Vitamin K supplementation and bone mineral density in dialysis: results of the double-blind, randomized, placebo-controlled RenaKvit trial.

BACKGROUND: Vitamin K deficiency is highly prevalent in patients on dialysis and may contribute to their low bone mineral density (BMD) and increased risk of fracture. This study investigated the effect of menaquinone-7 (MK-7) supplementation on BMD in patients on chronic dialysis. METHODS: In a multicentre, double-blind, placebo-controlled intervention trial, 123 patients on chronic dialysis were randomised to a daily oral supplement of either MK-7 360 µg or placebo for 2 years. BMD of the distal radius (1/3, mid, ultradistal and total), femoral neck, lumbar spine (L1-L4) and whole body was assessed by dual-energy X-ray absorptiometry. Serum levels of vitamin K1 and MK-7 and plasma levels of total osteocalcin, dephosphorylated-uncarboxylated matrix Gla protein and protein induced by vitamin K absence II were measured to assess vitamin K status. RESULTS: After 2 years, an accelerated BMD loss of the 1/3 distal radius was found with MK-7 supplementation {mean difference of changes relative to placebo -0.023 g/cm2 [95% confidence interval (CI) -0.039 to -0.008]}, whereas the decrease in lumbar spine BMD seen in the placebo group was prevented [mean difference of changes between groups 0.050 g/cm2 (95% CI 0.015-0.085)]. No significant effects were observed at the remaining skeletal sites. Vitamin K status strongly improved in MK-7-supplemented participants. CONCLUSION: Compared with placebo, an accelerated BMD loss of the 1/3 distal radius was found after 2 years of MK-7 supplementation, whereas a decline in lumbar spine BMD was prevented. As such, MK-7 supplementation might modify BMD site-specifically in patients on dialysis. In aggregate, our findings do not support MK-7 supplementation to preserve bone in patients on dialysis.

Multiplex proteomics as risk predictor of infection in patients treated with hemodialysis-A prospective multicenter study.

INTRODUCTION: Severe infection is a major problem in hemodialysis patients. Multiplex proteomics might reveal novel insights into disease mechanisms increasing the risk of infection and might also be used as a risk prediction tool. The aims of this study were (1) to evaluate associations between 92 proteins assessed by a proximity extension assay and the development of severe infection in patients on hemodialysis and (2) to develop a risk prediction model for severe infection using prespecified clinical variables and proteomics. METHODS: Prospective, observational multicenter cohort study with 5-year follow-up. Patients receiving in-center hemodialysis in five facilities in Denmark were included. The primary composite endpoint was death caused by infection, bacteremia, and infections requiring hospitalization of at least 2 days or prolonging a hospital stay. FINDINGS: Of 331 patients included 210 patients reached the primary endpoint during follow-up. In adjusted Cox regression analyses, 14 plasma proteins were associated with severe infection. Correcting for multiple testing revealed only cathepsin-L1 and interleukin-6 significantly associated with the primary outcome. Cathepsin-L1-hazard ratio: 1.64 (95% confidence interval [CI] 1.24-2.17) and interleukin-6-hazard ratio: 1.16 (95% CI 1.05-1.29). Apparent C-statistics of the risk prediction model using clinical variables was 0.605, addition of cathepsin-L1 and interleukin-6 to the model improved discrimination slightly: C = 0.625. DISCUSSION: Proteomic profiling identified cathepsin-L1 and interleukin-6 as markers for infectious risk in hemodialysis patients. Further studies are needed to replicate the results and to examine possible causality. The developed risk prediction models need considerable improvement before implementation in clinical practice is meaningful.

High-sensitivity Troponin T in hemodialysis patients: a randomized placebo-controlled sub-study investigating angiotensin-II-blockade, variation over time and associations with clinical outcome.

