Selection of stable reference genes for qPCR expression of Colletotrichum lindemuthianum, the bean anthracnose pathogen.

Phaseolus vulgaris L., commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat". However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), ß-tub (ß-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha-l-Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF, GAPDH, and ß-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes.

Morphological, pathogenic and genetic diversity in Diplodia seriata associated with black rot canker of apple in India.

Apple cankers are extremely destructive diseases threatening the global apple industry through direct and indirect losses. The population structure of the pathogens is of paramount significance for the development of efficient management strategies. Therefore, phenotypic, pathogenic, and genetic diversity of Diplodia seriata causing black rot canker of apple was investigated in this study. All the isolates were included for investigating the in vitro mycelial growth, conidial dimensions, and pathogenic variability on two-year-old potted apple seedlings. The ISSR approach was used to investigate the molecular diversity of D. seriata. Mycelial growth rates were found to vary significantly amongst the isolates; however, there were no major variations seen between the different geographical groupings of isolates. Pathogenicity tests revealed variations in the size of cankers among the isolates indicating the presence of virulence variability. The isolates were segregated into three virulence groups based on canker length. The Bayesian analyses of ISSR data divided the isolates into two genetic clusters. The genetic clustering of the isolates revealed no relationship with geographical origin of the isolates. Furthermore, no direct relationship of genetic clustering was observed with morphological or pathogenic variability. The ISSR primers revealed very high level of variability in D. seriata; however, no distinct populations of the pathogen existed which is an indication of high level of gene flow between the diverse geographical populations. According to our knowledge, this is the first thorough investigation on the diversity of D. seriata associated with apple black rot canker in India.

Genome-wide characterization and development of SSR markers for genetic diversity analysis in northwestern Himalayas Walnut (Juglans regia L.).

In the present study, we designed and validated genome-wide polymorphic SSR markers (110 SSRs) by mining the walnut genome. A total of 198,924 SSR loci were identified. Among these, successful primers were designed for 162,594 (81.73%) SSR loci. Dinucleotides were the most predominant accounting for 88.40% (175,075) of total SSRs. The SSR frequency was 377.312 SSR/Mb and it showed a decreasing trend from dinucleotide to octanucleotide motifs. We identified 20 highly polymorphic SSR markers and used them to genotype 72 walnut accessions. Over all, we obtained 118 alleles that ranged from 2 to 12 with an average value of 5.9. The higher SSR PIC values indicate their robustness in discriminating walnut genotypes. Heat map, PCA, and population structure categorized 72 walnut genotypes into 2 distinct clusters. The genetic variation within population was higher than among population as inferred by analysis of molecular variance (AMOVA). For walnut improvement, it is necessary to have a large repository of SSRs with high discriminative power. The present study reports 150,000 SSRs, which is the largest SSR repository for this important nut crop. Scientific communities may use this repository for walnut improvement such as QTL mapping, genetic studies, linkage map construction, and marker-assisted selection. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03563-6.

Unravelling rutin content of tartary buckwheat of north western Himalayas and insights into nucleotide polymorphisms in PAL gene to infer the associations with rutin biosynthesis.

