues in the smutty stem often became sunken and stems became twisted and thinner than normal due to the propagation of sori in the stem over time. Stem smut has been reported on crested wheatgrass and slender wheatgrass in other parts of Canada (2) and on T. intermedium in the United States (3). This is the first report of stem smut affecting commercial grass seed production in Alberta, Canada. This disease could also have a significant impact on the seed production of intermediate wheatgrass elsewhere. References: (1) G. W. Fischer. 1953. Manual of the North American Smut Fungi. Ronald Press, New York. (2) B. D. Gossen and D. Regnier. Can. Plant Dis. Surv. 71:88-89, 1991. (3) J. F. Karn and J. M. Krupinsky. Phytopathology 73:1152-1155, 1983.

Responses of giant freshwater prawn (Macrobrachium rosenbergii) to challenge by two strains of Aeromonas spp.

The virulence of two Aeromonas strains (A. veronii and A. caviae) isolated from the hepatopancreas of apparently healthy giant freshwater prawns (Macrobrachium rosenbergii) was compared using a challenge by injections. For the A. veronii strain, challenge with 3.7 x 10(5) cells/g of body weight led to 100% mortality; for the A. caviae strain, 3.8 x 10(6) cells/g produced 100% mortality. The 50% lethal doses (LD50) were 2.0 x 10(3) cells/g for A. veronii and 51.2 x 10(3) cells/g for A. caviae. Use of different culture media (trypticase soy broth vs prawn muscle extract) did not significantly affect the virulence of A. veronii. Injection of a sublethal dose (1 x 10(3) cells/g) of A. veronii led to a significant decrease in the total hemocyte count (THC) between 4 and 24 h after injection. Saline injections also caused a similar though less decrease in THC. In the first 24 h after injection of A. veronii (1 x 10(3) cells/g), the change in the percentages of granulocytes (both granular cells and semigranular cells) in the hemolymph was significantly different. After a significant initial increase, the percentage of hyaline cells fell by a factor of 4, from 9 to 2%. Phenoloxidase activity increased fourfold immediately after injection and returned to preinjection levels at 24 h.

Molecular identification and relatedness of potato witches'-broom phytoplasma isolates from four potato cultivars.

Four isolates of potato witches'-broom phytoplasma, designated as PW1, PW2, PW3 and PW4, were established on four potato cultivars. The identity of each isolate was confirmed by PCR using two universal primer pairs and one specific primer set derived from phytoplasma of 16S rDNA sequences. The four isolate samples formed similar RFLP patterns after digestion of 1.2 kb PCR products with restriction endonucleases AluI, HhaI, RsaI and Sau3A. The direct DNA sequencing with the specific primer pair showed that there are no differences in the base sequences among PW1, PW2, and PW3 phytoplasma isolates and that PW4 is closely related to them. Thus, the four isolates were identified as members of the clover proliferation group.

First Report of Stevia as a Host of Sclerotinia sclerotiorum.

Stevia (Stevia rebaudiana Bertoni; Asteraceae), an annual plant originating from Paraguay, contains glucosides of a diterpenoid (2), which is used as a low-caloric sweetener in some South American and southeast Asian countries. The main active ingredient, stevioside, is 100 to 300 times as sweet as sucrose. Stevia has been experimentally grown under field conditions in central and western Canada and has the potential to become a commercially viable alternative crop. In August 1996, a previously undescribed stem rot disease was observed on stevia plants at the Crop Diversification Centre South, Brooks, Alberta. The disease was found in research plots where 4-month-old plants were growing in loam soil. Diseased stems showed dark brown lesions above and at soil level when plant height reached approximately 30 cm. Under dry conditions, mild stem lesions caused plant stunting with lower leaves turning black and curling downward. Wilted leaf symptoms gradually spread upward in affected plants. Partial wilting symptoms appeared when girdling was restricted to branches. The entire plant collapsed when girdling of the crown and roots occurred. Superficial white mycelium developed over the basal part of affected stems under moist conditions, especially after rainy periods. Black, round to oblong sclerotia, 3.5 to 10.1 mm in size, formed externally on the crown areas after plant death. Sclerotinia sclerotiorum (Lib.) de Bary (1) was consistently isolated from the diseased plants. To confirm pathogenicity, 4-week-old stevia seedlings were obtained from shoot cuttings and grown in 12-cm pots of soilless mix. Sclerotia produced on potato dextrose agar were inserted into the mix 0.5 cm deep and 0.5 cm from the stems of test plants. Plants were placed in a growth chamber at 22°C with a 12-h photoperiod and 95% relative humidity. Two weeks after soil infestation, plants wilted and S. sclerotiorum was reisolated from the diseased crown tissues. This is the first report on stevia of sclerotinia stem rot, a disease that could significantly reduce foliar growth and stevioside production in field plantings. References: (1) L. H. Purdy. Phytopathology 69:875, 1979. (2) T. Robinson. 1991. The Organic Constituents of Higher Plants: Their Chemistry and Interrelationships. 6th ed. Cordus Press, North Amherst, MA.

