Puccinia graminis f. sp. tritici Population Causing Recent Wheat Stem Rust Epidemics in Kazakhstan Is Highly Diverse and Includes Novel Virulence Pathotypes.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a reemerging disease that caused severe epidemics in northern Kazakhstan and western Siberia in the period of 2015 to 2019. We analyzed 51 stem rust samples collected between 2015 and 2017 in five provinces in Kazakhstan. A total of 112 Pgt races were identified from 208 single-pustule isolates. These races are phenotypically and genotypically diverse, and most of them are likely of sexual origin. No differentiation of phenotypes and single-nucleotide polymorphism genotypes was observed between isolates from Akmola and North Kazakhstan provinces, supporting the idea of a wide dispersal of inoculum in the northern regions of the country. Similarities in virulence profiles with Pgt races previously reported in Siberia, Russia, suggest that northern Kazakhstan and western Siberia constitute a single stem rust epidemiological region. In addition to the races of sexual origin, six races reported in Europe, the Caucasus, and East Africa were detected in Kazakhstan, indicating that this epidemiological region is not isolated, and spore inflow from the west occurs. Virulence alone or in combination to several genes effective against the Ug99 race group was detected, including novel virulence on Sr32 + Sr40 and Sr47. The occurrence of a highly diverse Pgt population with virulence to an important group of Sr genes demonstrated the importance of the pathogen's sexual cycle in generating new and potentially damaging virulence combinations.

Primary alterations during the development of hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic, inflammatory disease of the apocrine gland-rich (AGR) skin region. The initial steps of disease development are not fully understood, despite intense investigations into immune alterations in lesional HS skin. OBJECTIVES: We aimed to systematically investigate the inflammatory molecules involved in three stages of HS pathogenesis, including healthy AGR, non-lesional HS and lesional HS skin, with the parallel application of multiple mRNA and protein-based methods. METHODS: Immune cell counts (T cells, dendritic cells, macrophages), Th1/Th17-related molecules (IL-12B, TBX21, IFNG, TNFA, IL-17, IL10, IL-23A, TGFB1, RORC, CCL20), keratinocyte-related sensors (TLR2,4), mediators (S100A7, S100A8, S100A9, DEFB4B, LCN2, CAMP, CCL2) and pro-inflammatory molecules (IL1B, IL6, TNFA, IL-23A) were investigated in the three groups by RNASeq, RT-qPCR, immunohistochemistry and immunofluorescence. RESULTS: Epidermal changes were already detectable in non-lesional HS skin; the epidermal occurrence of antimicrobial peptides (AMPs), IL-1ß, TNF-α and IL-23 was highly upregulated compared with healthy AGR skin. In lesional HS epidermis, TNF-α and IL-1ß expression remained at high levels while AMPs and IL-23 increased even more compared with non-lesional skin. In the dermis of non-lesional HS skin, signs of inflammation were barely detectable (vs. AGR), while in the lesional dermis, the number of inflammatory cells and Th1/Th17-related mediators were significantly elevated. CONCLUSIONS: Our findings that non-lesional HS epidermal keratinocytes produce not only AMPs and IL-1ß but also high levels of TNF-α and IL-23 confirm the driver role of keratinocytes in HS pathogenesis and highlight the possible role of keratinocytes in the transformation of non-inflammatory Th17 cells (of healthy AGR skin) into inflammatory cells (of HS) via the production of these mediators. The fact that epidermal TNF-α and IL-23 appear also in non-lesional HS seems to prove these cytokines as excellent therapeutic targets.

Influence of ionizing radiation on the antimicrobial activity of the antibiotics sulfamethoxazole and trimethoprim.