Buckwheat (Fagopyrum spp.) has immense nutritional and nutraceutical potential. All the plant parts of buckwheat possess various metabolites, such as rutin, quercetin, vitexin etc. The high content of rutin in this pseudo cereal crop strongly adapts it to grow under adverse environments. In the present study 50 germplasm lines of Fagopyrum tataricum were used for estimation of seed endosperm rutin content through HPLC. Furthermore, molecular analysis of PAL gene (Phenylalanine Ammonia Lyase), an upstream gene in rutin biosynthesis pathway was targeted for detection of SNPs to understand the variations in the concentrations of seed endosperm rutin content, among tartary buckwheat genotypes with highest and lowest seed endosperm rutin content. Three primer pairs were employed for amplification of PAL gene for F. tartaricum (covering whole gene) followed by sequencing. Rutin concentration in seed endosperm of F. tartaricum ranged from 194.86 to 1403.22 ppm with an average of 617.06 ppm. Highest rutin concentration was found in genotype BWZ90 and lowest in BWZ16. Significant variations were observed in the seed endosperm rutin content among the genotypes of tartary buckwheat. Furthermore, alignment of PAL gene sequences of genotypes with high seed endosperm rutin content and low seed endosperm rutin content revealed variations at 21 polymorphic sites. The amino acid sequences obtained from the nucleotide sequences were also aligned and the variations were detected at 19 positions. The putative protein structure showed conformational changes among predicted proteins from two contrasting genotypes for endosperm rutin content. We here established an inventory of seed endosperm rutin content of tartary buckwheat. This study also provided insights about role of these SNPs in rutin biosynthesis. Furthermore, this information can be used for breeding buckwheat for high metabolite contents. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03218-y.

Dynamics, phylogeny and phyto-stimulating potential of chitinase synthesizing bacterial root endosymbiosiome of North Western Himalayan Brassica rapa L.

The less phytopathogen susceptibility in Himalayan Brassica rapa L. has made it an exceptional crop eluding synthetic pesticide inputs, thereby guarantying economically well-founded and ecologically sustainable agriculture. The relevance of niche microflora of this crop has not been deliberated in this context, as endosymbiosiome is more stable than their rhizosphere counterparts on account of their restricted acquaintance with altering environment; therefore, the present investigation was carried out to study the endophytic microfloral dynamics across the B. rapa germplasm in context to their ability to produce chitinase and to characterize the screened microflora for functional and biochemical comportments in relevance to plant growth stimulation. A total of 200 colonies of bacterial endophytes were isolated from the roots of B. rapa across the J&K UT, comprising 66 locations. After morphological, ARDRA, and sequence analysis, eighty-one isolates were selected for the study, among the isolated microflora Pseudomonas sp. Bacillus sp. dominated. Likewise, class γ-proteobacteria dominated, followed by Firmicutes. The diversity studies have exposed changing fallouts on all the critical diversity indices, and while screening the isolated microflora for chitinase production, twenty-two strains pertaining to different genera produced chitinase. After carbon source supplementation to the chitinase production media, the average chitinase activity was significantly highest in glycerol supplementation. These 22 strains were further studied, and upon screening them for their fungistatic behavior against six fungal species, wide diversity was observed in this context. The antibiotic sensitivity pattern of the isolated strains against chloramphenicol, rifampicin, amikacin, erythromycin, and polymyxin-B showed that the strains were primarily sensitive to chloramphenicol and erythromycin. Among all the strains, only eleven produced indole acetic acid, ten were able to solubilize tricalcium phosphate and eight produced siderophores. The hydrocyanic acid and ammonia production was observed in seven strains each. Thus, the present investigation revealed that these strains could be used as potential plant growth promoters in sustainable agriculture systems besides putative biocontrol agents.

Phaseolus vulgaris-Colletotrichum lindemuthianum Pathosystem in the Post-Genomic Era: An Update.

Phaseolus vulgaris-Colletotrichum lindemuthianum is one among the oldest host and pathogen interface. Researchers have taken painstaking efforts across the world for understanding the dialogue during early and late phases of interaction. Collectively, these efforts resulted in the deluge of information that helped the researchers to underpin the interface. The latest molecular biology techniques furnished novel detection methods for the anthracnose pathogen, refined the understanding of pathogen population dynamics, and provided the insights on co-evolutionary common bean resistance and C. lindemuthianum virulence dynamics. One of the important breakthroughs came when the Phaseolus vulgaris and its corresponding anthracnose pathogen (C. lindemuthianum) genomes were decoded in 2014 and 2017, respectively. Availability of both the genomes yielded a significant genomic information that helped bean communities to fine map the economically important traits and to identify the pathogenicity determinants and effector molecules. The interface is in a continuous development as knowledge of the anthracnose resistance genes, their precise physical locations, and the identification of effector proteins; the fungus arsenals are being routinely updated. Hence, we revisited the interface and tried to provide an overview of host pathogen dialogue in the genomic era. Additionally, we compiled the sporadic information on this pathosystem from India and provided its futuristic road map to shape its research in the world and northern India, the major dry bean area in the country.

