Molecular Identification of Mutations Conferring Resistance to Azoxystrobin in Cercospora nicotianae.

Cercospora nicotianae, the causal agent of frogeye leaf spot (FLS) of tobacco, has been exposed to quinone outside inhibitor (QoI) fungicides for more than a decade through azoxystrobin applications targeting other major foliar diseases. From 2016 to 2018, a total of 124 isolates were collected from tobacco fields throughout Kentucky. Sensitivity of these isolates to azoxystrobin was previously characterized by determining the effective concentration to inhibit 50% conidial germination (EC50). Based on azoxystrobin EC50, isolates were categorized into three discrete groups: high sensitivity (<0.08 µg/ml), moderate sensitivity (0.14 to 0.64 µg/ml), and low sensitivity (>1.18 µg/ml). Variability in sensitivity in a limited number of C. nicotianae isolates was previously shown to be a result of resistance mutations in the fungicide target gene. To improve understanding of C. nicotianae cytochrome b (cytb) structure, the gene was cloned from three isolates representing each EC50 group, and sequences were compared. Our analysis showed that cytb gene in C. nicotianae consists of 1,161 nucleotides encoding 386 amino acids. The cytb sequence among the cloned isolates was identical with the exception of the F129L and G143A point mutations. To more rapidly determine the resistance status of C. nicotianae isolates to azoxystrobin, a polymerase chain reaction (PCR) assay was developed to screen for mutations. According to this assay, 80% (n = 99) of tested C. nicotianae isolates carried an F129L mutation and were moderately resistant to azoxystrobin, and 7% (n = 9) carried the G143A mutation and were highly resistant. A receiver operating characteristic curve analysis suggested the PCR assay was a robust diagnostic tool to identify C. nicotianae isolates with different sensitivity to azoxystrobin in Kentucky tobacco production. The prevalence of both the F129L and G143A mutations in C. nicotianae from Kentucky differs from that of other pathosystems where resistance to QoI fungicides has been identified, in which the majority of sampled isolates of the pathogen species have a broadly occurring cytb mutation.

Cytochrome b Mutations F129L and G143A Confer Resistance to Azoxystrobin in Cercospora nicotianae, the Frogeye Leaf Spot Pathogen of Tobacco.

Azoxystrobin is the only synthetic, systemic fungicide labeled in the United States for management of frogeye leaf spot (FLS) of tobacco (Nicotiana tabacum L.), caused by Cercospora nicotianae. Though traditionally considered a minor disease in the United States, FLS has recently become yield and quality limiting. In 2016 and 2017, 100 C. nicotianae isolates were collected from symptomatic tobacco from eight counties in Kentucky, United States. Prior to azoxystrobin sensitivity testing, some C. nicotianae isolates were found to utilize the alternative oxidase pathway and, after assay comparisons, conidial germination was utilized to evaluate sensitivity in C. nicotianae as opposed to mycelial growth. Azoxystrobin sensitivity was determined by establishing the effective concentration to inhibit 50% conidial germination (EC50) for 47 (in 2016) and 53 (in 2017) C. nicotianae isolates. Distributions of C. nicotianae EC50 values indicated three qualitative levels of sensitivity to azoxystrobin. Partial cytochrome b sequence, encompassing the F129L and G143A mutation sites, indicated single-nucleotide polymorphisms (SNPs) conferring the F129L mutation in C. nicotianae of moderate resistance (azoxystrobin at 0.177 ≤ EC50 ≤ 0.535 µg/ml) and the G143A mutation in isolates with an azoxystrobin-resistant phenotype (azoxystrobin EC50 > 1.15 µg/ml). Higher frequencies of resistant isolates were identified from greenhouse transplant (4 of 17) and conventionally produced (58 of 62) tobacco samples, as compared with field-grown tobacco (<4 weeks prior to harvest; 4 of 62) or organically produced samples (1 of 7), respectively. Together, these results suggest that resistance to azoxystrobin in C. nicotianae occurs broadly in Kentucky, and generate new hypotheses about selection pressure affecting resistance mutation frequencies.

