First report of Colletotrichum circinans Causing Anthracnose in Allium fistulosum L. var. giganteum Makino in Gansu Province, China

In September 2018, severe symptoms and high incidence (about 60%) of an onion anthracnose disease attributable to infection by Colletotrichum spp. was observed in production fields of Dingxi city, Gansu Province, China. The mature onion plants near to harvest (Allium fistulosum L. var. giganteum Makino) expressing necrotic symptoms had oval lesions with dark spots that were made up of stromatic masses that had formed beneath the cuticle of the plant base. Twenty symptomatic plants were sampled. Symptomatic tissues were surface sterilized with sodium hypochlorite, transferred aseptically to 20 mL potato dextrose agar in a petri plate and incubated at 30 ± 2 °C. After seven days, cultures produced acervuli and setae, a characteristic sign of Colletotrichum spp. Acervuli were observed with a black, bulbous base and acicular setae while conidia were elliptical (14-25 x 3-6 µm), unicellular hyaline and slightly falcate (Figure 1). DNA was extracted from a 7-day old culture and the ITS region was amplified using primers D1 (5’-GCATATCAATAAGCGGAGGAAAAG-3’) and D2 (5’-GGTCCGTGTTTCAAGACGG-3’) (Kurtzman and Robnett, 1997). The resulting sequence (548 bp) was deposited with NCBI GeneBank under accession number MW127281. The fungus was confirmed as Colletotrichum circinans after conducting a BLAST search with the ITS sequence that reported a highest homology (99% similarity) with MH81329.1 (C. circinans). The speciation was further confirmed by amplifying regions of the TUB2, GAPDH, and ACT genes using primers given in Table S1 and sequences were also submitted to NCBI GeneBank under accession numbers MZ456033, MZ456032 and MZ456031, respectively. The subsequent BLAST results for these three additional gene regions were consistent with the results of the ITS region and fungus was identified as C. circinans. The isolated pathogen was tested for its pathogenicity on onion plants (Allium fistulosum L. var. giganteum Makino). A conidial suspension 30 ml (5 × 105 conidia/ml) was mixed with 1 kg sterilized potted soil in 15 cm diameter plastic pots. Un-inoculated, sterilized soil was used as control. Three green onion plants per pot were planted (Figure S2). The experiment was repeated three times with 15 replications in each experiment. Plants were maintained for 120 days under greenhouse conditions and were monitored for the development of anthracnose symptoms. Symptoms recorded previously on onion plants in field (i.e. necrosis, sunken oval lesions and dark spots) were observed after 30 days on plants grown in inoculated soil while control plants remained asymptomatic (Figure S1). Three samples from symptomatic tissues of each plant were used for re-isolation of the pathogen on PDA, as described above. Cultures grown on PDA were confirmed both on a morphological and molecular basis as Colletotrichum circinans.These morphological, molecular, and pathogenicity tests of the isolated fungus confirm that the anthracnose symptoms observed on onion in Gansu Province, Beijing, China was caused by Colletotrichum circinans. Six different Colletotrichum spp. have been reported to cause diseases on onion worldwide (Rodriguez et al. 2012). C. circinans, which causes smudge, is an occasional onion pathogen was previously recorded as C. dematium (Pers.) Grove f. circinans Arx, which is specific to Allium spp. (von Arx 1970; von Arx 1981). However, Sutton (1992) described C. circinans as a distinct species from C. dematium. The fungus causes smudge or leaf spot of Allium spp. (Farr et al. 1989) and has been reported from Korea, Japan, Argentina, India, the UK and most other regions of the world (Cho & Shin 2004; Kiehr et al. 2012). Smudge may be a disease of concern post-harvest as fungal growth compromises the onion scales and bulb (Walker 1921). In China, this is the first report of C. circinans causing anthracnose in Allium fistulosum L. var. giganteum Makino in Gansu Province.