Optimizing the Agrobacterium tumefaciens-mediated transformation conditions in Colletotrichum lindemuthianum: a step forward to unravel the functions of pathogenicity arsenals.

Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes bean anthracnose and it is rated among the top 10 important diseases infecting beans. Currently our knowledge on molecular mechanisms underlying C. lindemuthianum pathogenesis is limited. About five pathogenicity genes have been identified in C. lindemuthianum using Restricted Enzyme Mediated Integration and the transformation using Agroinfection has not been optimized. In this study, a series of experiments were conducted to optimize the key parameters affecting the Agrobacterium tumefaciens-mediated transformation for C. lindemuthianum. The transformation efficiency increased with increase in spore concentration and co-cultivation time. However, the optimum conditions that yielded significant number of transformants were 106 ml-1 spore concentration, co-cultivation time of 72 h, incubation at 25°C and using a cellulose membrane filter for the co-cultivation. The optimized protocol resulted in establishment of large mutant library (2400). A few mutants were melanin deficient and a few were unable to produce conidia. To determine the altered pathogenicity, two new approaches such as detached leaf and twig techniques proved reliable and require fewer resources to screen the large mutant libraries in a short time. Among the 1200 transformants tested for virulence, 90% transformants were pathogenically similar to wild type (race 2047), 96 and 24 were reduced and impaired, respectively. The altered avirulent transformants can prove vital for understanding the missing link between growth and developmental stages of pathogen with virulence. This platform will help to develop strategies to determine the potential pathogenicity genes and to decipher molecular mechanisms of host-pathogen interactions in more detail.

Population structure of Venturia inaequalis, a hemibiotrophic fungus, under different host resistance specificities in the Kashmir valley.

Venturia inaequalis is a notorious fungal pathogen and show classical gene for gene interaction with its apple host. Neutral markers provide clues about history, evolutionary potential, genetic diversity and population structure of V. inaequalis. The genetic diversity and population structure of fungus indicates that the pathogen is highly diverse with the capacity to breach the scab resistance genes. In the present study, we collected 108 V. inaequalis isolates from three apple cultivars differing in Rvi1 resistance gene. Based on the AMOVA, the variation was mostly distributed among the isolates, providing evidence of non-existence of subpopulation in orchards thus founder population is difficult to arise in Kashmir apple orchards. Pair wise genetic differentiation is less due to regular occurrence of gene flow between the populations residing on different orchard as infected material is transported without stringent quarantine measures. Based on principal coordinate analysis and clustering algorithm as implemented in STRUCTURE, we observed admixture between the two subpopulations, which is quite low, suggesting the existence of pre-zygotic and post-zygotic barriers to gene flow and we cannot rule out the existence of other structures shared by accessions belonging to different varieties. Due to the continuous increase in introduction and monoculture of apple varieties, mixed orchard with different host resistance specificities are more suitable for managing the apple scab in Kashmir valley.

Why I use both prospective randomized trials and registry data when choosing the personalized treatment of an AAA patient.

Randomized controlled trials (RCTs) have been the core of level 1 data in medical and surgical science for at least the last three decades. However, frequently patient selection is very narrow, anatomic criteria do not match real-world experience, and much of the work is done in selected academic centers. We use RCTs to help explain the rational for intervention and then rely on longitudinal registries and single center data to give the patients a real-world expectation concerning outcomes and complications in our hands.

A comparison between methadone and buprenorphine for perioperative analgesia in dogs undergoing ovariohysterectomy.

