Molecular characterization and vegetative growth of pathogenic seed-borne fungus, Curvularia lunata of tomato and its in vitro control measures

Present studies were conducted to isolate and identify the seed-borne pathogenic fungus from the selected tomato variety through morphological and molecular techniques based on the sequencing of internal transcribed spacer (ITS) region of 18S rDNA. According to the colony and conidial features, the fungus was identified as Curvularia sp. The obtained ITS sequencing showed above 99% similarity with Curvularia lunata in the NCBI database. The sequence of the fungus was deposited in NCBI GenBank under the accession number: ITS, MH382879.1. Besides, the phylogenetic tree further confirmed the taxonomic position of the studied fungus. Growth characteristics of the fungus on nine different fungal culture media were evaluated, in which Honey peptone agar, Carrot agar, Potato sucrose agar, and Kauffman’s agar were found the most suitable. The maximum vegetative growth of the fungus was recorded at 30°C temperature and pH conditions. The bio-control potential of five different antagonists against the studied fungus was assessed, in which Trichoderma harzianum showed the better performance to restrict mycelial growth. Three ethanolic plant extracts were also evaluated, in which Lowsonia inermis L. exhibited above 60% mycelial growth inhibition of the fungus. Among three tested fungicides, Tilt 250 EC was found as an excellent fungicide to inhibit mycelial growth of C. lunata under in vitro conditions. Int. J. Agril. Res. Innov. Tech. 11(2): 124-132, Dec 2021

Reviewing the Clinical Spectrum Related to Kmt2b Gene Mutations: Unusual Clinical Presentation and a Possible New Pathogenic Mutation

Introduction KMT2B related dystonia is a childhood onset generalized dystonia. Since its first description in 2016, different phenotypic spectrum have been reported. The aim of the case report is to provide data that may help to understand the spectrum of KMT2B-related disorders. We present two members of a family with a possible non-previously described pathogenic mutation and an unusual KMT2B related dystonia presentation: an adult onset and focal dystonia.Case Presentation The index patient is a 32 year-old woman with a generalized dystonia. Her maternal uncle presented a focal dystonia. Next-generation sequencing revealed a heterozygous missense mutation in KMT2B gene (19q13.12), described as a variant of uncertain significance (VUS). Although characteristic phenotype of KMT2B dystonia is a childhood onset generalized dystonia, different phenotypes have been related according to the kinds of mutations in this gene, also varying the age of symptom onset and the penetrance of the mutation. Asymptomatic or sub-clinical carriers and adult onset has been described. Due to the low prevalence of this variant in the general population and the low penetrance and high intrafamilial variability of this entity, we suggest that this mutation might be a pathogenic variant.ConclusionsKMT2B related dystonia is an emerging and prevalent monogenic dystonia whose incidence, genetic variability and clinical spectrum remain unknown. Despite the study of this gene is indicated in childhood onset dystonia, description of cases such as ours shows that its sequencing in patients with an adult-onset dystonia with family history can be useful for the diagnosis.

Pathogenic Fungi Diversity of ‘CuiXiang’ Kiwifruit Black Spot Disease during Storage

Kiwifruit black spot disease has become increasingly widespread in many ‘CuiXiang’ kiwifruit plantings regions. This research was aimed at the pathogenic microorganisms of black spot of the ‘CuiXiang’ cultivar. Physiological, morphological and transcriptional characteristics between black spot fruit and healthy fruits were evaluated. Then, it applied a high-throughput internal transcribed spacer (ITS) sequencing to analyze the black spot disease microbial community. The cell structure showed that mycelium was attached to the surface of the kiwifruit through black spot, and that consequently the mitochondria were damaged, starch particles were reduced, and shelf life was shortened. Transcriptome revealed that different genes in kiwifruit with black spot disease were involved in cell wall modification, pathogen perception, and signal transduction. ITS sequencing results described the disease-causing fungi and found that the microbial diversity of black spot-diseased fruit was lower than that of healthy fruit. We predict that candidate pathogenic fungi Cladosporium cladosporioides, Diaporthe phaseolorum, Alternaria alternata, and Trichothecium roseum may cause black spot. This study was to explore the pathogenic fungal community of ‘CuiXiang’ kiwifruit black spot disease and to provide essential information for field prevention.