BACKGROUND: Troponin T (TnT) is a well-known risk factor for negative outcome in hemodialysis (HD) patients, but little is known about variation over time, and the impact of clinical and dialysis specific factors. This study investigated the effect of angiotensin II receptor blockade (ARB), short and long-term variation in TnT and associations with clinical parameters. METHODS: In this analysis based on the SAFIR-cohort (Clinical Trials ID: NCT00791830) 81 HD patients were randomized double-blind for placebo (n = 40) or angiotensin II receptor blocker (ARB) treatment (n = 41) with irbesartan (150-300 mg) and followed for 12 months with six serial measurements of TnT using a high-sensitivity assay. RESULTS: Fifty-four patients (67%) completed follow-up. Baseline TnT-medians (min-max) were (placebo/ARB): 45(14-295)/46(10-343) ng/L. ARB-treatment did not significantly affect mean TnT-levels over the 12-month study period. Median week-to-week and one-year TnT-variation (5th-95th-percentile range) using all samples regardless of intervention were: 0(- 14-10) ng/L (week-to-week) and 3(- 40-71) ng/L (12 months). Median TnT-amplitude, capturing the change from the lowest to the highest TnT-value observed during the one-year study period was 38% or 20.5 ng/L. Median ratios with 95% limits of agreement were: 1.00(0.73-1.37); P = 0.92 (1 week/baseline; n = 77) and 1.07(0.52-2.25); P = 0.19 (12 months/baseline; n = 54). Baseline TnT was positively correlated with diabetes, ultrafiltration volume, arterial stiffness, change in intradialytic total peripheral resistance and N-terminal pro b-type natriuretic peptide (NT-proBNP) and negatively correlated with hematocrit, residual renal function and change in intradialytic cardiac output. High baseline TnT was associated with a higher risk of admission and cardiovascular (CV) events during follow-up. Increase in TnT over time (ΔTnT = 12-months-baseline) was significantly associated with increase in left ventricular (LV) mass and NT-proBNP and decrease in LV ejection fraction and late intradialytic stroke volume. ΔTnT was not significantly associated with admissions, CV or intradialytic hypotensive events during follow-up. Admissions were significantly more likely with a high (TnT-amplitude> 20.5 ng/L) than a low TnT-amplitude. Peaks in TnT were less frequent in aspirin-treated patients. CONCLUSION: ARB-treatment had no significant effect on TnT-levels. Week-to-week variation was generally low, yet over 12 months individual patients had considerable TnT fluctuations. Rise in TnT over time was significantly correlated with markers of cardiac deterioration. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00791830 . Date of registration: November 17, 2008. EudraCT no: 2008-001267-11.

Author Correction: Genomic prediction and GWAS of yield, quality and disease-related traits in spring barley and winter wheat.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic prediction and GWAS of yield, quality and disease-related traits in spring barley and winter wheat.

Genome-wide association study (GWAS) and genomic prediction (GP) are extensively employed to accelerate genetic gain and identify QTL in plant breeding. In this study, 1,317 spring barley and 1,325 winter wheat breeding lines from a commercial breeding program were genotyped with the Illumina 9 K barley or 15 K wheat SNP-chip, and phenotyped in multiple years and locations. For GWAS, in spring barley, a QTL on chr. 4H associated with powdery mildew and ramularia resistance were found. There were several SNPs on chr. 4H showing genome-wide significance with yield traits. In winter wheat, GWAS identified two SNPs on chr. 6A, and one SNP on chr. 1B, significantly associated with quality trait moisture, as well as one SNP located on chr. 5B associated with starch content in the seeds. The significant SNPs identified by multiple trait GWAS were generally the same as those found in single trait GWAS. GWAS including genotype-location information in the model identified significant SNPs in each tested location, which were not found previously when including all locations in the GWAS. For GP, in spring barley, GP using the Bayesian Power Lasso model had higher accuracy than ridge regression BLUP in powdery mildew and yield traits, whereas the prediction accuracies were similar using Bayesian Power Lasso model and rrBLUP for yield traits in winter wheat.

