Sterile Discs Research Articles

Antimicrobial activity of red wine and oenological extracts against periodontal pathogens in a validated oral biofilm model

BackgroundPrevious research findings support an antimicrobial effect of polyphenols against a variety of pathogens, but there is no evidence of this effect against periodontal pathogens in complex biofilms. The purpose of this study was to evaluate the antimicrobial activity of red wine and oenological extracts, rich in polyphenols, against the periodontal pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum and total bacteria growing in an in vitro oral biofilm static model.MethodsA previously validated biofilm model, including Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was developed on sterile hydroxyapatite discs. Red wine (and dealcoholized wine), and two polyphenols-rich extracts (from wine and grape seeds) were applied to 72 h biofilms by dipping the discs during 1 and 5 min in the wine solutions and during 30 s and 1 min in the oenological extracts. Resulting biofilms were analyzed by confocal laser scanning microscopy and viable bacteria (colony forming units/mL) were measured by quantitative polymerase chain reaction combined with propidium monoazide. A generalized linear model was constructed to determine the effect of the tested products on the viable bacterial counts of A. actinomycetemcomitans, P. gingivalis and F. nucleatum, as well on the total number of viable bacteria.ResultsThe results showed that red wine and dealcoholized red wine caused reduction in viability of total bacteria within the biofilm, with statistically significant reductions in the number of viable P. gingivalis after 1 min (p = 0.008) and in A. actinomycetemcomitans after 5 min of exposure (p = 0.011) with red wine. No evidence of relevant antibacterial effect was observed with the oenological extracts, with statistically significant reductions of F. nucleatum after 30 s of exposure to both oenological extracts (p = 0.001).ConclusionsAlthough moderate, the antimicrobial impact observed in the total bacterial counts and counts of A. actinomycetemcomitans, P. gingivalis and F. nucleatum, encourage further investigations on the potential use of these natural products in the prevention and treatment of periodontal diseases.

Survival of acid-adapted and non-adapted Shiga toxin-producing Escherichia coli using an in vitro model

Shiga toxin-producing Escherichia coli (STEC) are known to adapt and survive acidic conditions. Acid-adaptation also may result in cross-protection against other stressors present in foods, such as reduced water activity (aw), refrigeration conditions, or the presence of antimicrobials. Three independent experiments were conducted with non-adapted (control) and acid-adapted E. coli O157:H7 and non-O157:H7 serogroups (E. coli O26, O45, O103, O111, O121 and O145) in vitro and in situ. Experiment 1: STEC cocktails in media were left untreated (non-adapted; control) or adapted (pH 5.0), exposed to mild acidic conditions (pH 4.5) or reduced aw (0.88 and 0.75) for 4 days at 24 °C, and evaluated for survival. Experiment 2: Non-adapted and acid-adapted STEC cocktails were subjected to desiccation (28 days at 24 °C) on sterile paper disks, and remaining pathogen populations were determined. Experiment 3: Control and acid-adapted STEC were subjected to mild acidic conditions (pH 4.5) and low aw (0.88 or 0.78) in ground beef slurries (GBS; 4 days at 24 °C) and surviving populations enumerated. Results from experiment 1 demonstrated that populations of non-O157:H7 STEC serogroups O45, O103, O111 and O121 were significantly different from populations of E. coli O157:H7, and non-O157:H7 STEC serogroups O26 and O145. In contrast, when STEC serogroups were acid-adapted, all populations of non-O157:H7 STEC were significantly different from populations of E. coli O157:H7. Results of experiments in media with aw of 0.88 or 0.78 demonstrated that there was no significant difference between E. coli O157:H7 and any of the non-O157:H7 STEC, regardless of acid-adaptation. In experiment 2, exposure to desiccation resulted in no significant difference in survival between E. coli O157:H7 and non-O157:H7 STEC, regardless of acid-adaptation. Experiment 3 in acidified GBS demonstrated that all non-O157:H7 STEC behaved similarly to E. coli O157:H7, except for E. coli O145, which had a higher reduction than O157:H7. There were no significant differences in bacterial populations associated with GBS with modified aw. Collectively, these results suggest than non-O157:H7 STEC behave similarly to E. coli O157:H7 when exposed to low pH (<4.5) and low aw (0.88 or 0.78) under laboratory conditions.

The Effect of Cinnamon Essential Oil on Lactobacillus species

Antimicrobial properties of cinnamon essential oil solutions (EO) have been identified against pathogenic bacteria, but its effects on commensal microorganisms have not yet been described. In previous studies in our laboratory, cinnamon EO was tested against Salmonella Typhimurium and Escherichia coli utilizing the disc diffusion method. Zones of inhibition were measured, and cinnamon EO at 2 and 4% displayed great effectiveness against these microorganisms. Both microorganisms are Gram negative rods and have the capability to cause disease. Thus, the main objective of this study is to evaluate the effect of cinnamon EO on the commensal Gram positive microorganisms Lactobacillus acidophilus and Lactobacillus rhamnosus.The Lactobacillus acidophilus (ATCC® 4356™) and Lactobacillus rhamnosus (ATCC® 7469™) inoculums were prepared by growing the bacteria in MRS (De Man, Rogosa and Sharpe) broth for 24h under microaerophilic conditions (5% CO2) at 37°C, yielding 6.9 ×108 CFU/ml. One hundred microliters of starting bacterial culture was spread onto MRS agar plates and dried for 15 minutes. Treatments were formed by creating cinnamon EO solutions of 0, 0.5, 1, 2 and 4% using 70% ethyl alcohol as the diluent. Each EO was vortexed and filtered using a 0.2 microliter sterile syringe filter. Twenty microliters of each EO were pipetted into a sterile paper disc and left to dry for 1h. Five paper discs of each treatment containing the EO were placed on standardized locations on each inoculated plate in duplicate. Additionally, three manufactured standard antibiotic paper discs of 5 mcg ciprofloxacin, 10mcg ampicillin, and 10mcg streptomycin were placed on separate plates in duplicate as controls. The zone of inhibition created by each EO or antibiotic was measured using a calibrated digital caliper. All data were analyzed using GLM procedure for ANOVA and Person correlation analysis.The antimicrobial property of the EO observed as the zone of inhibition increased as the concentration of the EO increased for Lactobacillus acidophilus (r=0.86, p&lt;0.0001). EO at 4% showed the greatest antimicrobial effect against Lactobacillus acidophilus compared to 2.0, 1.0, and 0.5% (2.59, 2.11, 0.0 and 0.0mm, respectively; p&lt;0.0001). Lactobacillus rhamnosus showed resistance to EO at all concentrations studied with no zones of inhibition observed. All antibiotics had some effect against Lactobacillus rhamnosus, where ampicillin had the greatest effect compared to ciprofloxacin and streptomycin (6.62, 6.27, and 2.33mm, respectively; p&lt;0.0001). Ampicillin had the greatest effect against Lactobacillus acidophilus compared to streptomycin (9.72 vs. 2.8mm, respectively; p&lt;0.0001). However, L. acidophilus was shown to be resistant to ciprofloxacin with no zone of inhibition formed.In conclusion, Lactobacillus species were generally resistant to cinnamon EO. These Lactobacillus species are commonly used as probiotics and are often adversely affected by antibiotic intake. Using cinnamon essential oil as an antibiotic alternative has shown to be a promising option with limited detrimental effects against the commensal bacteria. Further evaluation of the effects of cinnamon EO on commensal bacteria is needed to further determine the benefits of utilizing a natural alternative.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

First Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in Italy

HomePlant DiseaseVol. 103, No. 4First Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in Italy PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in ItalyI. Camele and S. M. MangI. Camele†Corresponding author: I. Camele; E-mail: E-mail Address: ippolito.camele@unibas.ithttp://orcid.org/0000-0001-8099-8234School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, ItalySearch for more papers by this author and S. M. MangSchool of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, ItalySearch for more papers by this authorAffiliationsAuthors and Affiliations I. Camele † S. M. Mang School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, Italy Published Online:13 Feb 2019https://doi.org/10.1094/PDIS-09-18-1686-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In spring 2018, in a vineyard (7 ha) located in Matera province (southern Italy), during a survey to verify the sanitary status of grapevines in Basilicata Region, Vitis vinifera plants (25-year-old, cv. Sangiovese) were found with typical “esca disease” symptoms on the leaves (tiger stripes). In addition, trunk cankers and internal wood necrosis were observed. More than 30% of the living plants were symptomatic and 10% more were dead. To isolate the likely pathogen, small pieces from trunk discs of living, symptomatic plants were surface sterilized with 70% alcohol, plated in Petri dishes containing potato dextrose agar (PDA), and incubated at 24°C for 14 days in darkness. From all samples, only a single fungal species was isolated, and this was identified as Seimatosporium vitis X.P. Xiao, Camporesi E. & K.D. Hyde through morphological comparison (Senanayake et al. 2015) and a molecular analysis based on three genes including the rDNA-ITS region (ITS), β-tubulin-2 (TUB2), and translation elongation factor 1-α (TEF1-alpha). The sequences of four pure-culture isolates were deposited in EMBL GenBank under accession numbers LS991528 to LS991531 (ITS), LS997596 to LS997599 (TUB-2), and LS999502 to LS999505 (TEF1-alpha). All sequences showed 99 to 100% identity with authentic S. vitis sequences already present in the GenBank database under the following accession numbers: ITS (KY632661, KX555652, and KU721882), TUB-2 (KY706248, KY706254, and KY706257), and TEF1-alpha (KY06328, KY06331, and KY06332). A phylogenetic tree, based on combined ITS, TUB-2, and TEF1-alpha data, was obtained using MEGA version 6.0 with the maximum parsimony analyses revealing that all isolates fell in the S. vitis clade, confirming the species identification. Koch’s postulates were fulfilled using four S. vitis isolates inoculated on woody stems of 2-year-old potted cultivar Sangiovese. Shoots were surface sterilized with 70% alcohol and then wounded with a sterile scalpel. Discs (0.5-mm diameter) from 14-day-old agar cultures were applied to the wound and sealed with Parafilm, and then the plants were kept in a growth chamber at 24°C and 90% humidity and 16-h light. Sterile PDA discs were used as controls. Each isolate was inoculated onto 10 plants, and five others were used as controls (45 plants in total). The pathogenicity test was carried out twice. About 45 days after inoculation, each inoculated shoot presented a canker lesion about 3 to 4 cm long around the inoculation point and interveinal chlorosis on leaves. Control plants were always symptomless. Only S. vitis was consistently reisolated from inoculated tissues. Pathogenicity assays confirmed that S. vitis from V. vinifera plants is the causal agent of the observed symptoms in grapevine. S. vitis was reported to be associated with grapevine trunk diseases (GTDs) in Hungary (Váczy 2017) and in California (Lawrence et al. 2018). Senanayake et al. (2015) had earlier reported S. vitis from Italy but not in association with GTDs and without pathogenicity tests. This is the first report of S. vitis associated with the GTDs in Italy.

