Sterile Medium Research Articles

In vitro Callus Induction of Catunaregam spinosa Using Leaves as Explant

Aims: C. spinosa (Family Rubiaceae) is a valuable medicinal since years ago. A protocol was developed including best sterilization and best medium for callus induction of C. spinosa using leaf discs as explants.
 Methodology: Sterilization protocol optimized using different concentrations of Carbendazim® (0.2,
 0.3%) and Clorox (10, 15%) exposing to different time intervals (10, 15 min). Percentage survival and contaminations were calculated. Best medium was optimized using different concentrations of 6-Benzyl Amino Purine (BAP) and Naphthalene Acetic Acid (NAA) (1.0-6.0 mg L-1). Growth regulators free Murashige and Skoog (MS) medium was used as control. Completely randomized design was followed with ten replicates in each concentration. Days taken to initiate calli, morphological characteristics and mean dry weights of calli were evaluated after 3 months of incubation.
 Results: Leaf discs sterilization with 0.3% Carbendazim for 10 min, 10 % Clorox for 10 min and 70% ethanol for 30 sec followed by two washings in sterile distilled water was found to be best sterilization protocol. It recorded lowest percentage contamination (13.34%) and highest percentage survival (86.66%) after 8 weeks. No observable changes were found in calli grown in growth regulators free MS medium. Calli growth and morphologies were significantly affected by the type of growth regulator and their concentrations in MS medium. Color of the calli varied from white opaque to yellow brown to green and the texture from foamy, loose, and friable to compact. Best medium with 1.0 mg L-1 BAP and 3.0 mg L-1 NAA produced green friable calli with 0.0969±0.01 g mean dry weight after 3 months. Some traits were found to be depended on synergetic effect of growth regulators and genotypic characteristics of explant.
 Conclusion: The study provides a better sterilization protocol and medium for in vitro calli induction and growth of C. spinosa.

Microbiological Parameters of Different Waste Waters to Evaluate Their Potential to be Use as a Media for Chlorella sp. and Spirogyra sp. Cultivation

Globally there is severe water scarcity, and methods to reuse wastewater need to be adapted. The objective of this research was to determine the microbiological parameters of different wastewaters in order to study the potential of wastewater as a media to cultivate algae.Food, agriculture, drainage and municipal wastewater sample were collected in sterile bottles separately. Aerobic, anaerobic plate counts, yeast and mold counts were evaluated. Enumeration of coliform, fecal coliform, coliphages and microalgae was performed with all wastewater samples. Chlorella sp. and Spirogyra sp. were isolated from three different paddy field water samples, and growth studies were carried out in different wastewater media with standard sterile Chu’s medium and in sterile tap water medium as control.Heterotrophic, anaerobic bacteria, microalgae, yeast and molds were found in all types of wastewater. Agriculture wastewater had the highest value for aerobic, anaerobic, yeast and mold count in the highest dilution. Coliforms and fecal coliforms were absent in food and agriculture wastewaters on Eosin Methylene Blue (EMB) medium. Coliform and Fecal coliforms were only observed in drainage and municipal wastewater on Endo agar (EA) medium. Coliphages were absent only in agricultural wastewater and found in the highest numbers in municipal wastewater. Except for agricultural wastewater, other three types of wastewaters contain microbial contaminations. Agriculture wastewater medium significantly enhanced the growth of Chlorella sp. than the standard Chu’s medium. The growth of Spirogyra sp. was stimulated by the food wastewater medium than the other media.
 Keywords: Wastewaters, Microbiological parameter, Chlorella sp., Spirogyra sp., Coliforms

Occurrence of bacteria belonging to the genus Enterococcus and Staphylococcus on inanimate surfaces of selected hospital facilities and their nosocomial significance.