OBJECTIVE: To investigate whether preoperative methadone provides superior perioperative analgesia compared to buprenorphine in dogs undergoing ovariohysterectomy. METHODS: Eighty female dogs were recruited to an assessor-blinded, randomised, clinical trial. Dogs received a premedication dose of 0·05 mg/kg acepromazine or 10 µg/kg medetomidine combined with either 0·3 mg/kg methadone or 20 µg/kg buprenorphine intramuscularly. Anaesthesia was induced with propofol and maintained with isoflurane. Pain was assessed using two scoring schemes (a dynamic interactive visual analogue scale and the short form of the Glasgow Composite Pain Scale) before premedication, 30 minutes later and every hour for 8 hours after premedication. If indicated, rescue analgesia was provided with methadone. Meloxicam was administered after the final assessment. The area under the curve for change in pain scores over time and the requirement for rescue analgesia were compared between groups. RESULTS: Groups premedicated with buprenorphine had significantly higher pain scores than those premedicated with methadone. There was no interaction between opioid and sedative for any outcome measure. Rescue analgesia was required by significantly more dogs premedicated with buprenorphine (45%) than that of methadone (20%). CLINICAL SIGNIFICANCE: At the doses investigated, methadone produced superior postoperative analgesia compared to buprenorphine in dogs undergoing ovariohysterectomy.

Carotid interventions (CEA and CAS) in acute stroke patients: which procedure on which patient.

Treatment of carotid bifurcation disease in patients presenting with acute stroke has been a controversial issue over the past four decades. Classically, patients were asked to wait four to six weeks before intervention was entertained in order for the brain to stabilize and the risks of intervention to be minimized. Unfortunately, up to 20% of patients will have a secondary event after their index event and the window of opportunity to save, potentially salvageable ischemic tissue will be missed. Early reports had demonstrated poor results with intervention. However, more recently, institutions such as ours have demonstrated excellent result in early intervention in patients who present with stable mild to moderate stroke with an NIH stroke scale less than 15 and preferably less than 10, present with stroke and ipsilateral carotid artery lesion of 50% or greater. Also more recently, we have been aggressively treating patients with larger ulcerative plaques even if the stenosis approaches 50%. In our and others experiences, patients who are treated at institutions that have comprehensive stroke centers (CSCs) where they have a multidisciplinary system that consists of vascular surgeons, neuro interventionalists, stroke neurologists, specifically trained stroke nursing staff and a neuro intensive ICU have had optimal results. Early assessment, diagnosis of stroke with recognition of cause of embolization is mandatory but patient selection is extremely important; finding those patients who will benefit the most from urgent intervention. Most studies have demonstrated the benefit of carotid endarterectomy in these patients. More recent studies have demonstrated acceptable results with carotid stenting, especially in smaller lesions, those less than 1.2 centimeters. Early intervention should be avoided in most patients who are obtunded or with an NIH stroke scale greater than 15 or who do not have any "brain at risk" to salvage. These patients may be better served by being treated medically than those small group of patients that do have some improvement may benefit from interval intervention.

Platelet function defects in adolescents with heavy menstrual bleeding.

Platelet function defects (PFD) are reported to occur frequently in adult women with heavy menstrual bleeding (HMB). Few studies on adolescent HMB report varying incidence rates (2-44%) for PFD. We reviewed our institutional experience in detecting and managing PFD in adolescent HMB. Postmenarchial girls and adolescents with HMB seen at our institution undergo a comprehensive bleeding disorder work-up by paediatric haematology and paediatric gynaecology providers. Whole blood platelet aggregometry (WBPA) is performed as a second tier test after excluding thrombocytopaenia, coagulation factor deficiencies and Von Willebrand disease (VWD). We retrospectively reviewed the medical records of adolescents with HMB seen between June 2009 and November 2010, as approved by the Institutional Review Board. Patient demographics, clinical features, laboratory results, therapy details and patient outcome information were analysed. Overall, 114 postmenarchial girls and adolescents with HMB were evaluated; 68 patients (59%) had WBPA study performed. Nineteen patients (28%) had at least one aggregation or secretion defect; 12 (18%) had two or more such defects. Treatment included hormonal therapy (13/19; 68%), antifibrinolytic agents (8/19; 42%) and intra-nasal DDAVP (3/19; 16%). Thirteen patients (81%) had improved outcome (median follow-up--15.6 months; range of 1-66 months). In this study, PFD were identified in nearly one-third of girls with HMB, with the majority of these having two or more defects as identified by WBPA. Further prospective studies are needed to better define the prevalence and address appropriate management of HMB and other bleeding complications of PFD in adolescents.