Mn(I) in an extended oxide: the synthesis and characterization of La(1-x)Ca(x)MnO(2+δ) (0.6 ≤ x ≤ 1).

Reduction of La(1-x)Ca(x)MnO(3) (0.6 ≤ x ≤ 1) perovskite phases with sodium hydride yields materials of composition La(1-x)Ca(x)MnO(2+δ). The calcium-rich phases (x = 0.9, 1) adopt (La(0.9)Ca(0.1))(0.5)Mn(0.5)O disordered rocksalt structures. However local structure analysis using reverse Monte Carlo refinement of models against pair distribution functions obtained from neutron total scattering data reveals lanthanum-rich La(1-x)Ca(x)MnO(2+δ) (x = 0.6, 0.67, 0.7) phases adopt disordered structures consisting of an intergrowth of sheets of MnO(6) octahedra and sheets of MnO(4) tetrahedra. X-ray absorption data confirm the presence of Mn(I) centers in La(1-x)Ca(x)MnO(2+δ) phases with x < 1. Low-temperature neutron diffraction data reveal La(1-x)Ca(x)MnO(2+δ) (x = 0.6, 0.67, 0.7) phases become antiferromagnetically ordered at low temperature.

Suppression of magnetic order in Sr3Fe2O4Cl2 by Fe-site substitution by cobalt.

The low-temperature topotactic reduction of Sr(3)Fe(2-x)Co(x)O(5)Cl(2) oxychloride phases with LiH allows the preparation of phases of composition Sr(3)Fe(2-x)Co(x)O(4)Cl(2) (0 ≤ x ≤ 1). The reduced phases adopt body-centered tetragonal structures which are isostructural with Sr(3)Fe(2)O(4)Cl(2) and contain square-planar (Fe/Co)O(4) centers connected into apex-linked sheets, analogous to the CuO(2) sheets present in superconducting cuprate phases. As the cobalt concentration in Sr(3)Fe(2-x)Co(x)O(4)Cl(2) is increased the antiferromagnetic order of the Sr(3)Fe(2)O(4)Cl(2) host phase is suppressed, ultimately leading to spin-glass behavior, at low temperature, in Sr(3)Fe(2-x)Co(x)O(4)Cl(2) phases with x ≥ 0.8. The limited influence of cobalt substitution on the reactions which form the Sr(3)Fe(2-x)Co(x)O(4)Cl(2) phases is discussed and contrasted to that of the related SrFeO(3-δ)-SrFeO(2) system.

The topotactic reduction of Sr3Fe2O5Cl2-square planar Fe(II) in an extended oxyhalide.

The topotactic reduction of the oxychloride Sr(3)Fe(2)O(5)Cl(2) with LiH results in the formation of Sr(3)Fe(2)O(4)Cl(2). Neutron powder diffraction data show that Sr(3)Fe(2)O(4)Cl(2) adopts a body-centered tetragonal crystal structure (I4/mmm, a = 4.008(1) Å, c = 22.653(1) Å at 388 K) with anion vacancies located within the SrO layer of the phase. This leads to a structure consisting of infinite sheets of corner-sharing Fe(II)O(4) square planes. Variable-temperature neutron diffraction data show that Sr(3)Fe(2)O(4)Cl(2) adopts G-type antiferromagnetic order below T(N) ∼ 378(10) K with an ordered moment of 2.81(9) µ(B) per iron center at 5 K consistent with the presence of high-spin Fe(II). The observed structural and chemical selectivity of the reduction reaction is discussed. The contrast between the structure of Sr(3)Fe(2)O(4)Cl(2) and the isoelectronic all-oxide analogue (Sr(3)Fe(2)O(5)) suggests that by careful selection of substrate phases, the topotactic reduction of complex transition metal oxychlorides can lead to the preparation of novel anion-deficient phases with unique transition metal-oxygen sublattices which cannot be prepared via the reduction of all-oxide substrates.

Area Under the Curve of Somatosensory Evoked Potentials Detects Spinal Cord Injury.