This work aimed to determine the representation and resistance of bacteria belonging to the genus Staphylococcus and Enterococcus on inanimate surfaces of two selected workplaces of the University Hospital of L. Pasteur in Košice (UHLP) and to investigate their importance in the hospital environment. The men's ward of the Department of Internal Medicine (DIM) and the Department of Anaesthesiology and Intensive Care (DAIC) were chosen. Using sterile sampling kits, a total of 182 swabs were collected from the inanimate surfaces of both UHLP workplaces. The swabs were then transported to a microbiological laboratory and inoculated onto sterile culture media (blood agar containing 5% ram erythrocytes). After culturing (24-48 hours, in a thermostat at constant temperature 37 °C), bacterial colonies were identified by mass spectrometry on a MALDI TOF MS. Bacteria belonging to the genera Staphylococcus and Enterococcus were subsequently separated from the spectrum of identified bacteria. Nosocomial significant strains of staphylococci (Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus aureus) and all isolated enterococci were subjected to susceptibility testing for selected antibiotics using the disk diffusion method - E-tests. Several members of the genus Staphylococcus were identified from the inanimate surfaces of both workplaces. These were mainly coagulase-negative strains - Staphylococcus epidermidis (45), Staphylococcus capitis (34), Staphylococcus haemolyticus (20), Staphylococcus hominis (45), Staphylococcus pasteuri (2), Staphylococcus sroph (1), Staphylococcus simulans (3), and Staphylococcus warneri (4). Staphylococcus aureus strains were also identified (2). Nosocomial significant isolates were tested for susceptibility to the antibiotics cefoxitin (FOX) and oxacillin (OXA). Two members of the genus Enterococcus - Enterococcus faecium (7) and Enterococcus faecalis (8) were isolated. All strains were subject to vancomycin susceptibility testing using the disk method.

Escherichia coli Biomarker Types in Colorectal Cancer Patients

Colorectal cancer is the second leading cause of cancer in the globe; increasing evidence suggests that the intestinal microbiota is a key factor associated with carcinogenesis of colorectal cancer (CRC) and colonic polyps. This study was conducted on 50 adult patients of both gender. These patients were proved colorectal cancer of different stages, and they were attending gastrointestinal center in Ramadi city, Gastroenterology and Liver Disease Center at Medical City Hospital Baghdad, during the period extended from November to February 2021. Thirty-five biopsies were taken from patients with colorectal cancer diagnosed in colonoscopy, and fifteen (15) stool specimens were taken from them. Each specimen was cultivated on blood agar, MacConkey agar, at 37°C for 24 hours. Then bacterial colonies were studied grossly, their size, color, elevation, texture, and odor; dry, stained smear was stained with Gram stain. A part of the test colony was submitted to biochemical investigation using a suitable sterile medium for each test. The bacterial diagnosis was confirmed using the VITIC system. For molecular research, Escherichia coli isolates were purified and maintained frozen at -20°C in a brain heart infusion broth containing 20% glycerol. Another 50 control adult, with normal colonoscopy from both genders. A stool specimen was taken from each control individually and processed as soon as possible in the same way for the patient group. Test E. coli isolated from both patient group and control group were tested for virulence genes different Frequency of 16S ribosomal RNA (16SrRNA) gene, cytotoxic necrotizing factor (CNF) gene, cytolethal distending toxin (CDT) gene, and polyketide synthase (PKS) genes. The Tumor Necrosis Factor (TNF) was measured in sera from patients with CRC and compared to healthy individuals. The genes implicated in carcinogenesis in E. coli strains, such as 16SrRNA, CNF, and CTD, were only found in test isolates of E. coli from patients, indicating that local E. coli containing such genes could become pathogens in such persons, contributing to CRC induction.

Functional activity of peripheral blood mononuclear cells in patients with non-Hodgkin's lymphomas receiving treatment after COVID-19 disease.