The response of the antimicrobial compounds sulfamethoxazole (SMX) and trimethoprim (TMP) - individually and in mixtures - to ionizing radiation was investigated using laboratory prepared mixtures and a commercial pharmaceutical formulation. The residual antibacterial activity of the solutions was monitored using Staphylococcus aureus and Escherichia coli test strains. Based on antibacterial activity, SMX was more susceptible to ionizing radiation as compared to TMP. The antibacterial activity of SMX and TMP was completely eliminated at 0.2 kGy and 0.8 kGy, respectively. However, when SMX and TMP were in a mixture, the dose required to eliminate the antibacterial activity was 10 kGy, implying a synergistic antibacterial activity when these are present in mixtures. Only when the antibiotic concentration was below the Minimum Inhibitory Concentration of TMP (i.e., 2 µmol dm-3) did the antibacterial activity of the SMX and TMP mixture disappear. These results imply that the synergistic antimicrobial activity of antimicrobial compounds in pharmaceutical waste streams is a strong possibility. Therefore, antimicrobial activity assays should be included when evaluating the use of ionizing radiation technology for the remediation of pharmaceutical or municipal waste streams.

First Report of Puccinia veronicae-longifoliae on Veronica spicata 'Royal Candles' in Minnesota.

In September 2008, Veronica spicata 'Royal Candles' plants showing foliar symptoms typical of a rust infection were brought to the Plant Disease Clinic at the University of Minnesota. Plants were grown in a local nursery in Dakota County, Minnesota. A dark brown discoloration was apparent on the upper surface of the leaf with lighter brown pustules on the underside. Teliospores collected from the pustules were 2-celled with smooth walls and 36.35 to 48.87 µm long, 11.96 to 18.28 µm wide, and had a wall thickness of 1.33 to 2.61 µm, which is in accordance with type specimen of Puccinia veronicae-longifoliae (4). Pathogen identity was confirmed by comparison of the DNA sequence of nuclear ribosomal RNA region containing the internal transcribed spacer regions 1 and 2, 5.8S and the 5' end of the 28S subunits between herbarium samples from the U.S. National Fungus Collection (BPI 841971/GenBank Accession JQ627617 and BPI 871789/GenBank Accession JQ627618) and the collected specimen (BPI 882886/GenBank Accession JQ627616). P. veronicae-longifoliae was first reported in the United States in 2004 from a commercial nursery in Michigan (2). Veronica rust has also been found in Michigan in 2005 and more recently in 2011 (1). The only other known report of Veronica rust in the United States occurred in Connecticut in 2007 (3). P. veronicae-longifoliae is not considered a quarantine pest by The Animal and Plant Health Inspection Service due to the limited host range, the host not being on the threatened or endangered list and the host being of little economic or environmental importance (2). References: (1) T. A. Dudek et al. MSU Extension News. Retrieved from http://msue.anr.msu.edu/news/veronica_rust_observed_this_season/ , 2011. (2) North American Plant Protection Organization's Phytosanitary Alert System. Retrieved from http://www.pestalert.org/oprDetail.cfm?oprID=129 , 2004. (3) Pundt, L. Floriculture Greenhouse Update. Retrieved from http://www.negreenhouseupdate.info/index.php/july/194-rust-on-veronica , 2007. (4) D. B. O. Savile. Can. J. Bot. 46:631, 1968.

First Report of Garlic Rust Caused by Puccinia allii on Allium sativum in Minnesota.

