Ralstonia solanacearum differentially modulates soil physicochemical properties and rhizospheric bacteriome of resistant and susceptible tobacco cultivars.

Ralstonia solanacearum is a devastating soilborne pathogen which poses significant yield and economic losses to tobacco production globally. The impact of R. solanacearum on rhizosphere bacteriome and soil physicochemical characteristics in resistant and susceptible tobacco cultivars is poorly understood. This study aims to determine the effect of R. solanacearum on soil physicochemical parameters and rhizosphere bacteriome of resistant (K326) and susceptible (Hongda) tobacco cultivars at various growth stages. Results demonstrated that the contents of available potassium and phosphorus, as well as soil pH were significantly increased in K326 soils (CK and T2) compared with Hongda (T1) after 21, 42, and 63 days post-inoculation (dpi) of R. solanacearum except for available nitrogen which showed an opposite trend. The qPCR results showed a significant decrease in R. solanacearum population in rhizosphere of K326 (T2) compared to the Hongda (T1) at 21 and 63 dpi than that after 42 dpi. The rhizosphere bacteriome analysis through 16S rRNA amplicon sequencing revealed that rhizosphere bacterial community composition was significantly different between two tobacco cultivars (Hongda and K326) and this effect was more prominent after 63 dpi (93 days after post-transplantation), suggesting that each cultivar recruits a unique set of bacterial communities. There was no obvious difference observed in the rhizosphere bacteriome of CK (K326) and T2 (K326), which might be attributed to the same genetic makeup and inherent resistance of K326 to bacterial wilt infection. Analysis of co-occurrence networks revealed that the microbial network in T1 (Hongda) was more complex than those in T2 (K326) and CK (K326), while the networks in CK and T2 were almost identical. The present research highlights the time-course relationship between environmental factors and rhizosphere bacteriome of tobacco cultivars showing different levels of resistance against R. solanacearum. Conclusively, studying the plant-soil-microbe interaction system in susceptible and resistant tobacco cultivars may enable us to develop effective integrated disease control plans for the healthy production of tobacco crops.

Comparison of nebulised 3% hypertonic saline with ipratropium bromide in treatment of children with bronchiolitis: a randomized control trial.

Introduction: Several drugs are in use for nebulization in children with acute bronchiolitis and no study has yet been conducted to compare the treatment outcome of nebulized ipratropium bromide and nebulized 3% hypertonic saline in Pakistan. Objective: The objective was to compare the treatment outcome of nebulized hypertonic saline and ipratropium bromide in children with acute bronchiolitis. Setting: Department of Pediatrics. Study duration: October 2019 to March 2020. Subjects and methods: A total of one hundred (n=100) children of either sex diagnosed with acute bronchiolitis were enrolled and randomized either to be nebulized with 3% hypertonic saline or ipratropium bromide. Outcomes were assessed in terms of respiratory rate, heart rate, and SpO2 and respiratory distress assessment instrument score at different time intervals, length of hospital stay, and need of admission. Results: Respiratory rate and SPO2 improved significantly at 60 min and 24 h, respiratory distress assessment instrument improved significantly at 30 min, 60 min, and 24 h after the treatment in patients who were nebulized with hypertonic saline when compared to those nebulized with ipratropium bromide. The length of hospital stay was significantly shorter (2.63 vs. 3.82 days, P=0.008) and a lesser number of patients needed hospital admission (22% vs. 44%, P=0.019) in patients who were nebulized with hypertonic saline when compared to those nebulized with ipratropium bromide. Conclusions: Nebulization with 3% hypertonic saline resulted in significant improvement in symptoms, a shorter duration of hospital stay, and a lesser number of hospital admissions as compared to nebulization with ipratropium bromide in children with acute bronchiolitis.

Bacillus amyloliquefaciens AK-12 Helps Rapeseed Establish a Protection against Brevicoryne brassicae.