e19564 Background: Patients with non-Hodgkin's lymphomas (NHL) develop abnormalities in the structural and functional organization of the immune system leading to immune deficiency. Chemotherapy (CT) in patients after SARS-CoV infection is associated with a more severe disease course affecting the treatment results. The cytokine-producing activity (CPA) of blood cells is poorly studied, while it determines the effectiveness of antitumor and anti-infective functions of the immune system. The purpose of this study was to evaluate CPA of peripheral blood mononuclear cells in patients receiving treatment for NHL after COVID-19. Methods: The study included 8 patients with large B-cell NHL with PCR-confirmed COVID-19 infection in past medical history. All patients received from 3 to 4 chemotherapy cycles. K2EDTA blood samples obtained before and after 3-4 CT cycles were divided into 2 parts after dilution with a sterile nutrient medium solution: part 1, to assess spontaneous CPA; part 2, with addition of a sterile mitogen (phytohemagglutinin 4 μg, concanavalin A 4 μg, and lipopolysaccharide 2 μg) to assess stimulated CPA. The samples were incubated for 24 hours at 370C, and the levels of IL-1β, IL-6, IL-8, IL-10, IL-18, IL-4, IL-2, TNF- α, INF-ɣ, INF-α were determined in the obtained plasma. The stimulation coefficient (SC) was calculated as the ratio of stimulated CPA to spontaneous CPA. Results: 3-4 CT cycles in patients after COVID-19 was accompanied by an elevation of spontaneous CPA of the blood cells IL-6, INF-ɣ, TNF-α, IL-8, compared to the initial levels, by 678%, 127%, 64% and 57%, respectively. The ability of cells to spontaneous production of IL-10 and INF-α decreased by 30% and 100%. The mitogen-induced CPA of mononuclear cells in relation to IL-10, IL-6, IL-2, IL-1β and INF-α increased by 300%, 130%, 92%, 52% and 52%, respectively. Stimulated CPA in relation to INF-ɣ decreased by 21% compared to initial levels. As a result of the revealed CPA changes, SC in NHL patients after COVID-19 receiving CT increased, compared to the initial levels, by 465%, 92% and 48% respectively for IL-10, IL-2, IL-1β, as well as the appearing ability to INF-α production. SC for IL-6, INF-ɣ, TNF-α, and IL-8 decreased by 70%, 66%, 33% and 27% respectively. Conclusions: Certain features of spontaneous and mitogen-activated CPA of blood mononuclear cells were revealed in NHL patients after COVID-19, indicating a change in the functional activity of immune cells which could affect the development of the disease and the effectiveness of the therapy. The data obtained require additional studies and can be used to assess the condition of patients, as well as to predict the therapy efficacy.

Characterization and proteome profiling of extracellular vesicles in a murine model of Staphylococcus aureus endophthalmitis

Endophthalmitis is a vision-threatening complication of intraocular surgery or penetrating injury of which Staphylococcus aureus is an important etiological agent. Extracellular vesicles (EVs) hold a tremendous possibility for developing diagnostic and therapeutic biomarkers due to their role in the pathogenesis of various infections. The aim of this study was to characterise the protein cargo of EVs, isolated from a murine (C57BL/6) model of S. aureus endophthalmitis by LC–MS/MS. Contralateral eye injected with sterile media served as control and both eyes were enucleated after 24 h, followed by extraction of EVs by ultracentrifugation. EVs were characterized by DLS and western blotting with tetraspanin markers, CD9 and CD81 and quantified by ExoCet quantification kit. Proteomic analysis identified 1964 proteins (FDR ≤ 0.01) in EVs from infected mice eyes, of which 40 proteins varied significantly in their amounts in comparison to EVs obtained from control eyeballs (P-value ≤ 0.05). The results of this study provide insight into the global EV proteome of S. aureus endophthalmitis with their functional correlation and differential protein amounts between infected and control set. Annexin A5, cathepsin D and C5a play a pivotal role in disease pathogenesis and could possibly play a role as a prognostic marker in endophthalmitis.

Mitigation of microbial corrosion by Cu addition to X65 pipeline steel by Pseudomonas aeruginosa MCCC 1A00099.

Microbiologically influenced corrosion (MIC) is becoming a knotty problem for transmission pipelines. Developing MIC mitigation strategies for pipelines is increasingly urgent. In this study, MIC resistance against Pseudomonas aeruginosa of the X65 pipeline steels with (X65Cu) and without (X65) Cu addition was comparatively studied by electrochemical measurements and surface observation. Experimental results demonstrated that the corrosion rate of X65Cu steel was lower than that of X65 steel no matter in sterile or bacteria-containing media. Cu addition is helpful to the formation of the rust layer in the sterile medium. Surface observation showed that X65Cu steel exhibited a better MIC resistance against P. aeruginosa than that of X65 steel. Cu ions released from the X65Cu steel could effectively kill the P. aeruginosa attached on the steel surface, thus evidently decreased the pit depth and diameter.