Bloodstream infections caused by Magnusiomyces capitatus and Magnusiomyces clavatus: epidemiological, clinical and microbiological features of two emerging yeast species

Background: Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available. Objectives: To determine the epidemiology and susceptibility of Magnusiomyces isolates from bloodstream infections (BSI) isolated in Germany and Austria from 2001-2020. Methods: In seven institutions a total of 34 Magnusiomyces BSI were identified. Identification was done by ITS sequencing and MALDI-TOF MS. Antifungal susceptibility was determined by EUCAST broth microdilution and gradient tests. Results: Of the 34 isolates, M. clavatus was more common (N=24) compared to M. capitatus (N=10). BSI by Magnusiomyces spp. were more common in men (62%) and mostly occurred in patients with haemato-oncological malignancies (79%). The highest in vitro antifungal activity against M. clavatus / M. capitatus was observed for voriconazole (MIC 50 0.03/0.125 mg/L), followed by posaconazole (MIC 50 0.125/0.25 mg/L). M. clavatus isolates showed overall lower MICs compared to M. capitatus . With the exception of amphotericin B, low essential agreement between gradient test and microdilution was recorded for all antifungals (0-70%). Both species showed distinct morphologic traits on ChromAgar Orientation and Columbia blood agar, which can be used for differentiation if no MALDI-TOF or molecular identification is available. Conclusion: Most BSI were caused by M. clavatus. The lowest MICs were recorded for voriconazole. Gradient tests demonstrated unacceptably low agreement and should preferably not be used for susceptibility testing of Magnusiomyces spp.

Microbial Diversity and Characteristics of Kombucha as Revealed by Metagenomic and Physicochemical Analysis

Kombucha is a fermented tea made from a Symbiotic Culture of Bacteria and Yeast (SCOBY) with a long history of use as a health tonic. It is likely that most health benefits come from the tea and fermentation metabolites from specific microbial communities. Despite its growing importance as a functional health drink, the microbial ecosystem present in kombucha has not been fully documented. To characterize the microbial composition and biochemical properties of ‘The Good Brew’ original base kombucha, we used metagenomics amplicon (16S rRNA and ITS) sequencing to identify the microbial communities at the taxonomic level. We identified 34 genera with 200 microbial species yet described in kombucha. The dominance of organic acid producing microorganisms Acetobacter, Komagataeibacter and Starmerella are healthy for the human gut and their glucose metabolising activities have a putative role in preventing conditions such as diabetes and obesity. Kombucha contains high protein (3.31 µg/mL), high phenolic content (290.4 mg/100 mL) and low sugars (glucose: 1.87 g/L; sucrose 1.11 g/L; fructose: 0.05 g/L) as compared to green tea. The broad microbial diversity with proven health benefits for the human gut suggests kombucha is a powerful probiotic. These findings are important to improve the commercial value of kombucha and uncover the immense prospects for health benefits.

A Detailed Item Response Theory Analysis of Algorithms and Programming Concepts in App Inventor Projects

Teaching computing in K-12 is often introduced focusing on algorithms and programming concepts using block-based programming environments, such as App Inventor. Yet, learning programming is a complex process and novices struggle with several difficulties. Thus, to be effective, instructional units need to be designed regarding not only the content but also its sequencing taking into consideration difficulties related to the concepts and the idiosyncrasies of programming environments. Such systematic sequencing can be based on large-scale project analyses by regarding the volition, incentive, and opportunity of students to apply the relevant program constructs as latent psychometric constructs using Item Response Theory to obtain quantitative ‘difficulty’ estimates for each concept. Therefore, this article presents the results of a large-scale data-driven analysis of the demonstrated use in practice of algorithms and programming concepts in App Inventor. Based on a dataset of more than 88,000 App Inventor projects assessed automatically with the CodeMaster rubric, we perform an analysis using Item Response Theory. The results demonstrate that the easiness of some concepts can be explained by their inherent characteristics, but also due to the characteristics of App Inventor as a programming environment. These results can help teachers, instructional and curriculum designers in the sequencing, scaffolding, and assessment design of programming education in K-12.