The Effect of Cinnamon Essential Oil on Escherichia coli

Antimicrobial properties of cinnamon essential oil have been identified against foodborne bacteria, but its effectiveness has not yet been fully determined. In a previous study, cinnamon essential oil at concentrations of 2% and 4% showed to be effective against Salmonella enterica serovar Typhimurium. However, its effect on a commensal, opportunistic and specialized pathogen like E. coli has not been completely described. Thus, the main objective of this study is to evaluate the effectiveness of cinnamon essential oil on Escherichia coli.The Escherichia coli (ATCC 25404™) inoculum was prepared by growing the bacteria in Luria‐Bertani (LB) broth for 24 hours at 37°C, yielding 8.7×109 CFU/ml. One hundred microliters of the starting bacterial culture were spread onto LB agar plates and left to dry for 15 minutes. Treatments were formed by creating cinnamon essential oil solutions (EO) of 0, 0.5, 1, 2 and 4% using 70% ethyl alcohol as the diluent. Each EO was vortexed and filtered using a 0.2 microliter sterile syringe filter. Twenty microliters of each EO was pipetted into a sterile paper disc (weight ranged of 0.0086–0.0090g) and left to dry for one hour. Five paper discs of each treatment containing the EO were placed on standardized locations on each inoculated plate in duplicate. The plates were then incubated at 37°C for 24 hours. Additionally, three manufactured standard antibiotic paper discs of 5 mcg ciprofloxacin, 10mcg ampicillin, and 10mcg streptomycin were placed on separate plates in duplicate as positive controls. The zone of inhibition created by each EO or antibiotic was measured using a calibrated digital caliper. All data were analyzed using the general linear model procedure for analysis of variance (ANOVA) and Pearson correlation analysis.The antimicrobial property of the EO observed as the zone of inhibition increased as the concentration of the EO increased (r= 0.922, p&lt;0.0001). EO at 4% showed the greatest antimicrobial effect against Escherichia coli compared to 2, 1, and 0.5% (6.7, 5.0, 0.88 and 0.0mm, respectively; p&lt;0.0001). There was no significant difference (p&gt;0.05) between the zone of inhibition of 0% and 0.5% EO. However, 2% EO showed increasing effectiveness against Escherichia coli with a zone of inhibition of 5.1mm (p&lt;0.0001). The 2% EO concentration showed a greater zone of inhibition against E. coli than the ampicillin treatment (5.1 vs. 2.3mm, p&lt;0.0001). All antibiotics had some effect against E. coli, and ciprofloxacin had the greatest effect; its effect was even greater than the 4% EO (7.6 vs. 6.7mm, p&lt;0.0001). The 4% EO was not as effective as ciprofloxacin, but it was more effective than both streptomycin and ampicillin (6.7 vs. 6.1 and 2.3mm, respectively; p&lt;0.0001).In conclusion, cinnamon EO showed increasing antimicrobial properties against Escherichia coli as the concentration of oil increased. It proved to be more effective than two of the antibiotic controls at 4% concentration. Further evaluation of the effects of cinnamon EO is needed to determine the extent of its effectiveness against additional commensal and pathogenic organisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antimicrobial Properties of Cinnamon Essential Oil on Salmonella Typhimurium

Essential oils have a wide range of properties due to various chemical compounds in their composition. Potential applications have been identified in areas such as food preservation, natural alternatives to antibiotics, and disease prevention and treatments. More specifically, the antimicrobial properties of cinnamon (Cinnamomum sp.) oil have been established against foodborne bacteria, but the effectiveness has not yet been defined at various concentrations under standardized conditions. Therefore, the main objective of this study was to evaluate the effect of cinnamon essential oil on Salmonella enterica ser. Typhimurium (Salmonella Typhimurium) using the disc diffusion procedure.The Salmonella Typhimurium (ATCC® 53648™) inoculum was prepared by growing the bacteria in tryptic soy broth for 24 hours at 37°C, yielding 6.9 × 108 CFU/ml. One hundred microliters of the starting bacterial culture were spread onto Mueller Hinton agar plates and left to dry for 15 minutes. Treatments were formed by creating cinnamon essential oil solutions (EO) of 0, 0.5, 1, 2 and 4% using 70% ethyl alcohol as the diluent. Each EO was vortexed and filtered using a 0.2 microliter sterile syringe filter. Twenty microliters of each EO was pipetted into a sterile paper disc (weight ranged of 0.0086–0.009g) and left to dry for one hour. Five paper discs of each treatment containing the EO were placed on standardized locations on each inoculated plate in duplicate. The plates were then incubated at 37°C for 24 hours. Additionally, three manufactured standard antibiotic paper discs of 5 mcg Ciprofloxacin, 10 mcg Ampicillin, and 10 mcg Streptomycin were placed on separate plates in duplicate as controls. The zone of inhibition created by each EO or antibiotic was measured using a calibrated digital caliper. All data were analyzed using the general linear model procedure for analysis of variance (ANOVA).The antimicrobial property of the EO observed as the zone of inhibition increased as the concentration of the EO increased (R= 0.884, P &lt; 0.0001). EO at 4% showed the greatest antimicrobial effect against Salmonella Typhimurium compared to 2.0, 1.0, and 0.5% (10.5, 6.0, 4.2 and 4.6 mm, respectively; P &lt; 0.0001). There was no significant difference (P=0.4) between the zone of inhibition of 0.5% and 1% EO. However, 2% EO showed a significantly larger zone of inhibition than 0.5% and 1% EO (P&lt;0.01). The 2% EO concentration showed a greater zone of inhibition against Salmonella than ampicillin and streptomycin treatments (6.0 versus 4.7 and 3.4 mm respectively; P&lt; 0.0001). All antibiotics had some effect against Salmonella where ciprofloxacin had the greatest effect; its effect was even greater than the 4% EO (14.0 vs. 10.5 mm, P&lt; 0.0001).In conclusion, cinnamon EO showed increasing antimicrobial properties against Salmonella Typhimurium as the concentration of oil increased. It proved to be more effective than two of the antibiotic controls as low as 2% concentration. Further evaluation of the effects of cinnamon EO is needed to determine the extent of its effectiveness against additional foodborne pathogens.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

First record of Melampodium divaricatum (Asteraceae) in West Tropical Africa

A plant that showed morphological closeness to Aspilia africana (Pers) C. D. Adams (Asteraceae) was spotted and collected in 2015 along Afe Babalola University road, Ado‐Ekiti, Ekiti State, Nigeria with coordinates 7°36′59.99″N, 5°12′60.00″E. However, upon closer observation some distinct and peculiar characteristics that clearly distinguished it from Aspilia africana were revealed, e.g. sterility of the disc florets and production of achenes by ray florets only. Another striking character of the plant was total emptying of the capitulum after achene maturation, leaving an empty capitulum cup on the plant. Literature and herbarium searches revealed that the plant had neither been reported from West Tropical Africa nor collected in any herbarium in Nigeria before. The plant was eventually identified as Melampodium divaricatum (L.) which is an annual erect herb, distributed in tropical and subtropical regions but mostly restricted to Mexico, North America and Central America. Morphological, reproductive and cytological studies carried out on the plant revealed it to possess a highly branched erect pigmented stem, simple opposite sub sessile leaves with acute apex and distantly serrated margins, capitula with yellow unisexual disc and ray florets, sterile disc florets, fertile ray florets, relatively high pollen fertility (92.85%), a somatic chromosome number of 2n = 24 and regular formation of 12 bivalents, indicating the plant to be a diploid species. Further studies on Melampodium in Nigeria and a general revision of the flora of West Tropical Africa is suggested as well as the need to monitor M. divaricatum in the region since it appears to have the capacity to become invasive.