Staphylococcus aureus Bacteremia Risk in Hemodialysis Patients Using the Buttonhole Cannulation Technique: A Prospective Multicenter Study.

RATIONALE & OBJECTIVE: Arteriovenous fistulas are the preferred access type for hemodialysis. The buttonhole needling technique has become an alternative to stepladder or area puncture. However, an increased risk for infection has been described. The present study examined the risk for infectious complications with different needling techniques. STUDY DESIGN: Prospective multicenter observational cohort study with 5 years of follow-up. SETTING & PARTICIPANTS: In-center hemodialysis patients from 5 hemodialysis units in Denmark, dialyzed on a native arteriovenous fistula. 286 patients were included; 144 cannulated with the buttonhole technique. EXPOSURE: The buttonhole cannulation technique was compared to the stepladder or area puncture technique. OUTCOMES: Primary end points: event rates of access-related Staphylococcus aureus bacteremia and the HR for first access-related S aureus bacteremia. Secondary end points: local infections and access-related S aureus bacteremia-related metastatic infections and mortality. ANALYTICAL APPROACH: Time-to-event analysis using Cox proportional hazards regression to estimate the HR of access-related S aureus bacteremia in buttonhole cannulation compared to stepladder/area puncture. Poisson regression was used for incidence rate ratio calculations. RESULTS: S aureus caused 48 access-related bacteremias; 43 (90%) in the buttonhole group compared with 5 (10%) in the stepladder/area group. The HR for first access-related S aureus bacteremia was significantly higher for buttonhole cannulation compared to stepladder/area needling (unadjusted, 6.8 [95% CI, 2.4-19.1]; adjusted, 8.4 [95% CI, 2.9-24.2]). The incidence rate ratio for access-related S aureus bacteremia was 6.8 (95% CI, 2.9-16.1), and the incidence rate ratio of local cannulation-site infection without access-related S aureus bacteremia was 3.8 (95% CI, 1.3-15.4) for buttonhole cannulation compared to stepladder/area needling. LIMITATIONS: Nonrandomized observational design, prevalent hemodialysis patients. CONCLUSIONS: Access-related S aureus bacteremia rates were very high for buttonhole cannulation compared to stepladder/area needling, questioning the use of buttonhole cannulation in routine clinical practice. A restrictive approach to buttonhole use is recommended, with buttonhole cannulation only being used as a second alternative to area technique when stepladder cannulation is not feasible.

Magnetic resonance imaging biomarkers for chronic kidney disease: a position paper from the European Cooperation in Science and Technology Action PARENCHIMA.

Functional renal magnetic resonance imaging (MRI) has seen a number of recent advances, and techniques are now available that can generate quantitative imaging biomarkers with the potential to improve the management of kidney disease. Such biomarkers are sensitive to changes in renal blood flow, tissue perfusion, oxygenation and microstructure (including inflammation and fibrosis), processes that are important in a range of renal diseases including chronic kidney disease. However, several challenges remain to move these techniques towards clinical adoption, from technical validation through biological and clinical validation, to demonstration of cost-effectiveness and regulatory qualification. To address these challenges, the European Cooperation in Science and Technology Action PARENCHIMA was initiated in early 2017. PARENCHIMA is a multidisciplinary pan-European network with an overarching aim of eliminating the main barriers to the broader evaluation, commercial exploitation and clinical use of renal MRI biomarkers. This position paper lays out PARENCHIMA's vision on key clinical questions that MRI must address to become more widely used in patients with kidney disease, first within research settings and ultimately in clinical practice. We then present a series of practical recommendations to accelerate the study and translation of these techniques.

Long-term effects of angiotensin II blockade with irbesartan on inflammatory markers in hemodialysis patients: A randomized double blind placebo controlled trial (SAFIR study).