TESTING THE RESPONSIBILITY OF EGGPLANT THORN (SOLANUM CAROLINENSE) ETHANOL EXTRACT ON THE GROWTH OF BACTERIA (STREPTOCOCCUS MUTANS) IN VITRO

Streptococcus mutans is the main pathogenic bacteria causing caries and tooth enamel damage. Eggplant spines (Solanum carolinense) contains active substances that can inhibit the growth of Streptococcus mutans bacteria on teeth. The purpose of this study was to determine the inhibitory effect of the antibacterial activity of ethanol extract of thorn eggplant on the growth of Streptococcus mutans bacteria and to determine the optimal concentration of thorn eggplant extract on the growth of Streptococcus mutans bacteria in vitro. The type of method used in this study is an experimental study, with the dependent variable being the zone of inhibition of the growth of Streptococcus mutans, the independent variable being differences in the concentration of ethanol extract of thorn eggplant (Solanum carolinense) at concentrations of 20%, 40%, 60%, 80% and 96 ethanol. % as negative control and chloramphenicol as positive control, control variables for the presence of other microbial contamination, media thickness, turbidity of bacterial suspension, incubation temperature, disc distance, incubation time, sterilization of equipment, media and room. The results of this study were the effect of the ethanol extract of thorn eggplant on Streptococcus mutans and the optimal concentration of the ethanolic extract of thorn eggplant was 80% with an inhibition zone diameter of 11 mm (strong) which was indicated by the appearance of the diameter of the bacterial inhibition zone at each treatment

Nitrate-reducing-bacteria assisted hydrogen embrittlement of X80 steel in a near-neutral pH solution

The effect of nitrate-reducing bacteria (NRB) on the hydrogen embrittlement of X80 steel in a near-neutral pH solution was studied by various tests. NRB promote the formation of numerous pits under static load after 120 days and enlarge the polarization current at the same polarization potential. The increasing probability of hydrogen penetration into the biotic medium further leads to hydrogen embrittlement higher than 7–17% of the sterile medium. Under the same failure risk of 40%, NRB can cause hydrogen deterioration of steel, and the safe cathodic potential range will be narrowed from − 0.79–− 1.08 V to − 0.79–− 0.86 V (saturated calomel electrode).

Selected corrosion of X80 pipeline steel welded joints induced by Desulfovibrio desulfuricans

Sulfate-reducing bacteria (SRB) corrosion of X80 pipeline steel welded joints has been investigated. The larger diameter, deeper depth and higher density of the corrosion pits were detected in the heating affected zone (HAZ) than in the base metal (BM) and weld zone (WZ) in the sterile medium. However, in the inoculated medium, Desulfovibrio desulfuricans (D. desulfuricans) tended to attach primarily to the BM and WZ, and increased their local corrosion sensibilities. D. desulfuricans disliked to attach to the HAZ surface and only non-uniform corrosion dominated on the surface, which was an important phenomenon in D. desulfuricans corrosion of pipeline steel.

Peptide-Encapsulated Single-Wall Carbon Nanotube-Based Near-Infrared Optical Nose for Bacteria Detection and Classification

Sense of smell has been used as a diagnostic tool for almost entire human history. While successful examples of the use of the human nose for diagnostics are rare in modern history, there are ample reports of use of animals to diagnose various medical conditions. Bacterial infections often result in strong odors. In recent years, electronic noses (e-nose) and optical noses (o-nose) are of high interest in diagnostics and classification of bacterial infections. Artificial olfactory sensors can perform noninvasively, immediately at the point of care, do not require extensive sample handling, and promise to be highly cost-effective. This manuscript demonstrates the development of a near-infrared optical sniffer comprised of peptide-encapsulated (6,5) single-wall carbon nanotubes (SWCNTs) for bacteria detection and classification. Sixteen different peptides that include tyrosine in different proportions and positions were synthesized. The ability of these peptides to disperse SWCNTs in water was tested, and the intensity of the resultant optical signal was evaluated. Overall, longer peptides provided better dispersion as compared to shorter peptides. Addition of the fluorenylmethyloxycarbonyl chloride (Fmoc) group to positively charged peptides tested in the current study significantly improved SWCNT dispersion and signal intensity. The sensors successfully distinguished between the odor of sterile growth medium, Escherichia coli, and Klebsiella pneumoniae. Moreover, we demonstrated the possibility of using the developed sensors for antibiotics susceptibility testing. The sensors provided results in real-time, enabled multiple-usage, and operated at room temperature.