The effectiveness of red betel leaf (Piper crocatum) extract against periodontal pathogens

Introduction: Red betel (Piper crocatum) has long been known as one of herbs that has antibacterial properties as it contains several useful compounds such as essential oils, flavonoid, alkaloids and tannins.  The objective of this study is to learn more about the effectiveness of red betel leaf extract based on in vitro test against periodontal pathogen bacteria such as Aggregatibacter actinomycetemcommitans (Aa) and Porphyromonas gingivalis (Pg).Method: This study was conducted using diffusion method that has Kirby-Bauer test sensitivity. The experiment was performed eight times on four treatments consisting of 10% DMSO as negative control and 2.5%, 5% and 10% of red betel leaf extract. The sterile paper disc was soaked into sterile water and red betel leaf extract at each concentration before evaporation inside the incubator.  After that, the dry paper disc was placed in the Nutrient Agar (NA) that had previously been inoculated and incubated for 24 hours at 37° Celsius. ANOVA test was used to compare inhibition zone in four group intervention.Result:  The result of the study shows that the 2.5%, 5% and 10% concentration of red betel leaf extract respectively could inhibit the growth of bacteria Aa and Pg. The result of the study also confirms that the 10% red betel leaf extract concentration created the largest inhibition zone (10.5786 mm for bacteria Aa and 10.7638 mm for bacteria Pg).Conclusion: Red betel leaf extract, particularly at 10% concentration is significantly effective in inhibiting the growth of Aa and Pg bacteria.Â

Effects of Er: YAG, 980 nm and 810 nm Diode Lasers Irradiation on Biocompatibility of SLA Titanium Disks Using SaOs2 Cells Morphology

Objectives The use of lasers for the treatment of periimplant hard and soft tissues is now refmore than ever before. Achieving bone integrity. The aim of this study is to evaluate the effects of Er:YAG, 980 nm and 810 nm diode lasers irradiation on biocompatibility of SLA titanium disks by SaOs2 cells morphology.Methods In this in-vitro study sixty sterile titanium disks with SLA surface were divided into four equal subgroups. One subgroup was used as a control group and the remaining three groups were irradiated with Er:YAG laser 980nm and 810nm Doide lasers, separately. After laser irradiation, all discs were autoclaved at 121° C and placed in 24 appropriate plates. The SaOs2 cells were, then, added to the plates at a density of 2×104. The cells were incubated in DMEM, CO2, and penicillin-streptomycin medium at 37 ° C for 3 days. Then, the samples were extracted from the culture medium for scanning electron microscopy. Finally, the photograph was taken by SEM at magnifications of 750, 1000, 3000, and 5000. The analyses were performed through Kruskal-Wallis and Mann-Whitney tests.Results All three groups, irradiated by laser, and the control group have shown spreading cells with plentiful phylopodia, which means the morphology of a mature bone cell. The numbers were 20.7% (Er:YAG), 52.7% (980nm Diode), 48.8% (810nm Diode) and 38.7% (Control) groups, respectively, which were not statistically significant.Conclusion Er:YAG, 980nm and 810nm diode laser irradiations with the parameters mentioned in this study do not have any negative effects on osteoblast cells attachment and their maturity on titanium implants.

First Report of Curvularia asianensis Causing Leaf Blotch of Epipremnum pinnatum in Guangxi Autonomous Region of China

Epipremnum pinnatum Engl. is a flowering plant in the family Araceae. In southern China, it is often cultivated for medicinal and ornamental purposes. A distinct leaf blotch was observed on E. pinnatum in October 2017. Symptoms included gray or tan irregular blotches, with reddish to yellow margins. Portions of the symptomatic leaves were surface sterilized in 70% ethanol for 30 s and then in 2% NaClO for 1 min, rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). Seven strains (GUCC 8601 to GUCC 8607) from four diseased E. pinnatum plants were obtained by single spore selection. Fungal colonies on PDA reached 70 to 75 mm in diameter after 7 days, were velvety to slightly powdery, and were gray olivaceous to olivaceous black both top and reverse. Conidiophores were single or in groups, septate, straight or flexuous, and geniculate. Conidiogenous cells were smooth walled to finely verruculose, terminal or intercalary, proliferating sympodially, pale brown to brown, subcylindrical to swollen, and 6 to 14 × 3 to 5 μm (n = 30). Conidia were curved, pale brown to brown, 3 to 4 distoseptate, 23 × 9 μm (n = 50) with a protuberant hilum; morphology was consistent with Curvularia asianensis Manamgoda, L. Cai & K. D. Hyde, isolated from a dried Panicum sp. leaf in northern Thailand (Manamgoda et al. 2012). The internal transcribed spacer (ITS) region and partial sequences of two protein-coding genes, namely, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-α (TEF1), were amplified using primers ITS1 and ITS4 (White et al. 1990), gpd1 and gpd2 (Berbee et al. 1999), and EF983F and 2218R (Schoch et al. 2009). The ITS, GAPDH, and TEF1 sequences were compared with entries in GenBank database, through nucleotide BLAST search, and our isolates showed higher than 99% identity to the ex-type strain (MFLUCC 10-0711) of C. asianensis (ITS, JX256424; GAPDH, JX276436; and TEF1, JX266593) for these three gene regions. The three gene regions were combined into a multigene phylogenetic tree. On the basis of morphological characteristics and nucleotide homology, our isolates were confirmed as C. asianensis. For pathogenicity tests, mycelial disks (5 mm in diameter) cut from 1-week-old PDA cultures of two strains (GUCC 8601 and GUCC 8602) were placed on four healthy leaves (two inoculated points on every leaf) of an E. pinnatum plant under natural conditions on 20 November 2017 and removed after 48 h. Leaves inoculated with sterile PDA disks served as controls. Within 24 h after discs were removed, tan lesions began to appear on the leaves. Seven days after inoculation, all inoculated leaves developed blotch symptoms consistent with what was observed in nature. C. asianensis strains were reisolated from infected tissues and confirmed by the methods mentioned above. Controls remained healthy. To our knowledge, this is the first report of C. asianensis causing leaf blotch disease on E. pinnatum in China.