Morphological, pathological and molecular variability in Botryodiplodia theobromae (Botryosphaeriaceae) isolates associated with die-back and bark canker of pear trees in Punjab, India.

Thirteen isolates of Botryodiplodia theobromae collected from pear varieties grown in various regions of Punjab were studied for morphological, pathological and molecular characterization. The mycelial growth of B. theobromae isolates was classified as fluffy or depressed, uniform to irregular and cottony white turning to black. Colony growth rate varied from 19.1 to 24.9 mm per day. Pycnidia were produced either on the edge, centered or scattered on Petri dishes after 20 to 34 days of incubation. Pycnidia and pycnidiospores ranged in size from 118.0 to 240.0 microm and 14.5-35.5 x 6.5-14.5 microm, respectively. Lesion length produced by different isolates ranged from 1.9-7.2 x 0.8-3.3 cm with 49.4-90.9% infection. Using nine SSR and seven RAPD markers, amplified DNA bands ranged from 0.2 to 1.5 and 0.18 to 2.0 kb, respectively. Polymorphism information content values ranged from 0.44 to 0.71 and 0.63 to 0.93 for SSR and RAPD markers, respectively. A dendrogram based on molecular data, grouped the isolates into three major clusters with 65 to 79.5% genetic similarity; most of the isolates showed variety-specific grouping. The isolates prevalent on pear cultivars 'Patharnakh' and 'Baggugosha' in Ludhiana, Amritsar and Hoshiarpur districts were found to have a high degree of similarity; these isolates were also considerably distant from mango isolates and from other isolates on other pear cultivars. The isolates from cultivars Punjab Beauty, LeConte and Kieffer also had a high degree of similarity. Isolates from cultivar Smith were different from other pear isolates but showed more similarity with mango isolates.

The applied physiology of cough.

The cough reflex is such an important defense mechanism of the respiratory tract that a thorough knowledge of its applied physiology in respiratory disease is essential for clinicians. It is a well-integrated reflex, which has afferent limb consisting of receptors and afferent nerves, the central cough center in the brainstem and the efferent limb consisting of motor nerves supplying the muscles of coughing. The cough process consists of inspiratory phase, compressive phase and expiratory phase. Stimuli that can initiate the cough process can be central or peripheral in the lungs or outside the lungs. The important function of the cough reflex is to maintain the airways and alveoli clear and healthy. The mechanisms by which it achieves this are (i) high velocity of expiratory gas flows produced in the expiratory phase (ii) the compression of the lungs and airways by high positive pleural pressure generated in the compressive phase. Cough threshold can be determined by giving acetic acid, citric acid or capsaicin inhalation challenge test. This has practical application in clinical research. The pathophysiological basis of different types of cough encountered in clinical practice is discussed.

Effects of an aqueous extract of processed bidi tobacco on the growth of hamster tracheal epithelial cells.