Enhancement of Secondary Cell Wall Formation in Poplar Xylem Using a Self-Reinforced System of Secondary Cell Wall-Related Transcription Factors.

The secondary cell wall (SCW) in the xylem is one of the largest sink organs of carbon in woody plants, and is considered a promising sustainable bioresource for biofuels and biomaterials. To enhance SCW formation in poplar (Populus sp.) xylem, we developed a self-reinforced system of SCW-related transcription factors from Arabidopsis thaliana, involving VASCULAR-RELATED NAC-DOMAIN7 (VND7), SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN 1/NAC SECONDARY WALL THICKENING-PROMOTING FACTOR3 (SND1/NST3), and MYB46. In this system, these transcription factors were fused with the transactivation domain VP16 and expressed under the control of the Populus trichocarpa CesA18 (PtCesA18) gene promoter, creating the chimeric genes PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16. The PtCesA18 promoter is active in tissues generating SCWs, and can be regulated by AtVND7, AtSND1, and AtMYB46; thus, the expression levels of PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16 are expected to be boosted in SCW-generating tissues. In the transgenic hybrid aspens (Populus tremula × tremuloides T89) expressing PtCesA18pro::AtSND1:VP16 or PtCesA18pro::AtMYB46:VP16 grown in sterile half-strength Murashige and Skoog growth medium, SCW thickening was significantly enhanced in the secondary xylem cells, while the PtCesA18pro::AtVND7:VP16 plants showed stunted xylem formation, possibly because of the enhanced programmed cell death (PCD) in the xylem regions. After acclimation, the transgenic plants were transferred from the sterile growth medium to pots of soil in the greenhouse, where only the PtCesA18pro::AtMYB46:VP16 aspens survived. A nuclear magnetic resonance footprinting cell wall analysis and enzymatic saccharification analysis demonstrated that PtCesA18pro::AtMYB46:VP16 influences cell wall properties such as the ratio of syringyl (S) and guaiacyl (G) units of lignin, the abundance of the lignin β-aryl ether and resinol bonds, and hemicellulose acetylation levels. Together, these data indicate that we have created a self-reinforced system using SCW-related transcription factors to enhance SCW accumulation.

Xanthan gum and sodium hypochlorite in vitro rooting of Gerbera hybrida cv. Essandre

The aim of this study was to check the use of the following gelling agents: the xanthan gum “Adicel®” and the stabilizer “Super Liga Neutra®” to replace agar in the in vitro rooting phase of Gerbera hybrida cv. Essandre. Additionally, the possibility of using chemical sterilization of both culture media and glassware with sodium hypochlorite to replace autoclaving was analyzed. The gelling agents, xanthan gum “Adicel®” and the stabilizer “Super Liga Neutra®” were tested at the following concentrations (g L-1): 7; 9; 11; 13; 15; 17; 19; and 21. No concentrations of Super Liga Neutra® provided effective solidification. Concentrations of 17 and 21 g of Adicel® provided a good gelling of the culture medium, which was compared to the medium containing agar (control) with two types of sterilization: autoclaving (for 20 and 40 minutes) and chemical sterilization. Autoclaving for 20 minutes did not provide effective elimination of contamination in the culture medium containing xanthan gum; this only occurred when autoclaving time increased to 40 minutes. Plant development in culture media containing 17 and 21 g of xanthan gum, either sterilized by autoclaving for 40 minutes or at a concentration of 17 g xanthan gum using sodium hypochlorite, was statistically the same as the control that contained agar. However, plant development at a concentration of 21g of xanthan gum in a sterilized medium using sodium hypochlorite was lower than that observed in media containing agar.

