First Report of Clonostachys rosea Causing Root Rot of Soybean in the United States.

Multiple fungal species have been associated with root rot of soybean (Glycine max) in the United States, but root rot in Minnesota (MN) also occurs in plants not known to be infected with previously reported pathogens (1). Soybean plants that lacked foliar symptoms, but exhibited taproot and lateral root necrosis were observed in 15 fields from nine counties in MN during 2007 and 2008. Plants were arbitrarily dug up at the R3 growth stage in July as part of a root rot study. Roots were washed, surface disinfested in 0.5% NaOCl for 3 min, rinsed in deionized water, dried, and embedded in potato dextrose agar (PDA). Thirty isolates with morphological characteristics consistent with those of Clonostachys rosea were recovered in total from necrotic lesions on different plants from all fields (3). For further morphological characterization, cultures were grown on PDA for 1 week at 24°C in the dark. Colonies were 39 to 46 mm in diameter, yellowish-white, and the surface was felty to tomentose with thick aerial hyphae. Primary conidiophores were Verticillium-like with two to three levels. The stipe length measured 65 to 105 µm and the base width was 5 µm. Primary conidia were smooth, hyaline, slightly curved, with an average length and width of 7 to 9 × 2.6 to 3 µm. Secondary conidiophores were penicillate with two or three whorls of phialides. The stipe length measured 50 to 75 µm, base width was 5 µm, and penicillus height was 25 to 35 µm. Secondary conidia were 5 to 6 × 2.5 µm. Perithecia were not produced. The identity of isolates was confirmed by sequencing the internal transcribed spacer (ITS) locus using the primers ITS1F/ITS4. BLAST analysis of the sequences in the NCBI database resulted in a 99.8 to 100% match for both C. rosea and its teleomorph Bionectria ochroleuca (e.g., HM751081, GU256766). Each isolate was tested for pathogenicity on soybean by initially growing it on sterile sorghum grain for 2 weeks at 23°C. Sterile sorghum was used for control plants. Seeds of soybean 'AG2107' were planted in 11.4-cm square pots containing pasteurized potting mix and a 25-cm3 layer of infested or sterile sorghum placed ~1 cm below the seeds. Two replicate pots containing four plants each were used per treatment and the experiment was repeated once. Root rot was assessed 28 days after planting in a greenhouse at 23°C day and 18°C night with a 14-h photoperiod. Twenty-eight of 30 C. rosea isolates caused taproot necrosis on inoculated plants in both experiments, whereas control roots did not exhibit necrosis. Approximately 6% of inoculated plants also developed interveinal chlorosis and marginal necrosis on trifoliates. Isolations were attempted from roots of all plants, and the isolates recovered from inoculated plants were identified as C. rosea based on morphology and ITS sequences. This fungus was not isolated from control plants. C. rosea was also isolated from petioles of symptomatic trifoliates, indicating systemic colonization of the plants. To our knowledge, this is the first report of C. rosea causing root rot of soybean and systemically colonizing soybean. This fungus may have been previously isolated from asymptomatic soybean plants and identified as Gliocladium roseum (2). The impact of this fungus on soybean production is unknown. References: (1) G. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathological Society, St. Paul, MN, 1999. (2) J. D. Mueller and J. B. Sinclair. Trans. Brit. Mycol. Soc. 86:677, 1986. (3) H.-J. Schroers et al. Mycologia 91:365, 1999.

Soybean Rust Caused by Phakopsora pachyrhizi Detected in the State of Campeche on the Yucatan Peninsula, Mexico.