Aphids are a serious threat to rapeseed (Brassica napus L.) production, and cause unmanageable loss. Therefore, effective prevention and management strategies are urgently required to avoid losses. Bacillus amyloliquefaciens AK-12 isolated from a dead aphid with aphicidal activity was tagged with a green fluorescent protein through a natural transformation. The transformed strains were checked for stability and growth, and the best-performing strain was tested for its colonization inside and outside the rapeseed plant. The stability of AK-12-GFP reached more than 95%, and the growth curve was consistent with that of AK-12. After 30 days of treatment, the colonization of 1 × 106 CFU/g was recorded in rapeseed leaves. Interestingly, AK-12 reduced the aphid transmission rate compared with the control and improved the growth of the rapeseed seedlings. Meanwhile, the AK-12 strain also exhibited phosphorus, potassium-solubilizing, and nitrogen-fixing activity, and produced 2.61 µg/mL of IAA at 24 h. Regulation in the activity of four enzymes was detected after the AK-12 treatment. Phenylalanine ammonia lyase (PAL) was recorded at a maximum of 86.84 U/g after 36 h, and catalase (CAT) decreased after 48 h; however, peroxidase (POD) and polyphenol oxidase (PPO) reached the maximum within 12 h of AK-12 application. Additionally, important resistance genes related to these enzymes were upregulated, indicating the activation of a defense response in the rapeseed against aphids. In conclusion, defense enzymes and defense-related gene activation could improve the pest resistance in rapeseed, which has good application prospects for the future to be developed into biopesticide.

Potential biocontrol efficiency of Trichoderma species against oomycete pathogens.

Plant health is of utmost importance for optimal agricultural production and sustainability. Unfortunately, biotic and abiotic factors put a major constraint on crop safety and productivity. Plant diseases caused by oomycetes inflict serious damage to various crops. Moreover, the injudicious use of chemical pesticides poses threats related to pesticide resistance development in pathogens and environmental pollution. Biocontrol offers an effective solution for disease control; however, research on biocontrol of oomycete-related diseases is scarce. Thus, this study undertakes the screening of biocontrol resources for the effective management of oomycete-related plant diseases. In this regard, 86 isolates of Trichoderma spp. were assessed against Phytophthora nicotianae, P. capsici, Pythium vexans, P. ultimum, and P. dissotocum through dual culture assay. Furthermore, the antagonistic effect of selected isolates was studied against tobacco black shank disease and damping-off of cucumber seedlings in the greenhouse. The relative control effect of the three antagonistic Trichoderma strains AR-4, Tv-1, and ST4-1 on tobacco black shank was more than 60%, which was not significantly different from 6.88 gl-1 fluopicolide-propamocarb. Whereas, the relative control effect of Trichoderma AR-4 and ST4-1 on damping-off of cucumber seedlings was 80.33% and 82.67%, respectively, which were significantly higher than Trichoderma Tv-1 (35.49%) and fluopicolide-propamocarb (47.82%). According to the morphological and molecular characterization, the fungal strains AR-4, Tv-1, and ST4-1 were identified as Trichoderma koningiopsis, T. asperellum, and T. gamsii, respectively. In conclusion, the strains exhibited a strong antagonistic effect against oomycete pathogens and can be integrated into disease management strategies.

Plant-Microbe Interaction: Mining the Impact of Native Bacillus amyloliquefaciens WS-10 on Tobacco Bacterial Wilt Disease and Rhizosphere Microbial Communities.

Ralstonia solanacearum, the causative agent of bacterial wilt disease, has been a major threat to tobacco production globally. Several control methods have failed. Thus, it is imperative to find effective management for this disease. The biocontrol agent Bacillus amyloliquefaciens WS-10 displayed a significant control effect due to biofilm formation, and secretion of hydrolytic enzymes and exopolysaccharides. In addition, strain WS-10 can produce antimicrobial compounds, which was confirmed by the presence of genes encoding antimicrobial lipopeptides (fengycin, iturin, surfactin, and bacillomycinD) and polyketides (difficidin, bacilysin, bacillibactin, and bacillaene). Strain WS-10 successfully colonized tobacco plant roots and rhizosphere soil and suppressed the incidence of bacterial wilt disease up to 72.02% by reducing the R. solanacearum population dynamic in rhizosphere soil. Plant-microbe interaction was considered a key driver of disease outcome. To further explore the impact of strain WS-10 on rhizosphere microbial communities, V3-V4 and ITS1 variable regions of 16S and ITS rRNA were amplified, respectively. Results revealed that strain WS-10 influences the rhizosphere microbial communities and dramatically changed the diversity and composition of rhizosphere microbial communities. Interestingly, the relative abundance of genus Ralstonia significantly decreased when treated with strain WS-10. A complex microbial co-occurrence network was present in a diseased state, and the introduction of strain WS-10 significantly changed the structure of rhizosphere microbiota. This study suggests that strain WS-10 can be used as a novel biocontrol agent to attain sustainability in disease management due to its intense antibacterial activity, efficient colonization in the host plant, and ability to transform the microbial community structure toward a healthy state. IMPORTANCE The plant rhizosphere acts as the first line of defense against the invasion of pathogens. The perturbation in the rhizosphere microbiome is directly related to plant health and disease development. The introduction of beneficial microorganisms in the soil shifted the rhizosphere microbiome, induced resistance in plants, and suppressed the incidence of soilborne disease. Bacillus sp. is widely used as a biocontrol agent against soilborne diseases due to its ability to produce broad-spectrum antimicrobial compounds and colonization with the host plant. In our study, we found that the application of native Bacillus amyloliquefaciens WS-10 significantly suppressed the incidence of tobacco bacterial wilt disease by shifting the rhizosphere microbiome and reducing the interaction between rhizosphere microorganisms and bacterial wilt pathogen.