High-quality garlic is an emerging crop grown in Minnesota for local markets, community supported agriculture, and select restaurants. In July 2010, Allium sativum cv. German Extra Hardy Porcelain plants showing foliar symptoms typical of rust infection were brought to the Plant Disease Clinic at the University of Minnesota by a commercial grower from Fillmore County, Minnesota. Infected leaves showed circular to oblong lesions (1 to 3 mm long), which ranged in color from yellow-orange (uredinia) to black (telia). Urediniospores collected from uredinia were globoid to ellipsoid, yellowish in color, and measured 18 ± 1 × 30 ± 2 µm with a wall thickness of 2.4 ± 0.5 µm. Teliospores were two celled, 18 ± 3 × 47 ± 10 µm, with a projected cross-sectional area (1) of 826 ± 87 µm2; cell walls were smooth, brown, 1.6 ± 0.3 µm (proximal cell) to 2.1 ± 0.5 µm (distal cell) thick, and 4.2 ± 0.8 µm at the apex. The pathogen was identified as Puccinia allii (2) and a sample was deposited in the U.S. National Fungus Collection (BPI 884132). DNA was extracted from infected leaf tissue and the nuclear ribosomal internal transcribed spacer region (ITS) and 5' end of the large subunit (LS) was amplified and sequenced as described by Anikster et al. (1). The 1,257-bp sequence from the sample collected in Minnesota (GenBank Accession No. JX402206) was identical to ITS/LS sequence of a sample of P. allii collected from garlic in California (GenBank Accession No. AF511077), with the exception that MN sequence contained nine "A"s rather than 10 in the hyper-variable area at the 3' end of the ITS region. P. allii has been shown to be a species complex comprising at least two different types, "leek type" and "garlic type" (1). Based on the ITS sequence and the projected cross-sectional area of the teliospores, the sample of P. allii from MN is consistent with the garlic type. Garlic rust occurred in localized foci late in the growing season and therefore did not cause significant loss to the 2010 crop. Reoccurrence of garlic rust was not reported in either 2011 or 2012 growing seasons in Minnesota. P. allii all but eliminated commercial garlic production in California in the late 1990s (1) and has the potential to cause significant negative impact to the emerging garlic crop in Minnesota. However, the epidemiology of garlic rust in the northern U.S. is not well understood and therefore predicting the risk of the Minnesota garlic crop to rust is difficult. References: (1) Y. Anikster et al. Phytopathology 94:569, 2004. (2) L. J. Szabo et al, Rust. Pages 41-44 in: Compendium of Onion and Garlic Diseases and Pests, Second Edition. H. F. Schwartz and S. K. Mohan, eds. APS Press, St. Paul, 2008.

Metabolism of the Synthetic Cathinone Alpha-Pyrrolidinoisohexanophenone in Humans Using UHPLC--MS-QToF.

Alpha-pyrrolidinoisohexanophenone (α-PHiP/α-PiHP) is a synthetic drug structurally related to cathine, a natural psychoactive alkaloid, isolated from Khat plant. The α-PiHP is a structural isomer of α-PHP, and both α-PHP and α-PiHP could be considered an analog of α-PVP, a Schedule I drug under the Convention on Psychotropic Substances by the United Nations. This α-pyrrolidinophenone was first reported to European Monitoring of Drug and Drug Addiction by Slovenia in December 2016. In Hungary, it was initially reported in August 2016, and until 2021, it had been detected in seizures only twice and never been identified in biological samples. However, in 2021, its consumption became prevalent in Hungary. This study aims to investigate the α-PiHP metabolites by performing in vitro and in vivo metabolite identification studies of human liver microsome (pHLM), S9 fraction (pS9) and urine samples (from control and users), using liquid chromatography in conjunction with high-resolution mass spectrometry. Ten in vivo urinary metabolites of α-PiHP were tentatively identified and confirmed by in vitro metabolites detected in pHLM and pS9 samples. Among the eight Phase I and the two Phase II metabolites, five were more abundant in urine than the parent compound. The two major metabolites via reduction of the keto moiety (M01) and via oxidation of the pyrrolidine ring combined with aliphatic hydroxylation and keto reduction (M06) were identified. The metabolites via the combination of keto reduction and aliphatic hydroxylation (M04), via ring-opening followed by carboxylation (M09) and via glucuronidation of the keto reduced metabolite (M07) were also dominant. The minor metabolites were one Phase II metabolite (M08), two metabolites via aliphatic hydroxylation (M02 and M03), one metabolite via the combination of keto reduction and oxidation of the pyrrolidine ring (M05) and one metabolite via oxidation of the pyrrolidine ring (M10).

BAHA Attract to Osia conversion patients: comparison of the two systems and long-term outcomes.

