Morphological and Molecular Analysis of Fungal Species Associated with Blast and Brown Spot Diseases of Oryza sativa.

Fungal diseases blast and brown spot in rice cause severe yield losses worldwide. Blast is caused by Magnaporthe oryzae, and Bipolaris oryzae is reported as the main causal organism of brown spot. Both diseases cause leaf lesions that are difficult differentiate until the later stages. Early detection and differentiation of the lesions would help the adoption of disease management strategies specific to the pathogens and prevent reductions in the quality and quantity of rice yields. This study was conducted in the Northern Province of Sri Lanka over five consecutive rice cultivating seasons to characterize the causal fungi of rice blast and brown spot diseases by morphological and molecular means and to develop a visual guide to differentiate the two diseases. Disease incidence was recorded in 114 fields from 2017 to 2019, and fungal isolates associated with the lesions of both diseases were cultured and subjected to morphological and molecular characterization. Competitive growth interactions between M. oryzae and the more common individual fungal isolates of the brown spot lesions were evaluated. Fungal metagenomic analysis was conducted for the fungal species isolated from brown spot lesions. A suppression of blast accompanied by an increased incidence of brown spot disease was observed during the study period. M. oryzae was confirmed to be the causal organism of the blast, and >20 species of fungi were identified to be associated with brown spot lesions through morphological and molecular studies and metagenomic analyses. Fungal internal transcribed spacer region sequencing revealed genetic variation in the highly conserved region of DNA sequences of blast and brown spot fungal isolates. B. oryzae, Curvularia, and Microdochium species were commonly isolated from brown spot lesions. In vitro competitive growth interactions between the fungal isolates revealed growth suppression of M. oryzae by the fungal isolates associated with brown spot lesions. Similarly, it can be speculated that the abundance and severity of blast in the field may have an influence on brown spot-associated fungi. A simple visual guide was developed to differentiate blast and brown spot lesions. The findings would be highly useful in the timely management of these major fungal diseases affecting rice.

In silico molecular and morphological analysis of rice blast resistant gene Pi-ta in Sri Lankan rice germplasm.

BACKGROUND: Pi-ta is a major blast resistant gene, introgressed from indica rice varieties. In this study, diversity of the Pi-ta gene of 47 Sri Lankan rice accessions was studied by bioinformatics, and the results were validated with molecular and disease reaction assays. Sequences of rice accessions at the locus Os12g0281300 were retrieved from Rice SNP-Seek Database, and the coding sequence of reference Pi-ta gene of cultivar Tetep (accession no. GQ918486.1) was obtained from GenBank. Comparisons were made at nucleotide, amino acid, and protein structure level, and the 3D models predicted using Phyre2 software were superimposed using TM-align software. RESULTS: In silico analysis revealed that 10 accessions possessed resistant allele of the Pi-ta gene. The remaining accessions recorded high polymorphism in the leucine-rich domain resulting in 9 allele types, leading to single-amino acid substitutions at 27 different positions including a functional mutation of alanine to serine at the 918th amino acid position. None of the genotypes led to truncations in the amino acid sequence. The in silico analysis results were validated on 23 accessions comprising resistant and susceptible genotypes and another 25 cultivars from Northern Sri Lanka, by molecular assay using YL183/YL87 and YL155/YL87 resistant and susceptible allele-specific markers. Resistance of Pi-ta gene for the causal fungus, Magnaporthe oryzae, was further validated through pathogenicity assay. CONCLUSION: The Pi-ta gene, especially the LRD region, revealed significant variations within Sri Lankan rice cultivars leading to high levels of resistance against blast. This information would be highly useful in breeding programmes for resistance against rice blast.

A functional molecular marker for detecting blister blight disease resistance in tea (Camellia sinensis L.).

KEY MESSAGE: Identification of an EST-SSR molecular marker associated with Blister blight, a common fungal disease of tea, facilitating marker-assisted selection, marking a milestone in tea molecular breeding. lister blight (BB) leaf disease of tea, caused by the fungus Exobasidium vexans, results in 25-30% crop loss annually. BB is presently controlled by Cu based fungicides, but genetic resistance is the most viable option in disease management. Tea is a naturally out-crossing, woody perennial necessitating a long time for completion of a breeding programme. Marker-assisted selection (MAS) is vital to expedite breeding programmes and also for better accuracy in gene identification. The aim of the current research was to derive marker-trait associations using an F1 population segregating for BB. The population was genotyped at 11 expressed sequence tag simple sequence repeat loci followed by detecting the alleles by fragment analysis. The genotypic and phenotypic data were subjected to single-marker analysis resulting in the identification of EST-SSR073 as a diagnostic marker amplifying three alleles of the sizes, 168, 170 and 190 bp in F1. Of them, alleles 190 and 168 bp were confirmed to concur BB resistance and susceptibility, respectively. The alleles were validated in a panel of 64 tea cultivars, resulting in the amplification of 12 alleles at EST-SSR073. The EST-SSR073 allele sequences matched with Camellia sinensis photosystem-I reaction center subunit-II. The marker EST-SSR073 can be effectively used in breeding tea against BB, recording a milestone in MAS in tea.