Francisella noatunensis modulates the hepatic profile of fatty acids in Patagonian blennie Eleginops maclovinus

Fatty acids (FA) have been reported to play a role in the defense against bacterial pathogens. However, studies in fish are limited and no information exists on the modulation of FA of Eleginops maclovinus challenged with the pathogen of aquaculture relevance Francisella noatunensis. We inoculated 144 fish intraperitoneally with 3 doses (low dose [LD]: 1 × 101 bact/μL; medium dose [MD]: 1 × 105 bact/μL; high dose [HD]: 1 × 1010 bact/μL) and a control dose (sterile culture medium, [C]) of the bacteria F. noatunensis and took liver samples at 1, 3, 7, 14, 21, 28 days post-inoculation (dpi). The LD of F. noatunensis did not present significant differences to the C, except for the C20:4n6 (ARA) at day 28, while the MD and HD of F. noatunensis did generate a significant effect of the FA profile of E. maclovinus. We observed a greater modulation after administering the HD of bacteria and detected significant differences from day 3 post-inoculation (P < 0.05). Significantly high levels of C18:0 was detected at day 28 (P < 0.05), while significantly low levels of C14:0, C20:0, C16:1, C18:1n9, C18:2n6; C18:3n3, and C20:5n3 were detected at different dpi of F. noatunensis (P < 0.05). ARA and C22:6n3 (DHA) showed a significant decrease during the first dpi (P < 0.05) but later they showed an increase in their levels, with ARA increasing its levels from day 14 (P < 0.05), while DHA increased its levels only at day 28 (P < 0.05). A large number of FA were highly correlated with each other and multivariate statistical analyzes (PCA and random forest) were able to detect the HD of F. noatunensis as the dose that most affects the FA profile of E. maclovinus, while the LD, MD and C cannot be differentiated in their FA profile. The FA most affected by the challenge with F. noatunensis were C18:2n3 and C18:3n3 (P < 0.05). Our results suggest a relationship between the bioavailability of FA in the liver and the level of the physiological response of E. maclovinus to an experimental challenge with F. noatunensis and suggest that hepatic FA modulation may explain or be part of the high tolerance to bacterial infections reported for E. maclovinus, although more studies are necessary to clarify the roles of the different types of FA on pathogen-host dynamics and its potential use in combating fish pathogenic bacteria.

First Report of Pythium aphanidermatum Causing Crown and Root Rot on Industrial Hemp in China.

In July 2020, symptoms of crown and root rot were observed on about 10% of 4-month-old plants of industrial hemp Cannabis sativa cultivar Yunma-1 in Weifang City, Shandong Province in eastern China (Fig 1). During this month, the local temperature ranged from 19-32°C, and the total precipitation was 148mm. The disease symptoms included leaf chlorosis, crown and root rot, stunted growth, and wilting (Figs. 1 and 2). The diseased stem and root tissues were collected and cut into fragments of 0.5cm each. The fragments were surface-sterilized by dipping into 1% NaClO for 1 min, rinsed in sterile water and plated on potato dextrose agar (PDA) and on oomycetes-selective medium PARP (Jeffers and Martin 1986). The plates were incubated at 25°C in the dark for 3 days and 18 total single-hyphal purified isolates were obtained for further analyses with 8 from PDA and 10 from PARP. The colonies of all 18 isolates were white, had abundant aerial hyphae, and were cottony in appearance, resembling Pythium spp (Watanabe 2002). The grass-leaf method (Van Der Plaats-Niterink 1981) induced their sexual reproduction. The size and shape of hyphae, oogonia, antheridia, and oospores were all consistent with those of Pythium aphanidermatum (Fig 3). DNA was extracted from three isolates and their internal transcribed spacer (ITS) regions of rDNA were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990). The ITS sequences of all three isolates were identical to each other (GenBank accession OK091124.1) and showed a 100% query coverage and 99.88% nucleotide sequence identity with that of type strain of P. aphanidermatum (GenBank accession AY598622.2). Pathogenicity tests were performed with three isolates on hemp cultivar B1. Sterile substrates were prepared in 2L-pots containing peat soil and vermiculite in a 2:1 ratio, with test hemp plants grown from rooted stem cuttings. Plants were kept in a greenhouse at 22 to 27°C under 16 h photoperiod, watered every two days (about 200ml each time) and supplied commercial nutrient solution once a week. A month after transplanting to pots, a wound of 1 mm deep and 10 mm long (made by a sterilized needle) on the surface of the root crown area of the main stem was inoculated with an 8-mm-diameter agar disk of mycelia grown on PDA for 4 days. Six plants were tested for each isolate and three plants were inoculated with sterile agar medium without mycelia as negative controls. The experiment was repeated twice. After one month, plants inoculated with P. aphanidermatum isolates showed the same disease symptoms as observed on field plants while all negative control plants remained disease-free. P. aphanidermatum was reisolated from the diseased tissue and confirmed to be identical to those inoculated based on ITS sequencing and colony morphology. To our knowledge, this is the first report of P. aphanidermatum causing crown and root rot on hemp in China. With an estimated 66,700 hectares hemp cultivation in China producing over US$1 billion worth of hemp fiber (McGrath 2020), this pathogen represents a serious threat to the hemp industry. This pathogen has been reported on hemp in the US and Canada (Beckerman et al. 2017; Punja et al. 2018). The origin of P. aphanidermatum on hemp in China and its relationship to those in North America remain to be examined.