OBJECTIVE: Osia is a new, transcutaneous, active bone-conduction implant. This study aimed to compare the BAHA Attract and the first-generation Osia system after BAHA Attract to Osia conversion surgery. METHOD: Five patients who had previously used the BAHA Attract system were converted to the first generation of the Osia system. Surgical aspects of the two different systems, audiological performance and subjective opinions of the patients were investigated. Pure tone audiometry and speech audiometry in quiet was performed with each patient's BAHA 5 sound processor on Attract, and the test battery was repeated six weeks after the Attract to Osia conversion and at different time points after the first fitting. Details of the surgery and patients' feedback were analysed. RESULTS: Audiology tests showed significant improvement when using either system; however, the Osia system performance was better. Based on patient feedback, all the five implantees preferred the Osia system. CONCLUSION: The study results suggest that the Osia system is a safe and powerful hearing implant that provides good clinical outcomes.

Ovarian hyperstimulation syndrome is correlated with a reduction of soluble VEGF receptor protein level and a higher amount of VEGF-A.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening condition associated with increased vascular permeability. The vascular endothelial growth factor (VEGF) system and its receptors have been identified as the main angiogenic factors responsible for increased capillary permeability and are therefore discussed as crucial for the occurrence of OHSS. Recently, a number of soluble receptors for the VEGFs have been detected (sVEGF-Rs) and it has been shown that these sVEGF-Rs compete with the membrane-standing VEGF-R to bind VEGFs. METHODS: We analyzed the serum levels of soluble VEGF-R1, -R2 and -R3 in 34 patients suffering from OHSS and in 34 controls without this disease. In a subgroup analysis, we correlated the severity of the OHSS with the detected amounts of VEGF-R1, -R2 and -R3. In addition, we determined the amount of total VEGF-A in the samples. RESULTS: All the three soluble VEGF receptors tended to be higher in the control group compared with that in the OHSS group but this difference only reached significance for sVEGF-R2 (mean ± SEM: 15.5 ± 0.6 versus 13.8 ± 0.5 ng/ml, respectively, P< 0.05). In the subgroup analysis, sVEGF-R2 levels decreased as the severity of OHSS increased (OHSS-I: 16.8 ± 1.9 ng/ml and OHSS-III: 12.7 ± 1.0 ng/ml, P< 0.05) Moreover, the serum levels of total VEGF-A were higher in the OHSS group than those in the controls (537.7 ± 38.9 versus 351 ± 53.4 pg/ml, respectively P< 0.05). CONCLUSIONS: We propose that VEGF-A plays a role in the occurrence of OHSS, that the amount of biologically available VEGF-A is modulated by sVEGF-Rs and that different combinations of VEGF-A and sVEGF-R levels might contribute to the severity of OHSS.

First Report of a New TTKSF Race of Wheat Stem Rust (Puccinia graminis f. sp. tritici) in South Africa and Zimbabwe.