Effect of Plant-Products Fumigation on Air-Borne Microbes

Background and Objective: Purity of the ambient air is essential for our health and well-being. One of the main factors influencing air quality is the presence of microbes. Fumigation of herbs has been recommended in Unani medicine to purify the air. Hence, the present study was aimed to evaluate the effect of selected Unani herbs fumigation on air-borne microbes. Methods: In this study, the effect of fumigation with Unani Medicinal herbs powder on air-borne microbes was assessed using differences in total colony counts of microbes in pre and post fumigation samples. Microbial load in the air was quantified using the passive open-air petri plate method. Formalin and potassium permanganate served as positive control, while tamarind wood charcoal fumigation served as negative control. Results: Fumigation with Unani medicinal herbs powder at a dose of 45 grams was found to be the most effective in reducing the microbial load of the air. Significant reduction in aerial microbial colonies was observed with fumigation at 30 and 45 gms in the fumigated areas (P<0.05). Conclusion: It can be inferred from the findings of the present study that the test drugs fumigation efficiently reduces the air-borne microbes, hence may be recomanded for air disnfectant. However, various factors that were not considered in this study, such as the effect of temperature and humidity on disinfection efficiency of fumigants should be addressed in further studies.

The Antimicrobial Efficacy of four different Intracanal Medication to Microorganisms existing in the Failure of Endodontic Therapy: An in vitro study

Aim.This study aims to identify the efficacy of different intracanal medication formulations existing in end of endodontic therapy’s failure, and related to strains of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus in Petri plates. Materials and methods. It was used diffusion test in agar where each Petri plate with the inoculated bacteria. Perforations of approximately 4 mm deep by 5 mm in diameter were made to prepare where the intracanal drug (25 µl) to be tested. The diameters of the bacterial inhibition zones were measured and registered to each tested medication at the period of 24 hours, 48 hours, 7 and 14 days respectively. Results. All the medications promoted inhibition halos. The inhibition halos were represented in mm. A higher elimination of micro-organisms can be significantly achieved through the association of different substances in the formulation of an intra canal medication, with emphasis to Ca (OH)2 combined with nitrofurazone and magnesium oxide respectively. Conclusion. Cleaning and shaping of the root canal system associated with the chemical combination of Ca (OH) 2 with antiseptic pastes or solutions considerably reduce the bacterial load.

The Antimicrobial Efficacy of four different Intracanal Medication to Microorganisms existing in the Failure of Endodontic Therapy: An in vitro study

Aim.This study aims to identify the efficacy of different intracanal medication formulations existing in end of endodontic therapy’s failure, and related to strains of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus in Petri plates. Materials and methods. It was used diffusion test in agar where each Petri plate with the inoculated bacteria. Perforations of approximately 4 mm deep by 5 mm in diameter were made to prepare where the intracanal drug (25 µl) to be tested. The diameters of the bacterial inhibition zones were measured and registered to each tested medication at the period of 24 hours, 48 hours, 7 and 14 days respectively. Results. All the medications promoted inhibition halos. The inhibition halos were represented in mm. A higher elimination of micro-organisms can be significantly achieved through the association of different substances in the formulation of an intra canal medication, with emphasis to Ca (OH)2 combined with nitrofurazone and magnesium oxide respectively. Conclusion. Cleaning and shaping of the root canal system associated with the chemical combination of Ca (OH) 2 with antiseptic pastes or solutions considerably reduce the bacterial load.

The Antimicrobial Efficacy of four different Intracanal Medication to Microorganisms existing in the Failure of Endodontic Therapy: An in vitro study

Aim.This study aims to identify the efficacy of different intracanal medication formulations existing in end of endodontic therapy’s failure, and related to strains of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus in Petri plates. Materials and methods. It was used diffusion test in agar where each Petri plate with the inoculated bacteria. Perforations of approximately 4 mm deep by 5 mm in diameter were made to prepare where the intracanal drug (25 µl) to be tested. The diameters of the bacterial inhibition zones were measured and registered to each tested medication at the period of 24 hours, 48 hours, 7 and 14 days respectively. Results. All the medications promoted inhibition halos. The inhibition halos were represented in mm. A higher elimination of micro-organisms can be significantly achieved through the association of different substances in the formulation of an intra canal medication, with emphasis to Ca (OH)2 combined with nitrofurazone and magnesium oxide respectively. Conclusion. Cleaning and shaping of the root canal system associated with the chemical combination of Ca (OH) 2 with antiseptic pastes or solutions considerably reduce the bacterial load.