INTRODUCTION: Low-grade chronic inflammation is common in hemodialysis (HD) patients. Previous studies suggest an anti-inflammatory effect of angiotensin II receptor blocker (ARB) treatment. The aim of this study was to compare the effect of ARB vs. placebo on plasma concentrations of inflammatory markers in HD patients. METHODS: Adult HD patients were randomized for double-blind treatment with the ARB irbesartan 150-300 mg/day or placebo. At baseline, 1 week, 3, 6, 9, and 12 months plasma high sensitivity C-reactive protein (hsCRP), interleukin (IL)-1ß, IL-6, IL-8, IL-18, and transforming growth factor-ß (TGF-ß) were measured using Luminex and enzyme-linked immunosorbent assay (ELISA) technology. FINDINGS: Eighty-two patients were randomized (placebo/ARB: 41/41). The groups did not differ in initial levels of any of the inflammatory markers (placebo/ARB median(range)): hsCRP 3.3(0.2-23.4)/2.7(0.2-29.6) µg/mL; IL-1ß 1.1(0.0-45.9)/1.1(0.0-7.2) pg/mL; IL-6 10(1-90)/12(1-84) pg/mL; IL-8 31(9-134)/34(5-192) pg/mL; IL-18 364(188-1343)/377(213-832) pg/mL; TGF-ß 3.2(0.8-13.9)/3.6(1.3-3.8) ng/mL. Overall, there was no significant difference in hsCRP, IL-6, IL-8, and TGF-ß between placebo and ARB-treated patients during the study period, and hsCRP, IL-6, IL-8, and TGF-ß were relatively stable during the study period (P ≥ 0.18 in all tests for parallel curves, equal levels, and constant levels). The IL-1ß level was slightly different in the two groups over time, but not significantly (P = 0.09 in test for parallel curves) and it was also relatively stable during the study period (P ≥ 0.49 in tests for equal levels and constant level). IL-18 was the only inflammatory marker which was not constant during the study period (P = 0.001 in test for constant level), but there was no significant difference between placebo and ARB-treated (P ≥ 0.51 in tests for parallel curves and equal levels). DISCUSSION: Inflammatory biomarkers were neither acutely, nor in the long-term significantly affected by the ARB irbesartan. Our findings suggest that ARB treatment in HD patients does not offer protective anti-inflammatory effects.

Quality of life development during initial hemodialysis therapy and association with loss of residual renal function.

INTRODUCTION: Health related quality of life (HRQOL) is markedly reduced in hemodialysis patients compared to the general population. We investigated the course of self-reported HRQOL over time and the association with selected factors, focusing on changes in glomerular filtration rate (GFR). METHODS: Eighty-two newly started hemodialysis patients from the SAFIR cohort filled out the Kidney Disease Quality of Life Short Form Version 1.3 (KDQOL-SFTM ) questionnaire at baseline, 6 and 12 months. The SAFIR study was a randomized, placebo-controlled, double-blind intervention study, examining the effects of the angiotensin II receptor blocker irbesartan. HRQOL was a secondary outcome measure. Main inclusion criteria: Dialysis vintage <1 year, left ventricular ejection fraction >30% and urinary output >300 mL/day. GFR was measured with mean creatinine and urea clearance from 24-hour urine collections at baseline, 6 and 12 months. FINDINGS: Irbesartan treatment did not affect HRQOL. Patients were pooled into one group for further analyses. Decline in GFR correlated significantly with decreasing HRQOL over time. HRQOL was stable over time, with a slight nonsignificant tendency toward improved HRQOL. The largest HRQOL-differences (positive values equal improved HRQOL) observed during the 12 month study period were (mean[95% confidence interval]): Burden of kidney disease:6.4[-2.2;15.0], Role limitations-physical:12.7[-2.1;27.5], and Role limitations-emotional:9.7[-5.2;24.6]. Comorbidity, especially diabetes, hospital admissions, female gender, and age were strongly associated with lower HRQOL in cross sectional analysis. DISCUSSION: Preservation of residual renal function seems to be important for HRQOL. In newly started HD patients, HRQOL showed little change after 12 months. HRQOL was negatively affected by comorbidity, especially diabetes, hospital admissions, female gender, and age.