Identification of Gracilariaceae (Rhodophyta) of central Portugal by histological and genetic methods

This study aims to identify different populations of Gracilariaceae collected from the central coast of Portugal through light microscopy, anatomical observations and genetic tools, essential approaches to correctly assign species identity. Samples were obtained from Ria de Aveiro (AV), Figueira da Foz (FFBC, FFMD), and Lagoa de Óbidos (LOBR, LOEV, LOBS). Although histological observations offered a visual representation of the characteristic pseudoparenchymatous organization, they did not allow a clear distinction among the species. The amplification of a ∼700 bp fragment of the mitochondrial cytochrome oxidase I gene, and its sequencing enabled us to assign the populations FFBC and LOBS to Gracilaria gracilis, and the populations AV, FFMD, LOBR, and LOEV to Agarophyton vermiculophyllum. This contribution will help phycologists to correctly identify the Portuguese populations of Gracilaria sensu lato at the species level, which will be crucial in ensuring that future studies and industrial exploration accurately target the correct species.

A Systematic Study of the Antibacterial Activity of Basidiomycota Crude Extracts

The excessive consumption of antibiotics in clinical, veterinary and agricultural fields has resulted in tremendous flow of antibiotics into the environment. This has led to enormous selective pressures driving the evolution of antimicrobial resistance genes in pathogenic and commensal bacteria. In this context, the World Health Organization (WHO) has promoted research aiming to develop medical features using natural products that are often competitive with synthetic drugs in clinical performance. Fungi are considered an important source of bioactive molecules, often effective against other fungi and/or bacteria, and thus are potential candidates in the search of new antibiotics. Fruiting bodies of sixteen different fungal species of Basidiomycota were collected in the Italian Alps. The identification of fungal species was performed through Internal Transcribed Spacer (ITS) sequencing. Most species belong to genera Cortinarius, Mycena and Ramaria, whose metabolite contents has been scarcely investigated so far. The crude extracts obtained from the above mushrooms were tested for their inhibition activity against five human pathogens: Candida albicans ATCC 14053, C. glabrata ATCC 15126, Staphylococcus aureus NCTC 6571, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC 13883. Twelve crude extracts showed activity against P. aeruginosa ATCC27853. Highest activity was shown by some Cortinarius species, as C. nanceiensis.

Comparison of clinical and biological characteristics of HIV-infected patients presenting Cryptococcus neoformans versus C. curvatus/C. laurentii meningitis

Background Cryptococcal meningitis is mainly caused by Cryptococcus neoformans/C. gattii complex. We compared the clinical, biological, and antifungal susceptibility profiles of isolates from HIV-Infected Patients (HIVIP) with C. neoformans (Cn) versus C. curvatus/C. laurentii (Cc/Cl) meningitis. Methods Comparative analytical study were conducted. Apart from patients’ clinical data, the following analysis were performed and the results were compared in both groups: biochemical examination, cryptococcal antigen test, India ink staining, and culture on Cerebral Spinal Fluid (CSF), strains identification by mass spectrometry, ITS sequencing, PCR serotyping and antifungal susceptibility. The main outcome variable was the “species of Cryptococcus identified”, which was compared to other variables of the same type using the Pearson Chi-square test or the Fisher exact test. Results A total of 23 (79.3%) Cn meningitis cases versus 6 (20.7%) Cc/Cl meningitis were retained.Cn meningitis was more frequently associated with headache (100% vs 50%, p = 0.005) than Cc/Cl meningitis and meningeal signs were more frequent in Cn infected patients. Biologically, hypoglycorrhachia and low CD4 count were more observed in Cn group (90% vs 20% of patients, p = 0.01; 45.6 vs 129.8 cells/µL, p = 0.02, respectively). A higher proportion of Cn strains (91.3%) showed a low Minimum Inhibitory Concentration (MIC) (< 8 mg/L) for fluconazole compared to Cc/Cl strains (66.7%). Also, Cc/Cl strains resistant to 5-flucytosine and amphotericin B were found in 16.7% of cases for each of the two antifungal agents. Cryptococcus detection by routine analysis (India ink, culture, and antigens) was better for Cn samples than Cc/Cl. Except ITS sequencing, which identified all strains of both groups, mass spectrometry and serotyping PCR identified Cn strains better than Cc/Cl (100% vs 80%, p = 0.1; 100% vs 0%, p < 0.0001, respectively). After treatment with amphotericin B, 5-flucytosine, and fluconazole in both groups, the outcome was similar. Conclusions Clinical presentation of Cn meningitis is certainly more severe than that of Cc/Cl meningitis, but Cc/Cl infection should be considered in the management of HIVIP with meningeal syndrome because of the diagnostic difficulty and the high MICs of antifungal agents required for the treatment of meningitis due to these cryptococcal species.