Anthocyanin as potential source for antimicrobial activity in Clitoria ternatea L. and Dioscorea alata L.

PurposeThe purpose of this paper is to validate the antimicrobial activity (both antibacterial and antifungal) of in vivo and in vitro ethanolic anthocyanin extracts of Clitoria ternatea L. (vivid blue flower butterfly-pea) and Dioscorea alata L. (purple yam) against selected bacteria (Bacillus subtilis, Staphylococcus aureus and Escherichia coli) and fungi (Fusarium sp., Aspergillus niger and Trichoderma sp.).Design/methodology/approachThe freeze-dried samples (0.2 g) from in vivo vivid blue flowers of C. ternatea L. were extracted using 10 mL ethanol (produced ethanolic red extraction) and 10 mL distilled water (produced aqueous blue extraction) separately. Two-month-old in vitro callus samples (0.2 g) were only extracted using 10 mL ethanol. The anthocyanin extractions were separated with the addition (several times) of ethyl acetate and distilled water (1:2:3) to remove stilbenoids, chlorophyll, less polar flavonoids and other non-polar compounds. Furthermore, the antimicrobial properties were determined using agar diffusion technique. Three bacteria (B. subtilis, S. aureus and E. coli) and fungi (F. sp., A. niger and T. sp.) were streaked on bacteria agar and dextrose agar, respectively, using “hockey stick”. Then, the sterile paper discs (6 mm diameter) were pipetted with 20 µL of 1,010 CFU/mL chloramphenicol (as control for antibacterial) and carbendazim (as control for antifungal) in vivo and in vitro extracts. The plates were incubated at room temperature for 48 h, and the inhibition zones were measured.FindingsBased on the results, both in vivo and in vitro ethanolic extracts from vivid blue flowers of C. ternatea L. showed the best antibacterial activity against the same bacteria (B. subtilis), 11 and 10 mm inhibition zones, respectively. However, different antifungal activity was detected in in vitro ethanolic callus extract (12 mm), which was against T. sp., contrary to in vivo ethanolic extract (10 mm), which was against F. sp.; antibacterial activity of D. alata L. was seen against the same bacteria (E. coli) with the highest inhibition zone for in vivo extract (8.8 mm), followed by in vitro extract (7.8 mm).Research limitations/implicationsAnthocyanins are responsible for the water soluble and vacuolar, pink, red, purple and blue pigments present in coloured plant pigments. These pigments (pink, red, purple and blue) are of important agronomic value in many crops and ornamental plants. However, anthocyanins are not stable and are easy to degrade and fade whenever exposed to light.Social implicationsPlant extracts containing bioactive agents with antimicrobial properties have been found to be useful in treating bacterial and fungal infections, as well as showed multiple antibiotic resistance.Originality/valueBoth in vivo and in vitro extracts from vivid blue flower petals (C. ternatea L.) and purple yam (D. alata L.) have important applications as natural antimicrobial (antibacterial and antifungal) agents in the coating industry, instead of natural pharmaceutical products.

First Report of Fusarium Species Associated with Fusarium Wilt in Coffea canephora Plants in Brazil.