Study of the effect of microbial addition in seed germination and seedling growth of Cryptocarya densiflora L

Cryptocarya densiflora L belongs to the Lauraceae family that is famous for its utilization of its leaves, stems, roots, and fruit. These species are also widely spread. The hardness of its seed coats inhibits its germination. Various efforts to accelerate the germination process by immersion in water or chemicals have done. However, the application of microbes has never been reported. Naturally, C. densiflora seeds fall to the ground directly in contact with soil microbes that might play a significant role in the germination process. This research aims to reveal the effect of inoculants’ application originated from soil microbes, Aspergillus niger, and natural microbial consortia in germination and growth of seedling C. densiflora. The experiments employed a completely randomized design using two main treatments, the inoculant formula, and the growing media. Inoculant formula factors consist of Formula 1 (A. niger), Formula 2 (microbial consortia), and Control, while the growing media consists of 3 types, A (soil and sand/1:1), B (soil, sand, and compost/1:1:1), and C (sand). All growing media were treated with 2 different treatments, sterilized (S) and non-sterilized (N). The growth stage was observed for 12 weeks after sowing with the following observed parameters: pH of the media at the end of the experiment, the percentage of sprout growth, plant height, number of leaves, root length, wet and dry weight of roots, and shoots. The results showed that the addition of microbial inoculant significantly affected the germination of C. densiflora seeds in all parameters measured, both for media A and B (sterile and non-sterile). Formula 1 gave the best results on all media, especially sterile media with parameters of plant height, number of leaves, root length, wet and dry weight of roots and shoots. The presence of microbes significantly affected germination and C. densiflora seedling growth.

The effect of a biofilm-forming bacterium Tenacibaculum mesophilum D-6 on the passive film of stainless steel in the marine environment

The microbiologically influenced corrosion of 304 stainless steel in the presence of a marine biofilm-forming bacterium Tenacibaculum mesophilum D-6 was systematically investigated by means of electrochemical techniques and surface analyses to reveal the effect of the selective attachment and adsorption of the biofilms on the passivity breakdown of the stainless steel. It was found that the T. mesophilum D-6 was electroactive and could oxidize low valent cations and metal, facilitating the local dissolution of the passive film and the substrate in the film defects, nearly doubling the surface roughness. The biofilms of T. mesophilum D-6 with mucopolysaccharide secreta and chloride ions tended to preferentially adsorb at the defects of the passive film on the steel, yielding non-homogeneous microbial aggregates and local Cl− enrichment there. The adsorption of the bacteria and chloride ions reduced the thickness of passive film by 23.9%, and generate more active sites for pitting corrosion on the passive film and more semiconducting carrier acceptors in the film. The maximum current density of the 304 SS in the presence of T. mesophilum D-6 was over one order of magnitude higher than that in the sterile medium, and the largest pit was deepened 3 times.

Do Microbiota in the Soil Affect Embryonic Development and Immunocompetence in Hatchling Reptiles?