Seven races have been described in the Ug99 race group of Puccinia graminis f. sp. tritici (2). Ug99-related races previously recorded in South Africa are TTKSF, TTKSP, and PTKST (4). In December 2010, severe stem rust infection of the winter wheat cv. Matlabas was observed for the first time in South Africa. Race analysis using the 20 North American (NA) stem rust differential lines and letter code system classified the race as TTKSF. In comparative infection studies in a greenhouse, cv. Matlabas seedlings were susceptible (infection type [IT] 4) to isolate UVPgt61/1 (TTKSF+) collected from Afrikaskop in the eastern Free State, whereas the cultivar was resistant (IT 1 to 2) to stem rust isolates 2013 (TTKSF), UVPgt55 (TTKSF), UVPgt59 (TTKSP), and UVPgt60 (PTKST). Isolate 2013 represents the original collection of race TTKSF in South Africa (1). In addition to the NA differentials, no variation in the IT range of seedlings of lines with Sr7a, 8b, 12, 13, 14, 16, 18, 19, 22, 25, 26, 27, 28, 29, 32, 33, 34, 35, 39, 41, 42, 43, 44, Em, R, Tt2, and Satu was observed between UVPgt61/1 and UVPgt55. With the exception of cv. Matlabas, ITs of 106 South African cultivars likewise did not differentiate UVPgt61/1 and UVPgt55. Seedling IT studies were conducted at least twice. Microsatellite analysis (4) showed that all single pustule isolates established from the original Matlabas isolate formed part of the Ug99 group. When characterized with selected single nucleotide polymorphisms (SNPs), all single pustule isolates shared an identical genotype that differed from UVPgt55 (TTKSF), a foreign introduction into South Africa (1,3). SNP genotype analysis suggests that UVPgt61/1 is genetically dissimilar to UVPgt55, as is Zim1009, another TTKSF+ isolate that was collected from Birchenough in Zimbabwe. Studies are underway to determine the identity of the defeated Sr gene in Matlabas and the cultivar has been added to the South African stem rust differential set. TTKSF+ is the eighth race detected in the Ug99 group. Since no other cultivars or advanced lines were found to carry the Matlabas gene, it is unlikely that race TTKSF+ will threaten wheat production in South Africa. However, the occurrence of a new Ug99-related race emphasizes the variability within this internationally important group. References: (1) W. H. P. Boshoff et al. Plant Dis. 86:922, 2002. (2) R. F. Park et al. Euphytica 179:109, 2011. (3) B. Visser et al. Mol. Plant Pathol. 10:213, 2009. (4) B. Visser et al. Euphytica 179:119, 2011.

Predicting Soybean Rust Incursions into the North American Continental Interior Using Crop Monitoring, Spore Trapping, and Aerobiological Modeling.

Between 2005 and 2009, millions of U.S. and Canadian soybean acres that would have received fungicide application remained untreated for soybean rust due to information disseminated through the Integrated Pest Management Pest Information Platform for Extension and Education (ipmPIPE), increasing North American producers' profits by hundreds of millions of dollars each year. The results of our analysis of Phakopsora pachyrhizi urediniospores in rain collections, aerobiology model output, and observations of soybean rust spread in 2007 and 2008 show a strong correspondence between spore collections and model predictions for the continental interior of North America, where soybean is an important crop. The analysis suggests that control practices based on up-to-date maps of soybean rust observations and associated commentary from Extension Specialists delivered by the ipmPIPE may have suppressed the number and strength of inoculum source areas in the southern states and retarded the northward progress of seasonal soybean rust incursions into continental North America. The analysis further indicates that spore trapping and aerobiological modeling can reduce our reliance on the costly Sentinel Plot Network while maintaining the effectiveness of the ipmPIPE system for soybean rust management.

Space use of wild boar (Sus Scrofa) in Budapest: are they resident or transient city dwellers?

In our study, we examined the movement of two wild boars marked with GPS/GSM transmitters in city of Budapest. We hypothesised that: the wild boars do not leave the urban area (H1); the wild boars prefer places that are less disturbed by people, and which are rich in potential hiding places (H2); and their home ranges would be smaller than that of wild boars living in non-urban environment (H3). Based on our results, we accepted our first hypothesis, as the wild boars had not left the area of Budapest. However, we partly rejected our second hypothesis: the wild boars preferred urban areas that were forested and richly covered with vegetation; however, human presence therefore disturbance was also high in those areas. The home range sizes of both marked wild boar sows were remarkably smaller than those of the wild boars living in natural environment (H3). City habitat modification, e.g. clearing undergrowth vegetation, could result that wild boars cannot find any hiding places. The significant part of food sources will disappear with the elimination of these places. By eliminating the two main factors together could prevent wild boars finding their living conditions within the city.

Identifying and quantifying Phakopsora pachyrhizi spores in rain.