No significant effect of angiotensin II receptor blockade on intermediate cardiovascular end points in hemodialysis patients.

Agents blocking the renin-angiotensin-aldosterone system are frequently used in patients with end-stage renal disease, but whether they exert beneficial cardiovascular effects is unclear. Here the long-term effects of the angiotensin II receptor blocker, irbesartan, were studied in hemodialysis patients in a double-blind randomized placebo-controlled 1-year intervention trial using a predefined systolic blood pressure target of 140 mm Hg (SAFIR study). Each group of 41 patients did not differ in terms of age, blood pressure, comorbidity, antihypertensive treatment, dialysis parameters, and residual renal function. Brachial blood pressure decreased significantly in both groups, but there was no significant difference between placebo and irbesartan. Use of additional antihypertensive medication, ultrafiltration volume, and dialysis dosage were not different. Intermediate cardiovascular end points such as central aortic blood pressure, carotid-femoral pulse wave velocity, left ventricular mass index, N-terminal brain natriuretic prohormone, heart rate variability, and plasma catecholamines were not significantly affected by irbesartan treatment. Changes in systolic blood pressure during the study period significantly correlated with changes in both left ventricular mass and arterial stiffness. Thus, significant effects of irbesartan on intermediate cardiovascular end points beyond blood pressure reduction were absent in hemodialysis patients.

Reliability of endogenous markers for estimation of residual renal function in haemodialysis patients.

BACKGROUND: Residual renal function in haemodialysis patients is of increasing interest. However, reproducibility and agreement between methods to measure and estimate glomerular filtration rate (GFR) require further elucidation. The aim of this study was to evaluate the accuracy and reproducibility of GFR estimates based on endogenous markers in haemodialysis patients. METHODS: Twelve patients were examined twice. GFR was estimated by (i) urine clearances of creatinine, urea and the average of creatinine and urea clearance [U-Cl(crea-urea)]; (ii) an equation based on serum concentration of cystatin C [eGFR(CysC)]. These were compared to (51) Cr-EDTA clearance in plasma [P-Cl(EDTA)] and urine [U-Cl(EDTA)]. RESULTS: U-Cl(crea-urea) produced results similar to U-Cl(EDTA). eGFR(CysC) had a low week-to-week variability. Visually, eGFR(CysC) differed from y = x when compared to the other methods indicating bias, probably due to extrarenal elimination of cystatin C. Coefficients of variation were significantly different, P<0·001: P-Cl(EDTA), 10%; U-Cl(EDTA), 13%; and U-Cl(crea-urea), 13%. P-Cl(EDTA) was 2·1 ml min(-1) 1·73 m(-2) higher than U-Cl(EDTA) (mean). CONCLUSIONS: Glomerular filtration rate in haemodialysis patients can be estimated from U-Cl(crea-urea) when complete urine collection is performed. The available eGFR(CysC) in haemodialysis patients seemed to be biased, and further development and validation is desirable. P-Cl(EDTA) was the most reproducible method and might be useful in special situations.

Aortic pulse wave velocity results depend on which carotid artery is used for the measurements.

OBJECTIVE: Aortic pulse wave velocity (aPWV) is a gold standard noninvasive marker of arterial stiffness. aPWV is usually obtained as carotid-femoral pulse wave velocity by measurements on the common carotid artery and the femoral artery. The carotid arteries branch slightly differently from the aorta towards the right and left side of the neck. Theoretically, using the right or left carotid artery could influence aPWV results and there are no clear guidelines to support the choice of side. The aim of this study was to investigate whether aPWV results depend on right or left side carotid artery measurements in a group of healthy individuals. METHODS AND RESULTS: Two different observers examined 50 individuals without known cardiovascular disease between 23 and 66 years of age. The measurements were performed with the SphygmoCor equipment using both right and left carotid arteries. We found that use of the right carotid artery provided significantly higher aPWV values compared with the left carotid artery, also when using different methods to estimate the travel length of the pulse wave (pooled data, subtracted distance: 0.2 ±â€Š0.4 m/s, P = 0.003; direct distance: 0.2 ±â€Š0.5 m/s, P = 0.001). CONCLUSION: Using right or left carotid artery affects aPWV, as right-side measurements provided higher values. Attention to this side difference and use of the same carotid artery will increase the strength of intervention studies using aPWV as a surrogate endpoint.