In silico structural homology modelling of EST073 motif coding protein of tea Camellia sinensis (L).

BACKGROUND: Tea (Camellia sinensis (L). O. Kuntze) is known as the oldest, mild stimulating caffeine containing non-alcoholic beverage. One of the major threats in south Asian tea industry is the blister blight leaf disease (BB), caused by the fungus Exobasidium vexans Masse. SSR DNA marker EST SSR 073 is used as a molecular marker to tag blister blight disease resistance trait of tea. The amino acid sequences were derived from cDNA sequences related to EST SSR 073 of BB susceptible (TRI 2023) and BB resistant (TRI 2043) cultivars. An attempt has been made to understand the structural characteristics and variations of EST SSR 073 locus that may reveal the factors influencing the BB resistance of tea with multiple bioinformatics tools such as ORF finder, ExPasy ProtParam tools, modeler V 9.17, Rampage server, UCSF-Chimera, and HADDOCK docking server. RESULTS: The primary, secondary, and tertiary structures of EST SSR 073 coding protein were analyzed using the amino acid sequences of both BB resistant TRI 2043 and BB susceptible TRI 2023 tea cultivars. The coding amino acid sequences of both the cultivars were homologous to photosystem I subunit protein (PsaD I) of Pisum sativum. The predicted 3D structures of proteins were validated and considered as an acceptable overall stereochemical quality. The BB resistant protein showed CT repeat extension and did not involve in topology of the PsaD I subunit. The C terminal truncation of BB resistance caused the formation of hydrogen bonds interacting with PsaD I and other subunits of photosystem I in the modeled three-dimensional protein structure. CONCLUSIONS: Camellia sinensis EST 073 SSR motif coding protein was identified as the PsaD I subunit of photosystem I. The exact mechanism of PsaD I conferring the resistance for blister blight in tea needs to be further investigated.

Anterior segment imaging-based subdivision of subjects with primary angle-closure glaucoma.

PurposeThe purpose of this study was to identify whether it was possible to subdivide subjects with primary angle-closure glaucoma (PACG) based on anterior segment optical coherence tomography (ASOCT) imaging, and to determine the characteristics of such subgroups.MethodsWe evaluated 210 subjects with PACG. All subjects underwent gonioscopy and ASOCT imaging. Customized software was used to measure ASOCT parameters. An agglomerative hierarchical clustering method was first used to determine the optimum number of parameters to be included in the determination of subgroups. Then, the best number of subgroups was determined using Akaike Information Criterion (AIC) and Gaussian Mixture Model (GMM) methods.ResultsThe mean age of the subjects was 67.9 years, and 53.3% were female. Following the hierarchical clustering, four parameters (iris area, anterior chamber depth (ACD), anterior chamber width (ACW), and lens vault (LV)) were chosen to be representative of related parameters. The optimal number of subgroups using GMM analysis and AIC was 3. Subgroup 1 (N=89; 42.4%) was characterized by a large iris area, subgroup 2 (N=24; 11.4%) by a large LV and a shallow ACD, whereas subgroup 3 (N=97; 46.2%) displayed only intermediate values across iris area, LV, and ACD.ConclusionsWe identified three distinct subgroups of PACG subjects based on ASOCT imaging.

Imaging of blebs after phacotrabeculectomy with Ologen collagen matrix implants.