Reptile eggs develop in intimate association with microbiota in the soil, raising the possibility that embryogenesis may be affected by shifts in soil microbiota caused by anthropogenic disturbance, translocation of eggs for conservation purposes, or laboratory incubation in sterile media. To test this idea we incubated eggs of keelback snakes (Tropidonophis mairii, Colubridae) in untreated versus autoclaved soil, and injected lipopolysaccharide (LPS) into the egg to induce an immune response in the embryo. Neither treatment modified hatching success, water uptake, incubation period, or white-blood-cell profiles, but both treatments affected hatchling size. Eggs incubated on autoclaved soil produced smaller hatchlings than did eggs on untreated soil, suggesting that heat and/or pressure treatment decrease the soil’s suitability for incubation. Injection of LPS reduced hatchling size, suggesting that the presence of pathogen cues disrupts embryogenesis, possibly by initiating immune reactions unassociated with white-blood-cell profiles. Smaller neonates had higher ratios of heterophils to leucocytes, consistent with higher stress in smaller snakes, or body-size effects on investment into different types of immune cells. Microbiota in the incubation medium thus can affect viability-relevant phenotypic traits of hatchling reptiles. We need further studies to explore the complex mechanisms and impacts of environmental conditions on reptilian embryogenesis.

Autochthonous and allochthonous lactic acid bacteria: action on the hematological and intestinal microbiota for two species of Astyanax genus.

The objective of this work was to evaluate the effect of autochthonous and allochthonous lactic acid bacteria (LAB) in two species of lambaris (Astyanax bimaculatus and Astyanax fasciatus), and to investigate the effects on intestinal microbiota and hematological changes. Two experiments were carried out, one for each lambari species, both assays were divided into three treatments: autochthonous LAB, allochthonous LAB and control. The 10% inoculum was included on diet in the LAB treatments and sterile medium for control. After 30 days for A. bimaculatus LAB indigenous changes all bacteria groups analyzed, while allochthonous LAB just decrease Staphylococcus spp. count. Though for A. fasciatus his autochthone LAB reduced the staphylococcal count. In hematology, for A. bimaculatus autochthonous LAB showed a higher number of thrombocytes, lymphocytes, monocytes and total leukocytes in the circulatory system than the control. Though for A. fasciatus his autochthone LAB showed a higher number of total lymphocytes and leukocytes than the control, while Lactobacillus sp. acting as an allochthone, it did not differ among treatments. In conclusion, both LAB (Lactobacillus sp. and L. lactis) promoted more beneficial changes in the microbiota and hematological profile when they act as an autochthone probiotic, demonstrating a probiotic-associated host.

New insights into the role of phytopathogenic fungi vectored by pine bark beetles in pine decline

The study presented was carried out in response to the alarmingly increasing number of outbreaks of bark beetles and associated fungi in European forests including Ukraine. Our study was aimed to generate new knowledge on bark beetles and understand the possible role of vectored fungi. The obtained data can contribute to the increased knowledge and will allow adequately responsing to an expected intensification of bark beetle disturbances, as well as may allow better understanding tree diseases and how to cope with them. In total, eleven ophiostomatoid fungi (Graphium sp., Leptographium sosnaicola, Grosmannia olivacea, Grosmannia penicillata, Ophiostoma bicolor, O. ips, O. canum, O. piceae, O. minus, O. nigrocarpum Graphilbum rectangulosporium) were found here in association with five species of bark beetles (Hylurgus ligniperda, Hylastes ater, Hylastes angustatus, Ips acuminatus and Ips sexdentatus) on Scots pine trees infested by bark beetles. Scots pine seedlings were inoculated with ophiostomatoid fungi and sterile medium (control) to evaluate their pathogenicity. The inoculated seedlings were examined finally 6 months later after inoculation. Inoculation with Leptographium sosnaicola, Graphium sp and O. minus produced significantly large lesions and mortality of pine seedlings. In total, all the eight fungal-inoculated species caused resin exudation and staining on the bark around inoculations in Scots pine seedlings and different rate of the seedlings decline.&#x0D; It can be concluded that the studied bark beetles are vectors for different functional groups of fungi including aggressive pathogens, and that ophiostomatoid fungi are the most closely associated symbionts. Moreover, high virulence fungi can help bark beetles to overcome the tree resistance and may significantly contribute to tree mortality.