PURPOSE: Intraoperative neurophysiological monitoring using somatosensory evoked potentials has been linked to a reduction in the incidence of neurological deficits during corrective surgery. Nonetheless, quantitative assessments of somatosensory evoked potential waveforms are often difficult to evaluate, because they are affected by anesthesia, injury, and noise. Here, we discuss a novel method that integrates somatosensory evoked potential signals into a single metric by calculating the area under the curve (AUC). METHODOLOGY: Thirty-two Sprague-Dawley rats underwent a laminectomy procedure and were then randomly assigned to a control group or to receive a contusive spinal cord injury ranging from 100 to 200 kilodynes. Neurophysiological testing was completed at various points perioperatively and postoperatively. Somatosensory evoked potential traces obtained were processed and the AUC metric was calculated. RESULTS: The AUC significantly decreased to 11% of its baseline value after impact and remained at 25% baseline after 1 hour for the 200-kdyn cohort. Postimpact, AUC for the control versus the 150-kdyn and 200-kdyn groups, and the 150-kdyn versus 200-kdyn groups were significantly higher (P < 0.01, P < 0.001, and P < 0.05, respectively). Across days, the only significant parameter accounting for AUC variability was impact force, P < 0.0001 (subject parameters and number of days were not significant). CONCLUSIONS: The AUC metric can detect an iatrogenic contusive spinal cord injury immediately after its occurrence. Moreover, this metric can detect different iatrogenic injury impact force levels and identify injury in the postoperative period. The AUC integrates multiple Intraoperative neurophysiological monitoring measures into a single metric and thus has the potential to help clinicians and investigators evaluate spinal cord impact injury status.

Perioperative neurologic complications during spinal fusion surgery: incidence and trends.

BACKGROUND CONTEXT: Perioperative neurologic complications after spine surgery may increase morbidity and health-care costs related to the procedure. PURPOSE: We estimate the national incidence of perioperative neurologic complications following anterior cervical discectomy and fusion (ACDF), posterior cervical fusion, and thoracolumbar fusion procedures using the Nationwide Inpatient Sample (NIS) data from 1999 to 2011. Additionally, we identify risk factors for developing perioperative neurologic complications and the effects of these injuries on quantifiable patient outcomes. STUDY DESIGN: A cross-sectional study was carried out. PATIENT SAMPLE: All patients included in the NIS databases from 1999 to 2011 comprised the sample. OUTCOME MEASURES: The primary outcome evaluated was the incidence of new neurologic deficits following elective spine surgery. Secondary outcomes evaluated include length of hospital stay, total hospital charges, hospital mortality rate, and discharge disposition. METHODS: A retrospective analysis of the NIS databases from the years 1999 to 2011 was conducted to identify the proportion of patients who underwent ACDFs, posterior cervical fusions, and thoracolumbar fusions who also developed perioperative neurologic complications. Statistical analyses were also conducted to identify statistically significant differences in demographics and outcomes between patients who did and did not develop perioperative neurologic complications. RESULTS: From 1999 to 2011, the total national incidence of perioperative neurologic deficits following elective ACDFs, posterior cervical fusions, and thoracolumbar fusions was 0.82%, which equates to a total of 15,066 patients who experienced these complications. The annual incidence rate of perioperative neurologic deficits has increased 54.41%, from 0.68% in the year 1999 to 1.05% in the year 2011. Additionally, the total number of procedures performed increased from 82,167 in 1999 to 186,353 in the year 2011. Perioperative neurologic deficits were associated with longer lengths of stay (9.68 days vs. 2.59 days; p<.001), higher total charges ($110,326.23 vs. $48,695.93; p<.001), and higher in-hospital mortality (2.84% vs. 0.13%; p<.001). CONCLUSIONS: The incidence rate of perioperative neurologic deficits associated with elective spine surgery documented in the NIS has increased over the time period from 1999 to 2011. The number of elective spine procedures performed has also increased over the same time period. Finally, outcomes data indicate that occurrence of perioperative neurologic complications is associated with increased rates of morbidity and mortality, as well as increased health-care use and cost. These trends indicate that the perioperative neurologic complications following spine surgery represent a growing problem in today's health-care system; further study is warranted to prevent and treat these complications to improve patient care and reduce health-care use and cost.