OBJECTIVE: To analyse blebs of phacotrabeculectomies performed with Ologen collagen implants (ProTop & MediKing, Taipei, Taiwan) and to compare these with blebs of mitomycin C (MMC)­augmented phacotrabeculectomies. METHODS: 33 participants underwent phacotrabeculectomy with Ologen implants, and 33 controls underwent phacotrabeculectomy with MMC. Blebs were analysed for height and area using anterior segment optical coherence tomography (ASOCT) at 30, 60 and 90 days after surgery and were also graded clinically with the Moorfields bleb grading system (MBGS) 60 days after surgery. RESULTS: With ASOCT, there was no difference in mean bleb height at 30 and 60 days, but at 90 days, bleb height was lower in the Ologen group (Ologen vs MMC, 0.74±0.20 vs 1.00±0.28 mm, p<0.001). There was no difference in mean bleb area at 30, 60 or 90 days. Mean reduction in intraocular pressure at 90 days was greater in the MMC group (Ologen vs MMC, 2.18±4.93 vs 8.00 ±7.60 mm Hg, p<0.001). At 90 days, the Ologen implants were visible in ASOCT images in 13 (39.4%) of 33 participants. With the Moorfields bleb grading system at 60 days, there was no difference in maximal bleb area score between the groups, but bleb height score was lower (Ologen vs MMC, 1.53±0.51 vs 1.81±0.59, p=0.05) and central bleb vascularity score was higher in the Ologen group (3.88±0.55 vs 2.91±0.59, p<0.001). CONCLUSIONS: Within 3 months of surgery, mean bleb height was lower in the Ologen blebs compared with the MMC blebs. The Ologen implants had not degraded in a third of eyes.

Reactivity and isotype profiling of monoclonal antibodies using multiple antigenic peptides.

The characterisation of monoclonal antibodies (MAbs) is essential for the development of assay systems particularly where antigens have been developed using synthetic peptides. Indeed some peptide-carrier conjugates fail to induce immune responses and may not generate antibodies that bind to native protein. As an alternative to peptide-carrier conjugates, multiple antigenic peptides (MAPs) have been used for immunization strategies, but with little regard to the characteristics of the MAbs produced. In this study, we used 3 MAPs of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) to immunise BALB/c mice. Overall, the polyclonal antibody responses from tail bleeds showed that MAPs evoked B-cell responses. However, on screening 144 hybridomas, 24 MAb supernatants exhibited weak to moderate reactivity in enzyme-linked immunosorbant assay (ELISA) and against cell cytospin preparations (B95.8 and AG876 LCL), respectively. Isotype profiling of hybridoma supernatants also showed that 11 out of 24 were IgM. Further characterization of 6 MAbs in Western blotting showed reactivity to recombinant LMP1 and only one MAb (B28D) showed weak reactivity to the malignant cells (Hodgkin/Reed-Sternberg; HRS cells) of an EBV+ Hodgkin's lymphoma using paraffin-embedded tissue. It is probable that these MAPs failed to augment T-cell help and contributed to the production of low affinity (IgM) antibodies. These observations may be of importance to future immunization strategies, where MAPs are used in the production of monoclonal reagents.

Molecular investigations implicate human endogenous retroviruses as mediators of anti-retroviral antibodies in autoimmune rheumatic disease.

Polymerase chain reaction using specific primers, failed to detect HTLV-I amplicons in patients with rheumatic diseases previously shown to possess antibodies to retroviral products. However, by employing broad spectrum oligonucleotide primers, 135 bp amplicons were generated from peripheral blood mononuclear cells and synovial fluid cells. Subsequent cloning and DNA sequencing revealed homology to a number of exogenous and human endogenous retroviruses (HERVs). Furthermore, in combining the presence of type B and C related endogenous retroviruses, a significant association (p=0.014) was apparent for chronic autoimmune rheumatic diseases as compared to controls. Reverse transcription polymerase chain reaction of RNA derived from patients, healthy controls and cell lines (U937, BJAB, human endothelial lung fibroblasts) demonstrated ubiquitous expression of HERV-K10 and RTVL-H2. Furthermore messenger RNA expression of HERV-K10 was enhanced in fibroblasts infected with human cytomegalovirus. It is plausible that subsequent production of HERV peptides could explain the presence of anti-retroviral antibodies in cohorts of patients with autoimmune rheumatic diseases.

Polarized distribution of Bcr-Abl in migrating myeloid cells and co-localization of Bcr-Abl and its target proteins.

Bcr-Abl plays a critical role in the pathogenesis of Philadelphia chromosome-positive leukemia. Although a large number of substrates and interacting proteins of Bcr-Abl have been identified, it remains unclear whether Bcr-Abl assembles multi-protein complexes and if it does where these complexes are within cells. We have investigated the localization of Bcr-Abl in 32D myeloid cells attached to the extracellular matrix. We have found that Bcr-Abl displays a polarized distribution, colocalizing with a subset of filamentous actin at trailing portions of migrating 32D cells, and localizes on the cortical F-actin and on vesicle-like structures in resting 32D cells. Deletion of the actin binding domain of Bcr-Abl (Bcr-AbI-AD) dramatically enhances the localization of Bcr-Abl on the vesicle-like structures. These distinct localization patterns of Bcr-Abl and Bcr-Abl-AD enabled us to examine the localization of Bcr-Abl substrate and interacting proteins in relation to Bcr-Abl. We found that a subset of biochemically defined target proteins of Bcr-Abl redistributed and co-localized with Bcr-Abl on F-actin and on vesicle-like structures. The co-localization of signaling proteins with Bcr-Abl at its sites of localization supports the idea that Bcr-Abl forms a multi-protein signaling complex, while the polarized distribution and vesicle-like localization of Bcr-Abl may play a role in leukemogenesis.