SPIRO-the automated Petri plate imaging platform designed by biologists, for biologists

The imaging of plant seedlings, fungal mycelia and bacterial colonies grown on Petri plates is commonly used in phenotyping assays, and is typically done manually despite the procedures being time-consuming and laborious. The main reason for this is the still limited availability of existing automated phenotyping tools and facilities. Additionally, constructing a custom-made automated solution is a daunting task for most research groups specializing in biology. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment. SPIRO was designed for biologists by biologists; thus its assembly does not require experience in engineering or programming and its operation is sufficiently intuitive to be done without training. SPIRO has a small footprint optimal to fit into standard incubators for plants and microbes and is equipped with an LED light source for imaging in the dark, thus allowing acquisition of photos under optimal growth conditions. SPIRO's web-based user interface allows setting up experiments and downloading data remotely, without interfering with samples growth. The robots' 8 MP camera provides excellent image quality suitable for automated image processing, which we demonstrate on the example of two semi-automated assays for analysis of commonly used phenotypic traits: seed germination and root growth. Moreover, the robot can be easily customized for a specific use, as all information about SPIRO, including the models for 3D-printed structural components, control software, and scripts for image analysis are released under permissive open source licenses.

Making normal NGM for imaging plates (Cabreiro Lab) v1

C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar which has been aseptically poured into petri plates. The NGM agar medium can be poured into petri plates easily and aseptically using a peristaltic pump. This pump can be adjusted so that a constant amount of NGM agar is dispensed into each petri plate. A constant amount of agar in the plates reduces the need for refocusing the microscope when you switch from one plate to another. The imaging plates can be 35mm, 60mm or 90mm in diameter depending on the assay design.

Comparative analysis of the antimicrobial potential of different intra-canal medication to microorganisms present in the failure of the endodontic therapy: In vitro study

Microorganisms that infect the root canals system are the main etiologic factor of the periapical pathologies. Some microorganisms are resistant to the antimicrobial treatment and may survive in the root canal after the chemical mechanical preparation and intra-canal medication, characterizing a persistent infection. In cases of failure of the endodontic treatment, a new approach may be done using additional measures that involve this infectious process control through the elimination or maximum reduction of microorganisms. Therefore, this article aims to evaluate the antimicrobial potential of different formulations of intra-canal medication compared to strains of Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus in Petri plates. It was used diffusion test in agar where each Petri plate with the inoculated bacteria presented 5 wells that were filled with each medication. The diameters of the bacterial inhibition zones were measured and registered to each tested medication at the period of 24 hours, 48 hours, 1 week and 2 weeks. All the medications promoted inhibition halos; however, a higher elimination of micro-organisms can be significantly achieved through the association of different substances in the formulation of an intra-canal medication, with emphasis to HPG and Ca(OH)2 + CHX.

A Novel Culturing Chip (cChip) Can Facilitate Culturing of Unculturable Bacteria From Aquatic Environment

Background: Culturing the unculturable microorganisms is an important aspect of microbiology. Once cultured the unculturable microorganisms can be a source of useful antibiotics, enzymes etc. Several studies have been conducted on culturing the unculturable microorganisms from soil. But little knowledge exists about culturing such bacteria from aquatic environment. Therefore, in this study we designed a novel culturing chip (cChip) to facilitate the growth of unculturable aquatic bacterial community. cChip was optimized for microbial growth using known bacteria in the lab, later, microbes from a freshwater lake were concentrated (instead of using the traditional dilution method) and inoculated in cChip before incubating the chip in simulated lake environment. Then further sub-culturing was done on laboratory media. The field samples were also analyzed using traditional culturing and metagenomics for a comparison. Results: Metagenomics analysis showed that 832 microbial species were present in the samples belonging to five different phyla, that is, Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes and Verrucomicrobia. However, traditional culturing yielded only 36 isolates that belonged to four different phyla, that is, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Culturing through cChip yielded 154 isolates belonging to five different phyla, that is, Proteobacteria, Actinobacteria, Bacteroidetes, Verrucomicrobia and Firmicutes. Out of these 154 cChip isolates, 45 were previously uncultured bacteria having a 16S rRNA gene similarity from 91.35 % to 98.7 % to their closest relatives according to NCBI GenBank. Conclusion: This study shows that culturing microorganisms in the cChip from aquatic environment using concentration process before performing the traditional petri plate culturing can result in the successful growth of unculturable bacteria. To the best of our knowledge this is the first study conducted for successful exploration of unculturable aquatic microbial community. This study can have a significant impact on our understanding of the techniques that can be applied for exploring the unexplored microbiome from diverse environments. We also hypothesize that certain modifications in the manufacturing material, keeping the design and techniques of this study intact can result in the application of this technique from gut microbiome to extremophiles in the environment.