HomePlant DiseaseVol. 102, No. 9First Report of Fusarium Species Associated with Fusarium Wilt in Coffea canephora Plants in Brazil PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Fusarium Species Associated with Fusarium Wilt in Coffea canephora Plants in BrazilL. L. Belan, L. L. Belan, A. M. Rafael, R. M. Lorenzoni, F. B. Souza-Sobreira, T. C. B. Soares, F. L. de Oliveira, and W. B. MoraesL. L. Belanhttp://orcid.org/0000-0002-7966-4963Search for more papers by this author, L. L. Belanhttp://orcid.org/0000-0002-3741-4173Search for more papers by this author, A. M. RafaelSearch for more papers by this author, R. M. LorenzoniSearch for more papers by this author, F. B. Souza-SobreiraSearch for more papers by this author, T. C. B. SoaresSearch for more papers by this author, F. L. de OliveiraSearch for more papers by this author, and W. B. Moraes†Corresponding author: W. B. Moraes; E-mail: E-mail Address: willian.moraes@ufes.brhttp://orcid.org/0000-0001-7478-7772Search for more papers by this authorAffiliationsAuthors and Affiliations L. L. Belan L. L. Belan A. M. Rafael , Department of Agronomy, CCAE-UFES, 29500-000 Alegre-ES, Brazil R. M. Lorenzoni F. B. Souza-Sobreira , Graduate Program in Vegetal Production, CCAE-UFES, 29500-000 Alegre-ES, Brazil T. C. B. Soares , Department of Pharmacy and Nutrition, UFES, 29500-000 Alegre-ES, Brazil F. L. de Oliveira W. B. Moraes † , Department of Agronomy, CCAE-UFES, 29500-000 Alegre-ES, Brazil. Published Online:24 Jul 2018https://doi.org/10.1094/PDIS-01-18-0186-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Coffea canephora plants have been wilting and dying in major producing regions of Brazil; however, the disease etiology is unknown. Coffee clones differ in susceptibility, and disease occurs throughout the year. The symptoms include wilting, leaf yellowing, defoliation, vascular tissue darkening, and drying and death of plagiotropic and orthotropic branches and the plant. To identify the etiological agent, vascular tissue fragments from symptomatic plants collected in the Jerônimo Monteiro municipality (20°53′28″S, 41°25′59″W) were surface sterilized, placed on carrot baits, and incubated at 26°C. The resulting mycelia were transferred to potato dextrose agar (PDA) medium and incubated at 26°C in 12-h light/dark for 10 days. Monosporic cultures from four isolates were prepared and characterized (Booth 1971; Summerell et al. 2003). A TEF-1α gene fragment was amplified (O’Donnell et al. 2000). The polymerase chain reaction products were sequenced by capillary electrophoresis (ABI3730) using POP7 polymer and BigDye version 3.1 (Myleus Biotechnology). We identified three species of Fusarium using BLAST analysis: F. decemcellulare and F. lateritium with 100% sequence similarity to GenBank accessions numbers KC491872.1 and AY707155.1, respectively, and F. solani with 99% similarity (DQ247549.1). F. decemcellulare colonies were cottony and pinkish to yellowish, with yellow or cream globular sporodochia; macroconidia (70.4 to 99.3 × 7.2 to 8.1 μm) were fusiform, with a beaked apical cell, a distinct pedicellate foot cell and 6 to 9 septa; microconidia (7.3 to 12.5 × 3.6 to 6.1 μm) were hyaline, oval, with a flattened basal papilla and 0 to 1 septum. F. lateritium colonies showed slow growth, with white to peach aerial mycelium; macroconidia (33.5 to 46.0 × 3.9 to 5.4 μm) with a beaked apical cell and 3 to 5 septa; microconidia (8.6 to 17.4 × 2.7 to 4.3 μm) were fusiform with 0 to 1 septum. F. solani colonies showed rapid growth and white to cream colored aerial mycelium with flat and globular cream-colored sporodochia; macroconidia (29.5 to 50.3 × 5.0 to 8.1 μm) were hyaline with 3 septa; microconidia (9.6 to 14.9 × 4.0 to 6.3 μm) were hyaline, unicellular, and oblong, with 0 to 2 septa. Coffee plants (4 months old) of clone CV02 and plantlets (40 days old) of Robusta Tropical cultivar were inoculated. The inoculation was realized with a monosporic isolate of each species. Ten coffee plants were inoculated with PDA disks containing 10-day-old colony, or sterile PDA disk (control), inserted into a stem puncture. The plants were maintained in a humid chamber for 48 h (100% relative humidity). The roots of 10 coffee plantlets were immersed for 30 min in a conidia suspension (106 conidia/ml) or in sterile water (control) prior to transplantation. The first symptoms of impaired growth, vascular tissue darkening, leaf chlorosis, wilting, and defoliation appeared 45 days after inoculation of coffee plants. Inoculated plantlets wilted and died after 65 days. Control plants and plantlets did not develop symptoms. The fungi reisolated from diseased plants were similar to those isolated from the original host. Another Fusarium sp. has been reported with this host in Puerto Rico, Virgin Islands, Fiji, and Cote d’Ivoire, but to our knowledge, this is the first report of F. decemcellulare, F. lateritium, and F. solani as etiological agents of Fusarium wilt in C. canephora. Studies on this pathosystem are necessary considering that the size of the area affected and losses are increasing, and 100% damage can occur in crops of susceptible cultivars.

First Report of Sclerotium hydrophilum Causing Stem Rot of Rice in Northeast China

In August 2016, rice plants with brown to black lesions with irregular shape and water-soaked margins in leaf sheaths were observed in Baoqing county in Heilongjiang province, Northeast China. To isolate the causal pathogen, pieces of symptomatic leaf sheaths were treated with 1% NaClO for 1 min, rinsed with sterile distilled water for 2 min, and then transferred to potato dextrose agar (PDA) medium for incubation at 28°C. The cultivated mycelium was transferred to new PDA medium. Colonies of pure cultures on new PDA medium were initially white and turned brown about 3 weeks later. The hyphal width was measured with a range of 4.5 to 6.2 μm. Large numbers of small and globose sclerotia were observed on surface of the colonies at 3 days after subculturing. The sclerotia were white at first and then turned black over time. The diameters of sclerotia ranged from 0.31 to 0.54 mm with an average of 0.41 mm (n = 50). DNA of a representative isolate named Hbq001 was extracted, and the 18S rDNA region was amplified by PCR with universal primer pair NS1/NS6 (White et al. 1990). The 18S rDNA sequence of isolate Hbq001 (GenBank accession no. KY995575) shared 99.7% nucleotide identities with those of Sclerotium hydrophilum (GenBank accession no. KC354147). Therefore, based on these cultural characteristics and rDNA sequence, isolate Hbq001 was identified as S. hydrophilum (Demirci et al. 2009). To complete Koch’s postulates, 4 mm diameter PDA disks containing mycelium and sclerotia of S. hydrophilum isolate Hbq001 were placed on the stem of 4-week-old healthy rice seedlings and wrapped with Parafilm. Sterile PDA disks were used as controls. After 4 days at 28°C, symptoms similar to those in the field were observed on the inoculated seedlings but not on control plants. Finally, the fungus S. hydrophilum, which showed the same cultural characteristics as described above, was reisolated from the inoculated seedlings. In China, S. hydrophilum was first identified in Yunnan province and was found to be distributed in southern China (Hu et al. 2008; Ruan and Chen 2002; Wang et al. 2013). Northeast China is a major rice-producing region. Differing from the rice-producing areas of southern China, this region has specific climatic conditions with cold temperature and abounds in high-quality japonica rice. To our knowledge, this is the first report of S. hydrophilum in the cold northern region of China.