First Report of Botrytis Blight, Caused by Botrytis cinerea, on Coneflowers.

Coneflowers (Echinacea purpurea (L.) Moench and E. pallida (Nutt.) Nutt. var. angustifolia (DC.) Cronq.) are popular medicinal herbs in North America and Europe. In May 1997, a previously undescribed disease was observed in a commercial field of 3-year-old E. pallida var. angustifolia plants in Vernon, British Columbia, Canada. Diseased plants had small to large, brown or black lesions on leaves and stems. Botrytis cinerea Pers.:Fr. (1,2) was consistently isolated from affected tissues. The pathogen appeared to infect leaves along the margins and tips, and occasionally on other parts of the blade as well. Lesions expanded rapidly under cool, humid conditions. Once the pathogen had invaded the midrib or veins, it advanced rapidly to the petiole and stem, which resulted in collapse of the leaf. The pathogen produced profuse conidia and mycelia on the surface of dead and dying leaves, stems, and blossoms, which resulted in a moldy gray appearance. Under dry conditions, the disease developed slowly or even became quiescent. Large lesions often split and formed holes in leaves. The average size of the conidia produced on naturally infected leaves ranged from 5.5 to 10.5 × 6.8 to 18.3 µm (average 8.1 × 13.0 µm), and on potato dextrose agar (1-month-old culture) ranged from 5.5 to 10.0 × 7.5 to 16.3 µm (average 7.4 × 11.3 µm) based on 100 spore measurements, respectively. Microsclerotia were round, spherical or irregular in shape, and ranged from 1.1 to 3.6 × 1.0 to 3.0 mm. Koch's postulates were verified by spraying potted, 3-month-old, narrow-leaved coneflower (E. pallida var. angustifolia) and 2-year-old purple coneflower (E. purpurea) plants with a spore suspension (4 × 105 conidia/ml). Inoculated plants were enclosed in transparent plastic bags for 7 days at 15/22°C (night/day) with a 12-h photoperiod. Typical symptoms were produced 2 to 7 days after inoculation. Some infected leaves quickly twisted and dried after removal of the plastic bags. Botrytis cinerea was reisolated from the affected tissues. This is the first report of Botrytis blight on Echinacea spp. Although B. cinerea does not usually kill coneflower plants, it often heavily infects disc flowers and young shoots. Therefore, Botrytis blight could have a significant impact on the establishment and productivity of this crop in both the field and greenhouse, especially under cool, wet, growing conditions. References: (1) J. R. Coley-Smith et al. 1980. The Biology of Botrytis. Academic Press, New York. (2) D. J. Morgan. Trans. Br. Mycol. Soc. 56:319, 1971.

First Report of Sclerotinia sclerotiorum on Coneflower.