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Atrial natriuretic peptide replacement therapy in rats subjected to biatrial appendectomy.

The postoperative fluid retention found in some patients after the Cox maze procedure has been attributed to surgically induced loss of atrial natriuretic peptide. We postulated that exogenous atrial natriuretic peptide could reverse this antidiuresis. A rat model was used to investigate this hypothesis. In group I, the sham group, the atrial appendages were left intact and the animals were then subjected to a fluid challenge equivalent to 1% of the animal's body weight. In group II, after biatrial appendectomy, the animals were subjected to a fluid challenge similar to that in group I. Animals in group III underwent the same protocol as that for group II plus intravenous administration of atriopeptin III at varying concentrations. Urine output and plasma atrial natriuretic peptide levels were significantly decreased after biatrial appendectomies (p < or = 0.01). Urine output returned to control levels after biatrial appendectomies with low-dose atrial natriuretic peptide infusion (0.5 pmol/min = 25.5 pg/min), although circulating atrial natriuretic peptide levels were lower. Urine output and plasma atrial natriuretic peptide levels increased with atrial natriuretic peptide infusions between 0.5 and 50 pmol/min. Heart rate and mean blood pressure did not vary significantly with atrial natriuretic peptide infusions. Thus atrial natriuretic peptide can be used effectively in low doses to induce a diuresis after biatrial appendectomies. Atrial natriuretic peptide may have clinical application after the Cox maze procedure.

Leucocyte esterase test as rapid screen for non-gonococcal urethritis.

The two standard tests for the initial diagnosis of non-gonococcal urethritis (NGU), microscopic examination of gram stained urethral smears and the two glass urine test, have the disadvantage of being insensitive and subjective. The leucocyte esterase test detects enzymes specific to polymorphonuclear leucocytes and can therefore be used as a sensitive indicator of pyuria. This study sought to evaluate its use as a rapid, sensitive, and non-subjective method of screening for NGU. Of the 81 men with urethral symptoms in the study group, 26 had more than 5 polymorphonuclear leucocytes per high power field (x 1000) and all 26 were leucocyte esterase test positive; whereas 55 had fewer than 5 polymorphonuclear leucocytes per high power field, but 29 (53%) of them had a positive leucocyte esterase test result. In addition, 25 patients in the study group yielded Chlamydia trachomatis on culture. Of these 25, 24 (96%) were leucocyte esterase test positive, whereas only 11 (44%) were Gram stain positive. All 40 patients in the control group (without urethral symptoms or signs) were leucocyte esterase test negative. The leucocyte esterase test is thus a rapid, sensitive, and non-subjective screening aid in the diagnosis of NGU.

Use of Kova-Slide II with grid and uncentrifuged segmented urine specimens in the diagnosis of nongonococcal urethritis: a quantitative technique.

An attempt was made to use uncentrifuged segmented urine specimens and Kova-Slide II with grid as a quantitative technique for the diagnosis of nongonococcal urethritis. Of the 100 men with urethral symptoms, 54 had fewer than five polymorphonuclear leukocytes per high-power field (x1000) in their gram-stained urethral smear. On the basis of examination of segmented urine and Kova-Slide test, 41 of these 54 patients were diagnosed as having nongonococcal urethritis. None of the 13 patients considered to be free of urethritis on the basis of segmented urine tests had a Chlamydia trachomatis--positive culture, and all became asymptomatic without treatment. All 46 patients who had more than five polymorphonuclear leukocytes per high-power field (x1000) also had positive segmented urine tests. The rate of isolation of C. trachomatis was similar for both groups of tests. This study revealed that patients with lower polymorphonuclear leukocyte counts in the first 10 ml of urine passed (voided-bladder urine 1) also had cultures positive for C. trachomatis. None of the 50 patients in the control group had polymorphonuclear leukocytes in either voided-bladder urine 1 or in the midstream specimen (voided-bladder urine 2). All controls had cultures negative for C. trachomatis.