Inhalation of tobacco dust is responsible for elevated genotoxicity and pulmonary ailments in workers engaged in processing tobacco for the manufacture of bidis, the Indian version of cigarettes. Tracheal tissue being the major site of interaction with tobacco dust, the effects of different concentrations of an aqueous extract of bidi tobacco (ATE) on the growth of a hamster tracheal epithelial cell line (HTE) were investigated. Colony forming efficiency assay revealed that ATE was cytotoxic only at the highest concentration of 5.0 mg/ml. In cultures treated with 1.25 mg/ml ATE, the cell doubling time and growth rate were similar to that of the controls, while a significant increase in cell doubling time (29.4+/-0.3 h vs 14.0+/-3.75 h, P<0.001) was observed at 2.5 mg/ml ATE concentration. Exposure of HTE cells to the non-toxic ATE concentration of 2.5 mg/ml was found to stimulate ornithine decarboxylase (ODC) activity, incorporation of [3H] methyl thymidine into DNA and increase in the S phase fraction was seen by flow cytometry. However, a 56% reduction in the growth rate of cultures treated with 2.5 mg/ml ATE was related to the prolongation of the traverse of cells through S phase. ATE-induced growth suppression was reversed when cultures were grown in ATE-free medium or upon repeated exposure to ATE. The findings suggest that increased tracheal cell proliferation induced by chronic inhalation of tobacco dust may contribute to the development of pulmonary disorders and possibly neoplasia in exposed individuals.

Childhood asthma--office management.

Childhood asthma is a major problem in office practice. For an acute life threatening attack (which is indicated by presence of severe distress, pulsus paradoxus, oxygen saturation less than 93%, cyanosis, peak expiratory flow rate of less than 50% of predicted) child should be directly admitted in intensive care unit. If it is a non life threatening attack the child can be managed in the office. Initially, the child should be given b2 agonists by inhalation route with either metered dose inhaler (MDI), MDI with spacer or nebuliser. It there is severe bronchospasm or inhalation therapy is not possible then epinephrine of b2 agonists may be given subcutaneously. The medications can be repeated 2-3 times. If response is adequate the child may be sent home on b2 agonist by oral or inhalation route at an interval of 406 hours. In case of inadequate response the child is started on oral or parenteral corticosteroids. Even after steroids if inadequate response the child is started on intravenous theophylline. Once the acute exacerbation is controlled the child is assessed for starting maintenance therapy. For this purpose his illness is graded from stage I to V depending on the severity. For stage I and II b2 agonists are prescribed as and when required. For stage III sodium cromoglycate by inhalation should be prescribed. For stage IV inhalation steroids in usual doses and for stage V inhalation steroids in higher doses are prescribed along with a minimum dose of oral steroids is added. For symptomatic control slow release theophylline or long acting b2 agonists may be added along with maintenance therapy as and when required. Apart from medications a proper education of parents and patients is necessary to improve the outcome of asthma by increasing the compliance and better control of environment.

Pulmonary hypertension associated with portal hypertension in a child with Williams syndrome--a case report.

A 14-year-old white female with Williams syndrome and portal hypertension presented in shock; at autopsy she was found to have grade II to VI vascular changes of pulmonary hypertension. This case demonstrates the association of portal hypertension and pulmonary hypertension in a pediatric patient.

Endoscopic balloon dilation of esophageal strictures in children.

A total of 17 patients, ages 3 weeks to 14 1/2 years, had 20 esophageal strictures develop after repair of esophageal atresia (9 strictures); primary gastroesophageal reflux (3 strictures); Nissen fundoplication (4 strictures); epidermolysis bullosa congenita dystrophica (1 stricture); congenital esophageal stenosis (2 strictures); or colonic interposition (1 stricture). These strictures were treated with 132 endoscopic balloon catheter dilations (average, 6.6/patient, range, 1 to 24) during a period of 42 months (average, 8 months; range, 2 to 42 months). Fifteen of the 17 patients are now asymptomatic. Thirteen of the 17 patients had documented reflux esophagitis and were also medically treated. The two residually symptomatic patients included a patient with severe proximal esophagitis secondary to epidermolysis bullosa congenita dystrophica who had only a temporary response to dilations and another patient with a tight Nissen fundoplication who did not improve after dilation but is now asymptomatic after corrective surgery. The 10 patients with tracheoesophageal fistula repair were asymptomatic within 3 to 21 months (average, 10.8 months). Only one patient had perforation develop as a result of the procedure. Sixteen of 17 patients had the procedures performed as outpatients. All patients were given oral feedings throughout the course of dilation and had normal growth.