The effect of doxepin on Bacillus subtilis and Pseudomonas aeruginosa

Doxepin is a tricyclic antidepressant (TCA) used as a pill to treat major depressive disorder, anxiety disorders, chronic hives, and for short-term help with trouble remaining asleep after going to bed (a form of insomnia). As a cream it is used for short term treatment of itchiness due to atopic dermatitis or lichen simplex chronicus. The aim of the present study was to in vitro evaluate the antibacterial effects of doxepin using MIC, MBC and disk diffusion methods. The MIC and MBC was determined by dilution assay using different concentrations of doxepin on Bacillus subtilis and Pseudomonas aeruginosa. Disk diffusion assay were prepared under sterile conditions disks of drugs (three repeat for each concentration) containing three different doses (25, 50 and 150 μg) were prepared. MIC values of doxepin against Pseudomonas aeruginosa was 0.750 mg/ml, and MBC values was 4.5 mg/ml and MIC values of doxepin on Bacillus subtilis was 0.500 mg/ml, and MBC values was 4.5 mg/ml. The results obtained from disk diffusion assay supported that the doxepin has not antibacterial effect against Pseudomonas aeruginosa and Bacillus subtilis . It can be said that doxepin does not have antibiotic effect against gram negative and gram positive bacterial strain.

The comparison of antimicrobial effects of herbal and chemical agents on toothpaste: An experimental study

Nowadays, health-care companies use different antimicrobial agents in toothpastes to reduce oral microorganisms. The aim of this study was to investigate the antimicrobial effects of one Iranian herbal toothpaste in different concentrations compared with the chemical type on oral microorganisms in vitro. In this experimental study, the antimicrobial effect of one Iranian herbal toothpaste in comparison with its chemical type at three concentrations of 1, 1:1, and 1:3 on Streptococcus mutans (SM), Lactobacillus (LB), and Candida albicans (CA), respectively, were studied by agar disc diffusion method. The microorganisms were cultured on 21 plates. Then, four sterile paper discs were placed on each plate and the extracts were placed on them in prepared concentrations and incubated at 37°C ± 0°C for 24 h. The diameter of the inhibition zone around the discs was then measured in millimeters and recorded two-way ANOVA, one-way ANOVA tests, and regarding the difference variances, Tamhane supplementary tests were used at the significance level of P < 0.05. According to the results of this study, the full concentration of Iranian herbal toothpaste on SM, LB, and CA microorganisms had higher antimicrobial effect than the other two concentrations. This difference was statistically significant (P < 0.05). Furthermore, all the three toothpastes at full concentration had the same antimicrobial activity (P < 0.05). The antimicrobial effect of herbal toothpaste decreased significantly compared with the chemical toothpaste while the concentration decreased (P < 0.05). At full concentration, herbal and chemical toothpastes have the same antimicrobial effect, but by reducing the concentration, the antimicrobial effect of herbal toothpaste is reduced compared with the chemical one.

First Report of Citrus Black Spot Caused by Phyllosticta citricarpa in Angola

Citrus is an important subsistence crop in Angola, consumed mainly as fresh fruit. In April 2014, fruit symptoms reminiscent of citrus black spot (CBS) consisting of depressed circular spots of about 5 mm in diameter with a light-brown to gray center and black margins surrounded by green rind tissue were observed on a ‘Valencia’ sweet orange (Citrus sinensis) orchard in Bembe, Uige Province, Angola, with a disease incidence of 47%. Dark brown pycnidia were present in fruit lesions, which often coalesced forming sunken blemishes. Symptomatic fruits from this orchard were surface disinfected with 0.5% NaOCl for 2 min. Small fragments from lesions were plated onto potato dextrose agar (PDA) amended with 0.5 g of streptomycin sulfate/liter and incubated for 15 days at 23°C in the dark. Fungal colonies were transferred to PDA and oatmeal agar (OA) and incubated for 30 days at 23°C with 12-h photoperiod for morphological examination. Colonies were black with irregular margins in PDA and surrounded by a yellow pigment in OA. Pycnidia in PDA were aggregated, globose, dark brown, and 210 µm in diameter. Conidia were 9 to 12 × 5 to 7 µm, hyaline, aseptate, guttulate, obovoid to ellipsoid with truncate base, a mucoid layer and apical appendage. Pycnidia and conidia in PDA or fruit lesions were morphologically similar to those of Phyllosticta (Wikee et al. 2013). The ITS1-5.8S-ITS2 region, partial TEF1-alpha region, and LSU were amplified and sequenced as per Wikee et al. (2013) from DNA extracted from the single-spore isolate IIA-GC003NA, obtained from a Valencia sweet orange fruit from Bembe (GenBank accession nos. KY457232, MF693404, and MF693405, respectively). The sequences had 100% identity with those of P. citricarpa (McAlpine) van der Aa (synonym Guignardia citricarpa Kiely) epitype strain CBS 127454 (Glienke et al. 2011) (GenBank accession nos. JF343583, JF343604, and KF206306, respectively). Laboratory pathogenicity tests were performed by injecting the albedo of 30 mature detached fruit of Valencia sweet orange with 100 µl of a suspension of 3.0 × 10⁴ conidia/ml of water of the isolate IIA-GC003NA (Perryman et al. 2014). Fruits treated with sterile distilled water were used as controls. Fruits were maintained in humid chambers for 40 days at 24 to 26°C with 12-h photoperiod, and those inoculated with the fungus developed depressed necrotic lesions after 35 days. Field pathogenicity tests were performed on 12- to 14-week-old Valencia sweet orange fruits covered by waxed paper bags. Leaf disks colonized by the fungus were placed onto the rind of 60 fruits (Baldassari et al. 2009). Sterile leaf discs were used as controls. Symptoms of a false melanose-type consisting of dark brown stipplings were observed after 86 days on fruit inoculated in the field. No symptoms were observed on control fruit in either experiment. P. citricarpa, identified as described above, was reisolated from lesions on inoculated fruit in all experiments, but not from control fruit. The disease was verified as CBS caused by P. citricarpa. To our knowledge, this is the first report of CBS, caused by P. citricarpa, in Angola. P. citricarpa is responsible for large economic losses worldwide, so further studies are needed to map its geographic distribution, population structure, and impact in Angola.