Secretomics identifies Fusarium graminearum proteins involved in the interaction with barley and wheat.

Fusarium graminearum is a phytopathogenic fungus primarily infecting small grain cereals, including barley and wheat. Secreted enzymes play important roles in the pathogenicity of many fungi. In order to access the secretome of F. graminearum, the fungus was grown in liquid culture with barley or wheat flour as the sole nutrient source to mimic the host-pathogen interaction. A gel-based proteomics approach was employed to identify the proteins secreted into the culture medium. Sixty-nine unique fungal proteins were identified in 154 protein spots, including enzymes involved in the degradation of cell walls, starch and proteins. Of these proteins, 35% had not been identified in previous in planta or in vitro studies, 70% were predicted to contain signal peptides and a further 16% may be secreted in a nonclassical manner. Proteins identified in the 72 spots showing differential appearance between wheat and barley flour medium were mainly involved in fungal cell wall remodelling and the degradation of plant cell walls, starch and proteins. The in planta expression of corresponding F. graminearum genes was confirmed by quantitative reverse transcriptase-polymerase chain reaction in barley and wheat spikelets harvested at 2-6 days after inoculation. In addition, a clear difference in the accumulation of fungal biomass and the extent of fungal-induced proteolysis of plant ß-amylase was observed in barley and wheat. The present study considerably expands the current database of F. graminearum secreted proteins which may be involved in Fusarium head blight.

Reliability of 5¹Cr-EDTA plasma and urinary clearance as a measure of residual renal function in dialysis patients.

PURPOSE: In dialysis patients, longer survival is associated with a higher residual renal function. Randomized controlled trials are conducted to clarify how residual renal function can be preserved. However, existing methods for measuring residual renal function are uncertain and there is a need for establishing a standard for measurements of glomerular filtration rate (GFR) in dialysis patients. METHODS: 5¹Cr-EDTA clearances in plasma, urine, and dialysate were evaluated in a sample of 12 hemodialysis and 12 peritoneal dialysis patients. The patients' condition was generally stable, and all patients were investigated twice within 4-10 days. RESULTS: Plasma clearances of 5¹Cr-EDTA for all patients ranged between 2.1 and 30.8 mL/min/1.73 m², whereas urinary 5¹Cr-EDTA clearances ranged from 0.7-20.0 mL/min/1.73 m². This difference was statistically significant (p < 0.001). Week-to-week reproducibility expressed as coefficients of variation were below or equal to 10% for plasma clearances and 13% for urinary clearances in hemodialysis patients and 14% in peritoneal dialysis patients. CONCLUSIONS: This study demonstrated a difference between 5¹Cr-EDTA plasma and urinary clearances in dialysis patients. Plasma clearance of 5¹Cr-EDTA had the best reproducibility. For repeated measurements as in clinical prospective trials, we recommend 5¹Cr-EDTA plasma clearance based on blood sampling at 5 + 24 hours with subtraction of 5¹Cr-EDTA dialysate clearance in peritoneal dialysis patients. Further studies are needed to corroborate our findings.

Analysis of early events in the interaction between Fusarium graminearum and the susceptible barley (Hordeum vulgare) cultivar Scarlett.