Purple coneflower (Echinacea purpurea (L.) Moench; Asteraceae), a perennial herb originating from North America, is used as a garden ornamental and is grown commercially for use in medicinal preparations as an immunostimulant. In October 1996, a previously undescribed stem rot disease was observed in a research plot of 6-month-old echinacea plants at Brooks. Seedlings had been raised in small rockwool cubes (2 × 2 × 5 cm3) in a greenhouse, then transplanted into the field in early June. By late August, dead and dying plants were observed throughout the stand. They had dark brown to black stem lesions above and at the soil level and dead leaves with bleached petiole lesions that extended ca. 15 cm above the axil. Diseased stems and petioles often disintegrated, leaving only fibrous tissues intact. Roots were rotted and black. Superficial white mycelium developed over the basal part of affected stems. Black, oblong to irregular-shaped sclerotia, 5.1 to 17.6 mm in size, formed externally on the crown areas after plant death. Sclerotinia sclerotiorum (Lib.) de Bary (1) was isolated from the diseased plants. Five isolates were selected to fulfill Koch's postulates with 3-month-old echinacea seedlings grown in 12-cm pots of soilless mix. Sclerotia from wilted, field-grown echinacea plants were transferred onto potato dextrose agar medium for 2 days at 20°C. Agar disks were cut with a 1-cm cork borer and two plugs containing sclerotial and mycelial tissues were inserted into the soilless mix 0.5 cm deep and 0.5 cm from the opposite sides of stems of test plants. Inoculated plants were enclosed in transparent plastic bags for 5 days and incubated in a growth chamber at 15/18°C (night/day) with a 12-h photoperiod. One to four lower leaves per plant wilted within 1 week after inoculation and aerial mycelia appeared on the petioles. Infected leaves quickly withered, dried, and dropped off the plant after the bags were removed. Plants often died 3 weeks after inoculation and S. sclerotiorum was reisolated from infected crown tissues. This disease was also found on 3-year-old plants of E. pallida (Nutt.) Nutt. var. angustifolia (DC.) Cronq. in Vernon, British Columbia, Canada, in May 1997. This is the first report of sclerotinia stem rot on Echinacea spp., a disease that could have a significant impact on the longevity and productivity of this crop in the field and greenhouse. Reference: (1) L. H. Purdy. Phytopathology 69:875, 1979.

Metobromuron/metolachlor ingestion with late onset methemoglobinemia in a pregnant woman successfully treated with methylene blue.

Metobromuron, a substituted urea herbicide, is widely used for control of grasses and broad-leaved weeds in Taiwan. Major systemic toxicity has not been reported following poisoning. A 22-year-old woman at 36 weeks of gestation was admitted to the emergency department three hours after ingestion of a mixture of 25% metobromuron and 25% metolachlor. Though stable initially, she developed central cyanosis 12 hours later. Emergent cesarean section was considered but administration of intravenous methylene blue readily reversed the cyanosis and prevented the operation. Recurrent cyanosis did not develop. Normal vaginal delivery occurred 17 days after the poisoning. Follow-up for four years revealed normal growth of the child. Metobromuron poisoning, like other urea herbicides, may cause methemoglobinemia via its hydrolysis products. Administration of methylene blue is effective treatment and should be considered in the treatment of methemoglobinemia following urea herbicide poisoning.

Termination of pathological pregnancy in second and early third trimesters with extraamniotic instillation of 16-phenoxy-omega-tetranor prostaglandin E2 methylsulfonylamide.

OBJECTIVES: To evaluate the efficacy, safety and influence on subsequent fertility of sulprostone, a prostaglandin E2 analog, in terminating pathological pregnancies via the extraamniotic route. METHODS: Forty pregnant women with intrauterine fetal death or major congenital anomalies were enrolled. Sulprostone was instilled into the extraamniotic space through a silicon Foley catheter. The instillation rate was 0.5-1 microgram/min. Instillation was discontinued when the catheter was expelled or when rupture of the membranes occurred. The duration of instillation and the time interval to completion of abortion was recorded. Information about subsequent fertility was collected by telephone or at outpatient clinic visits. RESULTS: The mean duration of instillation was 7.0 h and the mean dose of sulprostone was 314.8 micrograms. The mean induction-to-abortion interval (IAI) was 17.0 h. In two of the 40 patients, the cervix was not adequately ripened after 48 h and these pregnancies were ultimately terminated by alternative methods. The success rate of termination in 48 h was 92.5% (37/40). No severe side effects were encountered. CONCLUSION: To the best of our knowledge, this is the first report in the English literature of administration of sulprostone by extraamniotic instillation for termination of pathological pregnancies. The method is effective and safe and has an insignificant influence on subsequent fertility.