Inhibitory effects of copper on bacterial and fungal growth

Introduction: Researchers conducted a literature, technology and patent search that traced the history of understanding the “bacteriostatic and sanitizing properties of copper and copper alloy surfaces” which demonstrated that copper, in very small quantities, has the Copper alloy surfaces have intrinsic properties to destroy a wide range of microorganisms. Today copper, in the form of plumbing tube, copper or copper-alloy surfaces proved to be a significant step in decreasing the fungal and bacterial infections in hospitals. Aims and objective: To know the bactericidal and fungicidal properties of copper for its implication in various areas in preventing nosocomial infection. Material and Methods: Eight sterile petri dishes (four for Blood agar media and four for MacConkey agar media) and two sterile 30 ml screw capped bottles (for Sabouraud’s Dextrose agar media) were taken. Sterile copper discs were placed in four plates of Blood agar and MacConkey agar media and other four plates were without copper discs. In the same way copper piece was placed in one of the bottle with Sabourauds Dextrose agar media. Pure growths of E. coli, Klebsiella and candida were inoculated and incubated. Results: The plates with bacterial and fungal growth were reported accordingly. The growth was significantly reduced in Plates and bottles with copper discs. Conclusion: Copper alloy surfaces have intrinsic properties to destroy a wide range of microorganisms so copper’s use in water supplies and surfaces are recommended.

EVALUATION OF ANTIBACTERIAL ACTIVITY OF ESSENTIAL OIL OF CINNAMOMUM ZEYLANICUM, EUGENIA CARYOPHYLLATA, AND ROSMARINUS OFFICINALIS AGAINST STREPTOCOCCUS ORALIS

Objective: Streptococcus oralis plays an important role in the biofilm formation of dental plaque and the occurrence of periodontal disease. Thepresent study was conducted to evaluate in vitro antibacterial activity of three essential oils, namely, Cinnamomum zeylanicum, Eugenia caryophyllata,and Rosmarinus officinalis against S. oralis.Methods: The antibacterial activity of essential oils was investigated by diffusion method using sterile discs (or aromatograms). The minimuminhibitory concentration (MIC) of essential oils showing important antibacterial activity was measured using the broth dilution method.Results: Evaluation of the antibacterial activity of three essential oils as determined by the aromatogram technique showed that the essential oilof R. officinalis had no effect on S. oralis, while the latter was extremely sensitive to the other two essential oils, but with a higher efficiency of theessential oil of C. zeylanicum (42 mm diameter) than E. caryophyllata (20 mm diameter). Similarly, the MIC and minimum bactericidal concentration(MBC) were higher for the essential oil of C. zeylanicum than the essential oil of E. caryophyllata. The MBC/MIC ratio is of the order of 2. The essentialoils studied therefore appear to exert bactericidal activity against S. oralis.Conclusion: The findings suggest that essential oils of C. zeylanicum and E. caryophyllata may be used as an alternative to synthetic antibiotics.Keywords: Essential oil, Cinnamomum zeylanicum, Eugenia caryophyllata, Rosmarinus officinalis, Antimicrobial activity, Streptococcus oralis.

Antibacterial Activity Of Shin’iseihaito (Xinyiqingfeitang) And its Components Against Methicillin-Resistant Staphylococcus aureus

Objective: Shin’iseihaito (xinyiqingfeitang in Chinese, SSHT), a formula in traditional Japanese Kampo medicine and Chinese medicine comprising nine crude drugs, Gypsum, Ophiopogon Tuber, Scutellaria Root (SR, root of Scutellaria baicalensis), Gardenia Fruit, Anemarrhena Rhizome, Lilium Bulb, Magnolia Flower, Loquat Leaf, and Cimicifuga Rhizome (CR, rhizome of Cimicifuga heracleifolia), is commonly used to treat sinusitis associated with purulent nasal discharge and reddish nasal mucosa. We evaluated anti-bacterial activity of SSHT extract on methicillin-resistant Staphylococcus aureus (MRSA), one cause of bacterial sinusitis. Materials and Methods: Sterile paper disks impregnated with SSHT extract, the combination of crude drugs composing SSHT according to the traditional pharmacological theory, or each component were placed on Mueller-Hinton agar plates inoculated with several strains of MRSA isolated from the patients. The diameter of inhibitory zone was measured after 18–24 h incubation. Results: SSHT extract showed antibacterial activity against 128/190 (66.8%) MRSA clinical isolates. The effect of the extract of SSHT without heat-clearing drugs (SSHT–HC) or without exterior-releasing drugs (SSHT–ER) were significantly lower than that of SSHT extract. Each water extract of SR, Loquat Leaf, Magnolia Flower and CR showed significant anti-MRSA activity, and SR extract exhibited the largest inhibitory zone. Conclusions: SSHT has antibacterial activity against MRSA clinical isolates, and SR mainly contributes to the antibacterial activity of SSHT against MRSA clinical isolates.

Anti-microbial and Anti-oxidant Activity of Watermelon (Citrullus lanatus) Fruit and Watermelon Seed

Watermelon ( Citrullus lanatus) flesh and seeds were dried and pulverized, and their extracts were diffused to sterile discs for the evaluation of anti-microbial activity. The disc-diffusion technique was used to assess anti-bacterial activity against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Pasteurella multocida, Yersinia enterocolitica, Serratia marcescens, Klebsiella pneumoniae, Xanthomonas campestris, Staphylococcus aureus . Anti-fungal activity against the yeasts Candida albicans and Rhodotorula glutinis was also examined. Standard anti-biotics were also tested as controls. Watermelon flesh and seed extracts were found to be effective against gram-positive and gram-negative bacteria and yeasts. The extracts were also screened for anti-oxidant activity using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free-radical-scavenging assay, total reducing ability using the Fe 3+ –Fe 2+ transformation method and ferrous ion (Fe 2+ )-chelating activity. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid and α-tocopherol were used as reference anti-oxidant radical scavenger compounds. The most potent anti-bacterial activity was demonstrated by watermelon–ethanol extract (inhibition zone 30 mm) against K. pneumoniae, and the most potent anti-fungal activity was demonstrated by watermelon–acetone extract (inhibition zone 26 mm) against R. glutinis. Watermelon–ethanol and watermelon seed–ethanol extracts both demonstrated marked anti-oxidant activity. These results highlight that watermelon fruit and seed extracts have potential for the development of efficient, safe and cost-effective natural anti-oxidant compounds for application in the functional food industries.