A proteomic analysis was conducted to map the events during the initial stages of the interaction between the fungal pathogen Fusarium graminearum and the susceptible barley cultivar Scarlett. Quantification of fungal DNA demonstrated a sharp increase in fungal biomass in barley spikelets at 3 days after inoculation. This coincided with the appearance of discrete F. graminearum-induced proteolytic fragments of ß-amylase. Based on these results, analysis of grain proteome changes prior to extensive proteolysis enabled identification of barley proteins responding early to infection by the fungus. In total, the intensity of 51 protein spots was significantly changed in F. graminearum-infected spikelets and all but one were identified. These included pathogenesis-related proteins, proteins involved in energy metabolism, secondary metabolism and protein synthesis. A single fungal protein of unknown function was identified. Quantitative real-time RT-PCR analysis of selected genes showed a correlation between high gene expression and detection of the corresponding proteins. Fungal genes encoding alkaline protease and endothiapepsin were expressed during 1-3 days after inoculation, making them candidates for generation of the observed ß-amylase fragments. These fragments have potential to be developed as proteome-level markers for fungal infection that are also informative about grain protein quality.

Investigation of the effect of nitrogen on severity of Fusarium head blight in barley.

The effect of nitrogen on Fusarium Head Blight (FHB) in a susceptible barley cultivar was investigated using gel-based proteomics. Barley grown with either 15 or 100kgha(-1)N fertilizer was inoculated with Fusarium graminearum (Fg). The storage protein fraction did not change significantly in response either to N level or Fg, whereas eighty protein spots in the water-soluble albumin fraction increased and 108 spots decreased more than two-fold in intensity in response to Fg. Spots with greater intensity in infected plants contained fungal proteins (9 spots) and proteolytic fragments of plant proteins (65 spots). Identified fungal proteins included two superoxide dismutases, L-xylulose reductase in two spots, peptidyl prolyl cis-trans isomerase and triosephosphate isomerase, and proteins of unknown function. Spots decreasing in intensity in response to Fg contained plant proteins possibly degraded by fungal proteases. Greater spot volume changes occurred in response to Fg in plants grown with low nitrogen, although proteomes of uninfected plants were similar for both treatments. Correlation of proteome changes with measurement of Fusarium-damaged kernels, fungal biomass and mycotoxin levels indicated that increased Fusarium infection occurred in barley with low N and suggests control of N fertilization as a possible way to minimise FHB in barley.

Effects of beta-1,3-glucan from Septoria tritici on structural defence responses in wheat.

The accumulation of the pathogenesis-related (PR) proteins beta-1,3-glucanase and chitinase and structural defence responses were studied in leaves of wheat either resistant or susceptible to the hemibiotrophic pathogen Septoria tritici. Resistance was associated with an early accumulation of beta-1,3-glucanase and chitinase transcripts followed by a subsequent reduction in level. Resistance was also associated with high activity of beta-1,3-glucanase, especially in the apoplastic fluid, in accordance with the biotrophic/endophytic lifestyle of the pathogen in the apoplastic spaces, thus showing the highly localized accumulation of defence proteins in the vicinity of the pathogen. Isoform analysis of beta-1,3-glucanase from the apoplastic fluid revealed that resistance was associated with the accumulation of an endo-beta-1,3-glucanase, previously implicated in defence against pathogens, and a protein with identity to ADPG pyrophosphatase (92%) and germin-like proteins (93%), which may be involved in cell wall reinforcement. In accordance with this, glycoproteins like extensin were released into the apoplast and callose accumulated to a greater extent in cell walls, whereas lignin and polyphenolics were not found to correlate with defence. Treatment of a susceptible wheat cultivar with purified beta-1,3-glucan fragments from cell walls of S. tritici gave complete protection against disease and this was accompanied by increased gene expression of beta-1,3-glucanase and the deposition of callose. Collectively, these data indicate that resistance is dependent on a fast, initial recognition of the pathogen, probably due to beta-1,3-glucan in the fungal cell walls, and this results in the accumulation of beta-1,3-glucanase and structural defence responses, which may directly inhibit the pathogen and protect the host against fungal enzymes and toxins.