EXPLANT STERILIZATION AND IN VITRO CULTURE OF PURPLE SWEET POTATO TO OPTIMIZE NORMAL SHOOT FORMATION

An in vitro plant regeneration protocol was successfully established for Cryptocoryne wendtii , an ornamental aquatic plant by culturing shoot tip explants. The shoot tips were surface sterilized using 8 % Clorox® (5.25 % sodium hypochlorite, NaOCl) for 15 minutes followed by rinsing three times with sterile distilled water. They were again surface sterilized for another 4 % Clorox® (5.25 % sodium hypochlorite, NaOCl) for 5 minutes. Of the two cytokinins, 6-benzyl-aminopurine (BAP) and Kinetin (Kin) were evaluated as supplements to Murashige and Skoog (MS) medium [1], BAP at an optimal concentration of 3.0 mg/l was effective in inducing multiple shoots. The highest shooting percentage was 100.0 % on MS medium. Shoot multiplication of C. wendtii can be induced from shoot tip explants cultured on MS medium supplemented with 3.0 mg/l BAP and significantly different from other treatments, with the highest number of 16.20 shoots per explant and number of leaves at 72.40 leaves per explant. Shoots produced an average of 36 roots per plantlet and root length at 26.02 mm when cultured on MS medium supplemented with 1.0 mg/l α-Naphthaleneacetic acid (NAA). Plants were successfully transferred into plastic pots containing a mixture of organic soil and sand (1:1) overlaid with tap water with 100 % survival rate under greenhouse conditions. The in vitro regenerated plantlets were hardened and acclimatized successfully. The current protocol is the first successful report of C. wendtii micropropagation

A NOVEL APPROACH OF BACTERIA-FREE RHIZOME PRODUCTION OF GINGER THROUGH BIOTECHNOLOGY

Bacterial blight is a serious disease of carrot production worldwide. Under favorable conditions, the causal organism Xanthomonas hortorum pv. carotae causes serious loss especially in seed production because of its seed-borne character. Unlike fungal diseases, the treatment of bacterial diseases is limited and methods such as hot water or sodium hypochlorite (bleach) treatment are mainly used by seed companies. Here, we compared the efficacy of hot water treatment, sodium hypochlorite treatment and treatment with three phenolic compounds-carvacrol, thymol and eugenol, to eliminate Xanthomonas growth in vitro and subsequently in vivo on seeds of Xhc low, medium and highly infested carrot seed lots. The complete elimination of Xhc from germinated plants was obtained only for Xhc low infested seed lot with 1% sodium hypochlorite and carvacrol solutions in concentrations of 0.0196%- 0.313%. The significant reduction of Xhc presence in germinated plants of Xhc medium infested seed lot was achieved with 1% sodium hypochlorite treatment and hot water treatment. However, hot water treatment resulted in a significant reduction of seed germination percentage as well. Considering the elimination of Xhc infection from germinated plants and the effect on seed germination and plant vigor, 0.0196% carvacrol solution was suggested as an alternative to 1% sodium hypochlorite treatment regarding additional costs related to the liquidation of used treated water and to hot water treatment that has been proved to be insufficient to obtain disease-free plants.

During 2013, mango (Mangifera indica) producers of the most important growing area of Iran (Hormozgan province), reported extensive damages to panicles of mango trees of cv. Lengra, consisting of necrosis of flowers and pedicels. A fungus was isolated from affected panicles and its morphological traits were determined on colonies grown on Synthetic Nutrient Agar (SNA) medium as described by Bensch et al. (2012). Conidia 3-6×5.5-20 μm in size were fusiform with visible scars extensions. A portion of the translation elongation factor 1-α gene was amplified (Bensch et al., 2012) and sequenced (GenBank accession No. KT247610). A BLAST search showed 99% similarity with GenBank sequences belonging to Cladopsorium tenuissimum. Four healthy inflorescences were selected on a mango tree and, after disinfection by spraying 1% sodium hypochlorite and thoroughly washing with sterile distilled water, were sprayed with a spore suspension. Inoculum was prepared by rinsing a 7-day-old culture on PDA with sterile water and adjusted to 106 spores/ml. The inoculated inflorescences were kept covered with thin polythene bags for two days. Four control panicles on the same experimental tree were similarly disinfected and kept covered with thin polythene bags, after spraying with sterile distilled water. Symptoms resembling those observed in the field appeared on inoculated panicles from which the pathogen was re-isolated. Control treatments developed no symptoms. To our knowledge, this is the first report of C. tenuissimum causing inflorescence infection on magno in Iran.

Comparison of inactivation of Listeria monocytogenes within a biofilm matrix using chlorine dioxide gas, aqueous chlorine dioxide and sodium hypochlorite treatments

Colour fading is a method that is used to achieve a vintage look in textile goods. It is desired by customers in the textile market. Additionally, customers demand that these types of products are produced by environmentally friendly methods. In this study, sodium hypochlorite and ozone were used as laboratory‐scale colour fading reagents on dyed cotton fabrics. Cotton fabrics were dyed with four different primary colours: red, yellow, blue and black. Dyed fabrics were subjected to ozone and sodium hypochlorite treatment under different treatment conditions. Ozone was chosen as an alternative for comparison and it was applied at fixed flow rate (5 L/min) and time (10 minutes). Colour differences, chemical oxygen demand, bursting strength and energy, water and chemical consumptions were measured. The surface morphology was characterised by scanning electron microscopy. We can conclude that ozonation is effective in discharging colour from dyed fabric samples, and the colour‐fading effect is uniform, like in sodium hypochlorite treatment. It was observed that both processes are similar in terms of strength and surface modification. Results showed a 90% cost reduction, 85% water conservation and a 26% chemical oxygen demand reduction.