In summers of 2005 and 2006, rain was collected weekly at over 100 selected National Atmospheric Deposition Program/National Trends Network sites across the soybean-growing region of the central and eastern United States. Rain samples were screened for Phakopsora pachyrhizi (causal agent of soybean rust) DNA using a nested real-time polymerase chain reaction assay. Over this time frame, P. pachyrhizi spores were detected in every state in the study, but more frequently in states along the Gulf and Atlantic coasts and along the Ohio River Valley westward to Kansas. A bimodal temporal distribution of samples testing positive for P. pachyrhizi was found in both years. However, there was a greater than threefold increase in the number of samples testing positive for P. pachyrhizi in 2006 compared with 2005, with the most significant increase in August. There was also an increase in the average number of spores per sample in 2006 relative to 2005. Sequence analysis of a subset of positive samples was used to validate the assay results. From the sequence analysis, two reliable polymorphic regions were found, resulting in six distinct genotypes. One genotype was found in 56% of the samples tested, whereas the other genotypes were found less frequently.

Development and characterization of expressed sequence tag-derived microsatellite markers for the wheat stem rust fungus Puccinia graminis f. sp. tritici.

Puccinia graminis f. sp. tritici is the causal agent of stem rust disease in wheat. The rust fungus has caused devastating disease epidemics throughout history and is still posing a potential threat to wheat production in some regions of the world due to the appearance of new races. To develop microsatellite or simple sequence repeat (SSR) markers for use in population genetics studies, a total of 60,579 expressed sequence tag (EST) sequences (reads) generated from P. graminis f. sp. tritici were screened for tandemly repeated di- and tri-nucleotide units using a bioinformatics approach and 708 unisequences containing putative SSR loci with six or more repeat units were identified. Flanking primers were designed for 384 unique SSR loci, which mapped to different locations of the draft genome sequence of the fungus. Of the 384 primer pairs tested, 72 EST-SSR markers were eventually developed, which showed polymorphism among 19 isolates of P. graminis f. sp. tritici and 4 isolates of P. graminis f. sp. secalis evaluated. Thirty-two of the SSR loci were also evaluated in three other rust fungi (P. triticina, P. hordei, and P. coronata f. sp. hordei) for cross-species transferability. These SSR markers derived from ESTs will be useful for characterization of population structures and for gene mapping in P. graminis.

Detection of Virulence to Resistance Gene Sr36 Within the TTKS Race Lineage of Puccinia graminis f. sp. tritici.

The stem rust resistance gene Sr36 confers a near-immune resistance reaction to many races of Puccinia graminis f. sp. tritici and is highly effective against race TTKSK (syn. Ug99), which possesses unusually broad virulence combinations. Because this gene is widely used in United States soft winter wheat germplasm and cultivars, it has been considered to be an important source of resistance to TTKSK. In 2007, moderately susceptible infection responses were observed on wheat lines and cultivars carrying Sr36 in a field screening nursery for stem rust at Njoro, Kenya. We derived 18 single-pustule isolates from stem rust samples collected from the 2007 Njoro nursery. The isolates were evaluated for virulence on 20 North American stem rust differential lines and on wheat lines and cultivars carrying Sr36, Sr31+Sr36, and Sr24+Sr31. Of the 18 isolates, 10 produced infection types 3+ to 4 on line W2691SrTt-1 (monogenic for Sr36) and other lines that carry Sr36 and belonged to a new virulence phenotype that was not detected in previous years. These isolates were identified as race TTTSK. The remaining eight isolates were identified as races TTKSK (five isolates) and TTKST (three isolates), with avirulence and virulence, respectively, to Sr24. Thirteen simple sequence repeat (SSR) markers were used to examine the genetic relationships among the three races in the TTKS lineage. All isolates in the lineage shared an identical SSR genotype and were clearly different from North American races. In all, 16 wheat cultivars and 60 elite breeding lines, postulated to possess Sr36, were susceptible to race TTTSK. The occurrence of race TTTSK with combined virulence on Sr31 and Sr36 has further broadened the virulence spectrum of the TTKS lineage and rendered an important source of resistance ineffective.

Older Donation After Circulatory Death Kidneys for Older Recipients: A Single-Center Experience.