Microbial Cross-Talk: Dissecting the Core Microbiota Associated With Flue-Cured Tobacco (Nicotiana tabacum) Plants Under Healthy and Diseased State.

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease of flue-cured tobacco production which poses significant yield losses all around the world. In this study, we evaluated the rhizosphere microbiome of healthy and bacterial wilt-infected (diseased) flue-cured tobacco plants through amplification of V3-V4 and ITS1-5f variable regions of 16S and internal transcribed spacer (ITS) rRNA. The study was based on the location (Qujing, Shilin, and Wenshan), plant components (rhizosphere soil and roots), and sample types (healthy and diseased) to assess the diversity of bacterial and fungal communities. Bacterial and fungal communities present in roots primarily emanated from rhizosphere soil. Healthy flue-cured tobacco plants exhibit high microbial diversity compared to diseased plants. Among three variables, plant components significantly influence the diversity of microbial communities, whereas rhizosphere soil harbors higher microbial diversity than roots. Bacterial phyla Cyanobacteria and Proteobacteria were found in high relative abundance in roots and rhizosphere soil samples, respectively. As far as fungi is concerned, a high relative abundance of Ascomycota and Basidiomycota was found in both rhizosphere soil and root. Bacterial genera such as Bacillus, Bradyrhizobium, Ensifer, Neorhizobium, and Lysobacter related to plant growth promotion and disease suppressing abilities were dominant than fungal genera. Analysis of relative abundance at specie-level revealed that most fungal species are pathogenic to flue-cured tobacco and could provide a conducive environment for wilt infection. In conclusion, R. solanacearum significantly influences the microbial diversity of flue-cured tobacco plants and negatively affects the bacterial community composition. Altogether, our study demonstrates the complexity of bacterial and fungal communities that possibly interact with each other (microbe-microbe) and host (host-microbe). This cross-talk could be helpful for healthy flue-cured tobacco plant growth and to induce resistance against bacterial wilt disease.

Deciphering the Bacillus amyloliquefaciens B9601-Y2 as a Potential Antagonist of Tobacco Leaf Mildew Pathogen During Flue-Curing.

Tobacco leaf mildew caused by Rhizopus oryzae (Mucorales, Zygomycota) is an important and devastating post-harvest disease during flue-cured tobacco period, and also is known to cause diseases of fruits and vegetables. In this study, assessment of several candidate biological control agents were first tested in vitro to determine their antifungal activities and potential strains were further applied to tobacco leaves to prevent pathogen colonization during the tobacco-curing process. In vitro screening of 36 bacteria and the isolates of one fungus were performed for their antifungal activities against R. oryzae using dual culture method. Potential five isolates viz. Bacillus amyloliquefaciens B9601-Y2 (Y2), B. amyloliquefaciens YN201728 (YN28), Pseudomonas sp. (Pb), and B. amyloliquefaciens YN201732 (YN32) and T. harzianum B (Th-B) from total screened isolates have shown remarkable results for controlling the mycelial growth of R. oryzae. Finally, out of these five isolates, B. amyloliquefaciens B9601-Y2 potentially reduced the mycelial growth of fungal pathogen with great inhibitory effect. In order to get a better understanding of the biocontrol effect of B9601-Y2 in a flue-curing barn, various suspension density tests with two application methods involving spraying and soaking were examined. Two application methods of B. amyloliquefaciens B9601-Y2 had 98.60 and 98.15% control effects, respectively. In curing barn, the incidence in the treatment group was significantly reduced and tobacco leaves did not get mildew. Altogether, the study demonstrated that candidate bacterial strain B. amyloliquefaciens B9601-Y2 is a potential antagonist for the management of tobacco leaf mildew during flue-curing.

Bacillus amyloliquefaciens YN201732 Produces Lipopeptides With Promising Biocontrol Activity Against Fungal Pathogen Erysiphe cichoracearum.