Soybean rust caused by Phakopsora pachyrhizi Syd. & P. Syd is a destructive foliar disease of soybean (Glycine max L), which was first confirmed in North America in Louisiana during 2004 (4). Soybean rust (SBR) has also been reported late in the growing season as far north as Illinois, Indiana, and Iowa. SBR was first confirmed in Mexico in 2005 in the state of San Luis Potosi on soybean (3) and subsequently reported in the states of Tamaulipas, Veracruz, and the southwestern coast of Chiapas. Symptoms of SBR were observed on leaves of multiple, nearly mature soybean plants near the city of Campeche (19.72796°N, 90.0771°W) on the Gulf Coast of the Yucatan Peninsula during November 2008. Angular and irregular chlorotic lesions on leaves contained necrotic spots and pale brown, erumpent, cone-like uredinia with a central opening. Ellipsoid to obovoid, echinulate, light tan urediniospores (10 to 13 × 16 to 18 µm) were observed microscopically. DNA was extracted from leaf tissue containing uredinia and from asymptomatic tissue with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA). P. pachyrhizi was confirmed in the symptomatic leaves by a PCR assay with Ppm1/Ppa2 primers, but not from the asymptomatic leaves (1). Subsequently, the DNA extracted from symptomatic and asymptomatic leaf tissues was tested again in another laboratory by a specific quantitative PCR assay (1), and positive results for the presence of soybean rust were obtained only from the symptomatic tissue. As a final confirmatory step, amplified DNA from the PCR assay was sequenced, and the results matched P. pachyrhizi sequences in the GenBank database. To our knowledge, these observations confirm for the first time the presence of P. pachyrhizi in the state of Campeche of southern Mexico. Although it was confirmed on soybean during 2008, it is not known how long the pathogen has been present or which other hosts may be infected there. The presence of SBR on the Yucatan Peninsula is significant because of its potential effects on local plant hosts. In addition, the climate allows possible year-round survival of the pathogen and long-distance transport of urediniospores to the United States. Potential transport of SBR spores from this part of Mexico to the United States has been reported through the application of NOAA's HYSPLIT (Hybrid Single Particle Lagrangian Integrated Transport) model and atmospheric back-trajectory analysis (2). References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) S. V. Krupa et al. Plant Dis. 90:1254, 2006. (3) A. C. Rodriguez et al. Plant Dis. 90:1260, 2006. (4) R. W. Schneider et al. Plant Dis. 89:774, 2005.

Comparison of commercially available media for detection and isolation of Ureaplasma urealyticum and Mycoplasma hominis.

The Mycotrim Triphasic flask system (Irvine Scientific, Irvine, Calif.) was compared with a system composed of Mycotrim GU broth (Irvine Scientific) and A7 or A8 agar (Remel, Lenexa, Kans.) for the ability to detect Ureaplasma urealyticum and Mycoplasma hominis from 129 genital specimens. Of the 64 specimens positive for U. urealyticum, 25, 98, and 100% were detected on Mycotrim Triphasic agar and A7 and A8 agars, respectively. All 18 specimens that grew M. hominis were detected by A7 and A8 agars, and 94% grew on Mycotrim Triphasic agar. Mycotrim GU broth detected all of the positive specimens, and Mycotrim Triphasic broth detected all but one. Mycotrim GU broth inoculated simultaneously with either A7 or A8 agar was found to be more sensitive and cost-effective than the Mycotrim Triphasic flask system.

von Willebrand factor interacts with malignant hematopoietic cell lines: evidence for the presence of specific binding sites and modification of von Willebrand factor structure and function.

von Willebrand factor (vWf) mediates the adherence of platelets to exposed vascular subendothelium. During hematogenous metastasis in certain model systems, platelets are also deposited at endothelial surfaces in heterotypic aggregates with tumor cells. The role of vWf in this metastatic event was investigated by examining the interaction between purified iodine 125-labeled vWf and the human tumor cell lines, U937 and CA46. vWf-tumor cell binding was specific, partially reversible, and saturable at 4 degrees C. At 37 degrees C, however, U937 cell binding sites could not be saturated, suggesting endocytosis as a possible mechanism of interaction. When constant amounts of vWf were added, interactions with tumor cells were complete after 60 minutes, but rapidly dissociated over the next 2 hours. With the addition of specific proteinase inhibitors, vWf remained in stable association with tumor cells throughout a 3-hour time course. Loss of vWf antigen in supernatant samples complemented the 125I-labeled vWf bound directly to tumor cells. However, disproportionately large reductions in vWf functional activity were observed and correlated with alterations in multimeric structure. These data suggest an initial binding of vWf to some tumor cells, followed by a time-dependent loss of structural and functional integrity. VWf may contribute to tumor cell arrest within the microvasculature and may influence other tumor cell-subendothelial interactions as the adhesive function of vWf is subsequently modified.

Critical role of the carbohydrate moiety in human von Willebrand factor protein for interactions with type I collagen.