The number of older patients is increasing on the transplant waiting list. Donation after circulatory death (DCD) kidney transplantation has increased, but there remains hesitancy in use of older DCD organs. The aim of this study was to evaluate outcomes of directing older DCD donor kidneys into older recipients. METHODS: Patients 60 years or older who received transplants from DCD donors 60 years or older, between February 2010 and January 2014, were identified from a prospectively maintained database. RESULTS: Over a 4-year period, 88 patients 60 years or older received DCD kidney transplants from donors 60 years or older. Of these 44 (55%) were 60 to 69 years old and 40 (45%) were 70 years or older. Median follow up was 63 months. Patient survival was 95% and 79% at 1 and 5 years, respectively, with a survival in those 70 years and older (69%) compared with those aged 60 to 69 (88%) years (P = .01). Censored for death graft survival was 94% and 80% at 1 and 5 years, respectively. Median estimated glomerular filtration rate at 12 months and 36 months was 36 mL/min (range, 11-70 mL/min) and 39.5 mL/min (range, 11-77 mL/min), respectively. CONCLUSIONS: Older DCD kidneys, when transplanted into older recipients, result in good patient and graft survival and an acceptable graft function, especially considering their age. This represents a good use of this organ resource.

A rapid method for detecting and quantifying bacterial DNA in rust fungal DNA samples.

Bacterial DNA contamination of rust fungal DNA can be a significant problem for sequencing the rust fungus. Sequence assembly is much more difficult if the sequence contigs are mixed with bacterial sequence. A quantitative real-time polymerase chain reaction (qPCR) assay was developed to quantify bacterial DNA within rust fungal DNA samples and the results were compared with those obtained from traditional CFU counts. Real-time PCR showed higher values of DNA contamination than CFU. However, the ranking of samples from low to high for bacterial contamination was consistent between the methods. Reasons for the differences between the methods are discussed. The qPCR assay was tested by adding known quantities of Escherichia coli DNA to Puccinia graminis DNA samples. The assay reliably quantified bacterial contamination at > or = 1.0% of the total sample DNA. When bacterial contamination was <1.0%, fungal DNA also occasionally was amplified, nullifying the quantification measurement. However, primer specificity was not simply the product of the ratio of bacterial DNA to fungal DNA. Bacterial contamination could be quantified below 1.0% if the bacterial DNA concentration was approximately 70 pg/mul or greater. Therefore, spiking the fungal samples with a known concentration of E. coli bacterial DNA successfully eliminated the amplification of fungal DNA, making quantification of contaminating bacterial DNA possible for samples with low contamination levels.

Detection of Virulence to Resistance Gene Sr24 Within Race TTKS of Puccinia graminis f. sp. tritici.

The stem rust resistance gene Sr24 is effective against most races of Puccinia graminis f. sp. tritici, including race TTKS (syn. Ug99), and is used widely in commercial wheat cultivars worldwide. In 2006, susceptible infection responses were observed on wheat lines and cultivars carrying Sr24 in a field stem rust screening nursery at Njoro, Kenya. We derived 28 single-pustule isolates from stem rust samples collected from the 2006 Njoro nursery. The isolates were evaluated for virulence on 16 North American stem rust differential lines; on wheat lines carrying Sr24, Sr31, Sr38, and SrMcN; and on a wheat cultivar with a combination of Sr24 and Sr31. All isolates were identified as race TTKS with additional virulence on Sr31 and Sr38. These isolates were divided into two groups: group A (seven isolates and the two control isolates), producing a low infection type, and group B (21 isolates), producing a high infection type on Sr24, respectively. Isolates of group B represented a new variant of race TTKS with virulence to Sr24. Eighteen simple sequence repeat (SSR) markers were used to examine the genetic relationship between these two groups of isolates in race TTKS and five North American races (MCCF, QCCQ, RCRS, RTHS, and TPMK) that are representative of distinct lineage groups. All isolates of race TTKS shared an identical SSR genotype and were clearly different from North American races. The virulence and SSR data indicated that the new variant of race TTKS with Sr24 virulence likely has arisen via mutation within the TTKS genetic lineage. We propose to revise the North American stem rust nomenclature system by the addition of four genes (Sr24, Sr31, Sr38, and SrMcN) as the fifth set. This revision recognizes the virulence on Sr31 and differentiates isolates within race TTKS into two separate races: TTKSK and TTKST, with avirulence and virulence on Sr24, respectively. The occurrence of race TTKST with combined virulence on Sr24 and Sr31 has substantially increased the vulnerability of wheat to stem rust worldwide.