Prenatal diagnosis of placental chorioangioma: contribution of color Doppler ultrasound.

A case of placental chorioangioma was diagnosed at 35 weeks' gestation using color Doppler ultrasound. Color flow imaging showed an intraplacental hypoechoic mass fed directly by the anomalous chorionic tumor vessels arising from the insertion of the umbilical cord. Hypervascularization was present in some areas of the tumor. The diagnosis of typical 'angiomatous' type was confirmed by histopathological examination following delivery.

Uptake of low density lipoproteins by rat tissues. Special emphasis on the luteinized ovary.

The aim of this study was to determine how luteal cells of the hormone-primed (luteinized) ovary process low density lipoproteins (LDL). Ovary uptake of perfused 125I-LDL was assessed by tissue levels of radioactivity; the distribution of LDL protein in cells was assessed on autoradiograms of the fixed tissue; and the level of stimulation of steroidogenesis, as well as degradation of LDL protein, was assessed on effluent perfusion samples. Human LDL ligand used in these studies was rigorously defined biochemically and physiologically. Homologous (rat) LDL was used as a special ligand control. Other tissue controls included the use of perfused or in vivo-infused luteinized ovaries from animals pretreated to reduce circulating lipoprotein levels, perfused ovaries from a second hormone-primed model, perfused liver from estrogen-treated rats, and isolated and cultured cells from the same ovarian tissues used in the perfusion experiments. The results show that perfused LDL promptly stimulates steroidogenesis. However, the labeled protein moiety of the LDL is not interiorized by the luteal cells, nor is there evidence of LDL protein degradation in the effluent samples. In contrast, internalization of the ligand occurs when luteal cells are incubated with the ligand in vitro. We have observed also that uptake of the 125I-LDL by the ovary can be displaced equally well by excess unlabeled LDL or HDL3. Overall, these experiments suggest that in the intact luteinized ovary, LDL binds to the same sites on the cell surface where HDL "binds," and that LDL cholesterol must be obtained by these steroid hormone-producing cells by a mechanism that does not require internalization of the intact lipoprotein particle.

Effects of antimicrotubule agents on phospholipid metabolism in rat hepatic subcellular membranes.

Treatment of animals with antimicrotubule drugs has been shown to cause a perplexing variety of cellular changes which, theoretically, could be the result of changes in endomembrane biosynthesis, composition or flow. In the current study we have focused on this possibility by identifying antimicrotubule drug-induced changes in the phospholipid metabolism of hepatic subcellular membranes. Young adult rats were pretreated with radiolabeled [32 P]orthophosphate for 12 hr, and subsequently given saline, colchicine (2.5 mg/kg body wt) or vinblastine (20 mg/kg body wt) for 4 additional hr. Afterwards, the livers were homogenized, and separate microsomal and Golgi membrane fractions were prepared and subjected to phospholipid extraction and identification using two-dimensional thin-layer chromatography. The results show that colchicine and vinblastine given in vivo caused specific, rapid and in some cases, dramatic changes in phospholipid turnover in different membrane fractions of rat liver. The drugs specifically increased labeling of phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-biphosphate and decreased the radioactivity associated with phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in all fractions examined. In contrast, the antimicrotubule drugs produced a differential effect on the labeling pattern of sphingomyelin and lysophosphatidylcholine, i.e. they stimulated labeling of these phospholipids in microsomes, produced no changes in heavy Golgi fractions, and markedly increased their labeling in light Golgi fractions. These data suggest that antimicrotubule drugs restrict the incorporation of certain precursor phospholipids into forming membranes but do not affect the subsequent metabolism of these phospholipids. At the same time, the drugs appear to retard the flow of membranes from one cellular compartment to another.