Bacillus amyloliquefaciens YN201732 is an endophytic bacteria with high biocontrol efficiency and broad-spectrum antimicrobial activities. In order to clarify the main active ingredients and their antifungal mechanisms against powdery mildew of tobacco, this study is focused on lipopeptide obtained through acid precipitation and organic solvent extraction. HPLC and LCMS-IT-TOF were used to separate and identify antimicrobial lipopeptides. Findings revealed that bacillomycin D plays an important role against surrogate fungal pathogen Fusarium solani. Synthetic pathways of sfp, bacillomycin D, and fengycin were separately disrupted. The sfp gene knockout mutant B. amyloliquefaciens YN201732M1 only showed minor antagonistic activity against F. solani. While Erysiphe cichoracearum spore germination was inhibited and pot experiments displayed a significant decrease in tobacco powdery mildew. The spore inhibition rate of YN201732M1 was only 30.29%, and the pot experiment control effect was less than 37.39%, which was significantly lower than that of the wild type. The inhibitory effect of mutant YN201732M2 (deficient in the production of bacillomycin D) and mutant YN201732M3 (deficient in the production of fengycin) on the spore germination of E. cichoracearum were 50.22% and 53.06%, respectively, suggesting that both fengycin and bacillomycin D had potential effects on spore germination of powdery mildew. Interestingly, in a greenhouse assay, both B. amyloliquefaciens YN201732M2 and YN201732M3 mutants displayed less of a control effect on tobacco powdery mildew than wild type. The results from in vitro, spore germination, and greenhouse-pot studies demonstrated that antimicrobial lipopeptides especially bacillomycin D and fengycin may contribute to the prevention and control of tobacco powdery mildew. In addition, gene mutation related to lipopeptide synthesis can also affect the biofilm formation of strains.

Megaloblastic anaemia in a 9-weeks old infant: A case report.

Megaloblastic anaemia due to vitamin B12 and folic acid deficiency is uncommon in infancy and rarely reported in infants below 3 months of age. We hereby report a case of megaloblastic anaemia in a 9-weeks old infant having fever from 7th week of life. Blood picture showed pancytopenia and diagnosis was confirmed on bone marrow biopsy and serum level of vitamins. Patient positively responded to vitamin B12 and folic acid supplementation. Infants with pancytopenia even younger than 2 months, should also be investigated for vitamin B12 and folate deficiency. Mother of the baby was not antenatally investigated for anaemia. Prompt antenatal diagnosis and treatment of mothers can reduce the incidence in the infants.

Cangrelor versus Ticagrelor in Patients Treated with Primary Percutaneous Coronary Intervention: Impact on Platelet Activity, Myocardial Microvascular Function and Infarct Size: A Randomized Controlled Trial.

BACKGROUND: Oral P2Y12 inhibitors take more than 2 hours to achieve full effect in healthy subjects and this action is further delayed in patients with acute myocardial infarction. Intravenous P2Y12 inhibition might lead to more timely and potent anti-platelet effect in the context of emergency primary angioplasty, improving myocardial recovery. OBJECTIVES: This article compares the efficacy of intravenous cangrelor versus ticagrelor in a ST-elevation myocardial infarction (STEMI) population treated with primary percutaneous coronary intervention (PPCI). MATERIALS AND METHODS: In an open-label, prospective, randomized controlled trial, 100 subjects with STEMI were assigned 1:1 to intravenous cangrelor or oral ticagrelor. The co-primary endpoints were platelet P2Y12 inhibition at infarct vessel balloon inflation time, 4 and 24 hours. Secondary endpoints included indices of coronary microcirculatory function: index of microvascular resistance (IMR), initial infarct size (troponin at 24 hours) and final infarct size at 12 weeks (cardiac magnetic resonance). Secondary endpoints included indices of coronary microcirculatory function (index of microvascular resistance [IMR]), initial infarct size (troponin at 24 hours), final infarct size at 12 weeks (cardiac magnetic resonance), corrected thrombolysis in myocardial infarction (TIMI) frame count, TIMI flow grade, myocardial perfusion grade, and ST-segment resolution (ClinicalTrials.gov NCT02733341). RESULTS: P2Y12 inhibition at first balloon inflation time was significantly greater in cangrelor-treated patients (cangrelor P2Y12 reaction unit [PRU] 145.2 ± 50.6 vs. ticagrelor 248.3 ± 55.1). There was no difference in mean PRU at 4 and 24 to 36 hours post-dosing. IMR, final infarct size, angiographic and electrocardiographic measures of reperfusion were all similar between groups. CONCLUSION: Cangrelor produces more potent P2Y12 inhibition at the time of first coronary balloon inflation time compared with ticagrelor. Despite this enhanced P2Y12 inhibition, coronary microvascular function and final infarct size did not differ between groups.