Detection and identification of four common rust pathogens of cereals and grasses using real-time polymerase chain reaction.

ABSTRACT Puccinia spp. are widespread pathogens of cereals and grasses that annually cause significant yield losses worldwide, especially in barley, oat, and wheat. Urediniospore morphology and early symptom development have limited usefulness for distinguishing Puccinia spp. Therefore, we developed real-time polymerase chain reaction assays for rapid detection of the four rust pathogen species, Puccinia graminis (Pers.:Pers.), P. striiformis (Westend.), P. triticina (Eriks.), and P. recondita (Roberge ex Desmaz.). Duplex assays were constructed for the nuclear rDNA gene, using the variable internal transcribed spacer 1 (ITS1) region to distinguish between species, and the conserved 28S region as an internal control. Species-specific ITS1 primer/probe sets were highly specific and could detect <1 pg of DNA. The species-specific primer/probe sets showed positive results over a linear range of DNA five orders of magnitude or greater. Specificity of the assays was tested using multiple collections representing a range of races and formae speciales within a species. Additionally, assay specificity was evaluated by testing a range of other grass rust pathogens, as well as other fungi. The 28S primer/probe combination was successful in detecting all Puccinia spp. tested within the duplex assays, validating the integrity of each assay. Finally, the assays were used to identify unknown rust fungi infecting pasture grasses.

Laparoscopic treatment of nonparasitic hepatic cysts.

BACKGROUND: We present our experience with laparoscopic deroofing of nonparasitic hepatic cysts. METHODS: Laparoscopic deroofing was performed due to a solitary hepatic cyst in 21 patients and polycystic liver in four patients. Laparoscopy was indicated when a cyst was larger than 5 cm (the general size of cysts was 6.9 cm) and caused complaints and was in a superficial position. In eight patients in whom the cyst was larger than 10 cm, omentoplasty was performed. RESULTS: Intraoperative complications were not detected. Two conversions were performed because of the deep position of the cyst. Postoperative bile leakage was detected in one case that was treated conservatively. The average hospital stay was 4.7 days. Relapse occurred in two patients (8%), but only one of them required a second operation. CONCLUSIONS: We recommend laparoscopic deroofing for treatment of nonparasitic liver cysts. This operation causes only slight discomfort for the patients, the intra- and postoperative morbidity is low, and relapses are rare.

Renal artery aneurysm at the anastomosis after kidney transplantation.

Vascular complications represent serious problems after kidney transplantation. An aneurysm of the transplanted renal artery is an extremely rare but potentially devastating complication that which occurs in fewer than 1% of recipients. It can cause hypertension, functional impairment, and even graft loss. A 49-year-old man was admitted 6 months after his second renal transplantation. Duplex ultrasonography demonstrated an aneurysm at the anastomosis of the transplanted renal artery. The patient has not had any complaints. The function of the graft was stable. A computed tomography scan confirmed the diagnosis. Because of the high risk of rupture we decided upon surgical repair. During the operation, blood flow to the kidney was occluded; the graft was cooled with Euro-Collin's solution and ice-cold saline. After the resection there was enough usable arterial wall to construct a new anastomosis. The patient had an uneventful postoperative period, the serum creatinine decreased to the preoperative level, and the function of the graft was stable. Renal artery aneurysms represent high-risk complications. We decided on surgical repair, which was performed with simultaneous perfusion and cooling of the graft. There are only a few similar cases in the literature; it was the first operation using this method in our practice. Surgical reconstruction of a renal artery aneurysm, if feasible, is a safe procedure that prevents aneurysm rupture and saves the graft.