The ability of von Willebrand factor protein (vWF) to agglutinate platelets with ristocetin depends upon the presence of its highest molecular weight multimers (HMWM) and its intact carbohydrate structure. Previously we demonstrated that the HMWM are preferentially adsorbed to purified fibrillar type I collagen. The role of the carbohydrate structure of vWF in this function has not been established. In these studies complete desialylation (greater than 95%) of the intact protein by neuraminidase did not interfere with the normal adsorption of vWF activity to type I collagen. In contrast, modification of the penultimate galactose of the desialylated protein with galactose oxidase or beta-galactosidase markedly reduced adsorption of vWF activity by collagen. Subsequent reduction of the oxidized desialylated protein with potassium borohydride completely regenerated the normal adsorption of vWF activity by collagen. Enzymatic modification of the penultimate galactose moiety of vWF resulted in a loss of the HMWM, as observed following SDS-glyoxyl agarose electrophoresis. This was in contrast to desialylated vWF, which appeared intact structurally and which predictably lost its HMWM upon exposure to collagen in a manner similar to native vWF. Therefore, the carbohydrate structure of vWF and, in particular, the penultimate galactose moiety, may be critical for vWF-collagen interactions and for the mediation of primary hemostasis.

Collagen-factor VIII/von Willebrand factor protein interaction.

Factor VIII/von Willebrand factor (FVIII/vWF) protein interaction with collagen was studied by incubating plasma or purified FVIII/vWF with purified type I fibrillar collagen. Collagen adsorbed FVIII/vWF activities in a similar time and concentration-dependent manner from normal plasma, plasmas from classical and variant type von Willebrand's disease (vWD), and from purified FVIII/vWF. Incubation with denatured collagen or fibrin, produced in the presence or absence of fibronectin, showed no adsorption of FVIII/vWF. Examination of the multimeric structure of the remaining unadsorbed FVIII/vWF protein by agarose gel electrophoresis and autoradiography showed that the largest multimers had been adsorbed to the collagen. Studies of the adsorbed FVIII/vWF protein when eluted from collagen showed that it complemented the alterations in multimeric structure observed in the supernatants following collagen exposure. The multimeric structure of normal plasma following collagen adsorption resembled that of unadsorbed type IIb plasma; however, the collagen-adsorbed normal plasma did not produce enhanced ristocetin-induced platelet aggregation ( RIPA ). This phenomenon, therefore, must not be due solely to absence of large multimers from type IIb FVIII/vWF protein. The adsorbed multimers of FVIII/vWF protein may act as a subendothelial collagen-platelet bridge to promote primary hemostasis.

Hemagglutination treponemal test for syphilis.

Sera from 290 hospital patients were tested to compare the sensitivity, specificity, and reproducibility of the hemagglutination treponemal test for syphilis (HATTS) with the fluorescent treponemal antibody absorption test (FTA-ABS). Complete agreement was obtained between the methods when 142 syphilitic sera from patients with various stages of syphilis were tested. By using clinical histories, the specificity with 148 nonsyphilitic sera was determined to be 100% for the HATTS and 96.6% (143 of 148) for the FTA-ABS. Satisfactory reproducibility was obtained with both methods. Compared with the FTA-ABS, the HATTS was more specific, easier, and more economical to perform. We therefore recommend the HATTS as a suitable alternative to the FTA-ABS.

Effect of serum source on protein-free diet lymphocyte blastogenesis.

The influence of protein depletion on serum factors in PHA lymphocyte blastogenesis was studied in a rat model. Buffalo rats were divided randomly into two groups and fed either a protein-free (PF) diet or a regular 25% protein diet (RD). At weekly intervals, lymph node lymphocytes were cultured with PHA in either autologous or pooled 10% rat serum. For weeks 2 through 6, PHA stimulated blastogenesis of lymphocytes from rats maintained on PF diet incubated with autologous serum decreased significantly compared with RD lymphocytes cultured with RD serum. At week 4, PHA blastogenesis of PF lymphocytes cultured with RD serum was similar to that of RD lymphocytes incubated with RD serum. AT weeks 5 and 6, PHA stimulation of PF lymphocytes assayed with RD serum was depressed compared with RD lymphocytes cultured with RD. For weeks 4 through 6, blastogenesis of RD lymphocytes assayed with PF serum decreased significantly compared with RD lymphocytes incubated with RD serum. Three weeks of protein repletion of rats previously on a PF diet for 6 weeks restored PHA blastogenesis to that observed for lymphocytes from animals in the RD group. The data suggested that suppression of PHA blastogenesis of lymphocytes from rats maintained on PF diet involved a serum factor in the early stages of protein depletion and an additional defect (not serum related) in lymphocyte blastogenesis after prolonged protein depletion. In addition, this defect was corrected after a period repletion.