Hippocampal neurotransplantation evaluated in the rat kainic acid epilepsy model.

OBJECTIVE: Neurotransplantation has focused on disorders that involve subcortical brain targets. We evaluated the concepts of epileptic focus repair and changes in animal behavior through replacement of lost hippocampal neurons. The safety of hippocampal neurotransplantation was assessed in the rat kainic acid (KA) epilepsy model. METHODS: Sixty-three rats were studied and classified into six groups: KA plus 40,000 LBS-Neurons (Layton BioScience, Sunnyvale, CA; n = 13); KA plus 80,000 cells (n = 12); KA plus media (n = 9); no-KA plus 40,000 cells (n = 12); no-KA plus 80,000 cells (n = 12); and no-KA plus media (n = 5). Clinical observation (2 h daily) and electroencephalogram recording (3 h every other week) were performed to check for seizures until Week 11 after KA injection. On Week 12, the Morris water maze test was performed to assess spatial learning and memory. RESULTS: Four rats were excluded because of intracranial hematoma or abscess. In the clinical observation of seizures, the no-KA plus media group had significantly fewer seizures than rats that received KA followed by injection of 40,000 cells, 80,000 cells, or media (P = 0.001, 0.0004, and 0.004, respectively). On electroencephalographic analysis, there was no significant difference between any of the groups. Transplanted rats with KA-induced epilepsy did not have an increased number of seizures. In the Morris water maze test, the hidden platform task showed that the KA plus 80,000 cell group had significantly longer swim latencies than groups with no-KA plus 40,000 cells (P = 0.035) or no-KA plus 80,000 cells (P = 0.015), demonstrating the behavioral deficits caused by KA injection. The probe trial showed no significant difference for the percentage of time in the target quadrant between any of the groups. Histological studies showed that 26 (59%) of 44 transplanted rats had evidence of graft survival. CONCLUSION: The safety of cortical neurotransplantation was demonstrated, even in an animal model predisposed to epilepsy. We did not find evidence for cessation of seizures or improvement in behavior using this model.

Field Resistance to Strobilurin (Q(o)I) Fungicides in Pyricularia grisea Caused by Mutations in the Mitochondrial Cytochrome b Gene.

ABSTRACT Gray leaf spot caused by Pyricularia grisea is a highly destructive disease of perennial ryegrass turf. Control of gray leaf spot is dependent on the use of preventative fungicide treatments. Strobilurin-based (Q(o)I) fungicides, which inhibit the cytochrome bc(1) respiratory complex, have proven to be very effective against gray leaf spot. However, in August 2000, disease was diagnosed in Q(o)I-treated perennial ryegrass turf on golf courses in Lexington, KY, Champaign, IL, and Bloomington, IL. To determine if resistance was due to a mutation in the fungicide target, the cytochrome b gene (CYTB) was amplified from baseline and resistant isolates. Nucleotide sequence analysis revealed an intronless coding region of 1,179 bp. Isolates that were resistant to Q(o)I fungicides possessed one of two different mutant alleles, each of which carried a single point mutation. The first mutant allele had a guanine-to-cytosine transition at nucleotide position +428, resulting in a replacement of glycine 143 by alanine (G143A). Mutant allele two exhibited a cytosine-to-adenine transversion at position +387, causing a phenylalanine-to-leucine change (F129L). Cleavable amplified polymorphic sequence analysis revealed that neither mutation was present in a collection of baseline isolates collected before Q(o)I fungicide use and indicated that suspected Q(o)I- resistant isolates found in 2001 in Indiana and Maryland possessed the F129L mutation. The Pyricularia grisea isolates possessing the G143A substitution were significantly more resistant to azoxystrobin and trifloxystrobin, in vitro, than those having F129L. DNA fingerprinting of resistant isolates revealed that the mutations occurred in just five genetic backgrounds, suggesting that field resistance to the Q(o)I fungicides in Pyricularia grisea is due to a small number of ancestral mutations.