Safety and efficacy of ketamine xylazine along with atropine anesthesia in BALB/c mice

Anesthetics are an indispensable prerequisite for surgical intervention and pharmacological animal studies. The objective of present study was to optimize the dose of ketamine (K) and xylazine (X) along with atropine sulfate (A) in order to achieve surgical tolerance in BALB/c mice. Several doses of ketamine (100, 150, 200 mg/kg) and xylazine (10, 15, 20 mg/kg) were mixed and combination of nine doses (K/X: 100/10, 100/15, 100/20, 150/10, 150/15, 150/20, 200/10,200/15,200/20) were evaluated (n=9 per combination). A constant dose of atropine (0.05 mg/kg) was also used to counter side effect. Time-related parameters were evaluated on the basis of reflexes. KX at dose 200/20 mg/kg produced surgical tolerance in all nine mice with duration 55.00±6.87 minutes. The induction time 0.97±0.09 minutes, sleeping time 90.67±5.81 minutes and immobilization time (102.23±6.83 minutes) were significantly higher than all combination. However, this combination was considered unsafe due to 11 % mortality. While, KX at dose 200/15 mg/kg results in none of the mortality, so was considered as safe. Moreover, this combination produces surgical tolerance in 89 % mice with duration (30.00±7.45 minutes). It was concluded that KX at dose 200/15 mg/kg along with atropine 0.05 mg/kg is safe for performing surgical interventions in BALB/c mice.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan.

OBJECTIVE: To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis (CL) is endemic and was thought to be caused by Leishmania tropica only. METHODS: Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011-2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. RESULTS: Leishmania amastigotes were detected by microscopy in 308 of 432 samples (71.3%) while 374 out of 432 samples (86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed L. tropica in 351 and L. major in 6 biopsy samples. CONCLUSIONS: This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of L. major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

Novel use of a guiding catheter to relieve left main stem occlusion complicating Stamford type A aortic dissection.

Acute ST elevation myocardial infarction occurring as a result of acute aortic dissection is a relatively rare complication with an incidence of 1-2%. However, despite this the outcome is frequently fatal. This report documents novel use of a percutaneous guiding catheter to relieve left main stem occlusion complicating Stamford type A aortic dissection, as a bridge to successful surgical treatment. A 62-year-old man presented with acute chest pain and electrocardiographic changes consistent with acute anterior myocardial infarction, and was sent for primary percutaneous coronary intervention. Angiography demonstrated an acute Stamford type A aortic dissection with associated secondary myocardial infarction caused by mechanical obstruction of the left main coronary artery by the dissection flap. Engagement of the coronary ostium with a guide catheter relieved the obstruction and allowed transfer of the patient to the operating theatre where surgery was successfully performed.

Cost analysis of different screening strategies for gestational diabetes mellitus.

OBJECTIVE: To analyze the findings of a survey in Tehran, Iran, to determine the screening method for gestational diabetes mellitus (GDM) best suited to the local population. METHODS: In four university teaching hospitals in Tehran, 2,416 pregnant women were classified into high-, intermediate-, and low-risk groups, on the basis of criteria established by the American Diabetes Association, and then screened for GDM. A two-step approach was implemented, with use of blood glucose thresholds of 130 mg/dL and 140 mg/dL and the previously advocated diagnostic criteria of two or more abnormal results of an oral glucose tolerance test. RESULTS: The prevalence of GDM is increasing globally, and the major determinants of screening programs for GDM are the cost-to-benefit ratio and the prevalence in the target population. The prevalence of GDM in our study sample was 4.7%. Changing from the 130 mg/dL to the 140 mg/dL blood glucose threshold decreased case-detection sensitivity by 12%, to 88%. With this approach, however, the cost of screening for GDM per pregnancy decreased from US dollars 3.80 to US dollars 3.21 (-15.5%), and the cost per detected case of GDM declined from US dollars 80.56 to US dollars 77.44 (-3.9%). CONCLUSION: We recommend universal screening for GDM in populations, such as ours, that have a substantial baseline prevalence of GDM and variable health-care coverage. In such a setting, a considerable proportion of cases of GDM may be missed. Moreover, universal screening is less expensive in developing countries than in more developed economies and leads to clearer long-term savings for a health service stretched to the limit.