Laboratory experiments were conducted to investigate the effects of Heterodera glycines cyst components and surface disinfestants on hatching of H. glycines eggs. In one set of experiments, H glycines eggs were incubated in H. glycines cyst wall fragments, cyst wall and egg rinsate, egg homogenate, sterile distilled water, zinc sulfate, and soybean root diffusate. Hatch in cyst wall and egg rinsate and egg homogenate was greater than hatch in sterile distilled water. Hatch in cyst wall fragments was not different from hatch in sterile distilled water. In another set of experiments, H glycines eggs were treated with chlorhexidine diacetate, streptomycin sulfate, mercuric chloride, or sodium hypochlorite, then incubated in sterile distilled water, zinc sulfate, soybean root diffusate, and H. glycines egg homogenate. Hatch of eggs treated with and chlorhexidine diacetate, mercuric chloride, and streptomycin sulfate was lower than hatch of untreated eggs, whereas hatch of eggs treated with sodium hypochlorite was greater than hatch of untreated eggs. Hatch in zinc sulfate was not affected by treatment of eggs with three of the four surface disinfestants tested, but hatch of eggs treated with mercuric chloride was lower than hatch of untreated eggs in zinc sulfate. Results of these experiments clearly indicate that cyst wall and egg rinsate and egg homogenate of H. glycines stimulated hatch, and treatment of eggs with surface disinfectants affected hatch in H. qlycines. Uptake of acridine orange by Globodera rostochiensis eggs, resulting from increased eggshell permeability, correlates with hatching. Experiments were conducted to determine whether fluorescence of H. qlycines eggs after staining with acridine orange correlated with hatching. Heterodera glycines eggs were stained with acridine orange or hatched in sterile distilled water, zinc sulfate, and soybean root diffusate. Fluorescence of mature eggs was positively correlated with hatching in zinc sulfate. However fluorescence of mature eggs correlated positively, negatively, and not at all with hatch in soybean root diffusate, depending on diffusate concentration. Although there were some significant correlations between fluorescence and hatching of H. glycines eggs, the relationships were consistent enough to be useful as a predictor of hatch in H. glycines.

Dear Editor, Despite major advances in dentistry and dental prostheses, complete removable dentures are still widely used for oral rehabilitation of edentulous people. Denture surfaces prepare a favorable environment for adherence and proliferation of oral endogenous microorganisms, which is a crucial first step in the initiation of biofilm formation, caused denture stomatitis.[1] Candida species are known as the predominant oral etiologic agent of denture stomatitis, a common mucosal inflammation usually seen on the denturebearing mucosa of denture wearers.[2][3] There are several physical and chemical methods for cleaning of denture plaques.[4] Overnight soaking of denture in chemical solutions' including denture cleansers and detergents is an applicable and inexpensive method for improving of denture hygiene.[5] Ideal denture cleansers and disinfectants should be bactericidal, fungicidal, nontoxic and harmless to denture’s structure, effective for removing of organic and inorganic deposits on the denture.[4] To compare the effectiveness of seven commercially available disinfectants for cleaning of complete denture, we performed an experimental study in Yazd Dentistry School. Sixty three complete maxillary dentures, which were used more than one year, were randomly divided to nine groups. Cultures from their internal surface were obtained by vigorous rubbing with a sterile cotton swab onto Sabouraud dextrose agar plates, incubated 48 hours at 30oC for quantification of isolated Candida colonies. Isolated Candida colonies were also identified using germ tube test. Denture were then immersed for 1 hour in sterile ‘ziplock’ bags filled with 200 ml of sodium hypochlorite (2%, 1% and 0.5%), 0.2% chlorhexidine, 4% benzalconium chloride, 1% deconex and normal saline. Sterile distilled water and 100,000 IU solution of nystatin were also used as negative and positive controls in the current study. After the disinfectant protocol, the dentures were washed with sterile distilled water, re-swabbed and cultured as previously described before the disinfection. All cultures were performed by a single operator using sterile gloves, which were disposed and replaced for each denture. Density of isolated Candida colonies before and after disinfection was compared with Wilcoxon statistical (P<0.05) test using SPSS software. All dentures were positive for Candida species in the initial culture before disinfection protocol (Table 1). Candida albicans was the most common isolated Candida species isolated from 71% of dentures, followed by non-albicans Candida species, isolated from 23% of dentures and 6% had mix species. All immersion solutions were found to reduce the growth of Candida in comparing with the initial culture. Two percent sodium hypochlorite (p=0.008), 1% sodium hypochlorite (p=0.016), 0.2% cholorhexidine (p=0.025) and 1% deconex (p=0.046) were known as the most effective disinfectants shown the maximum reduction in the load of isolated Candida species in current study (Table 2). Table 1 Positive cultures of Candida on dentures before and after protocol, the numbers indicate the positive growth from total of seven dentures Table 2 Load of Candida colonies (CFU) on the dentures The results of the current study showed that all the initial cultures obtained from dentures, which taken directly from the patients’ mouth were positive for Candida species as expected. The objective of denture disinfection by immersing in chemicals is to obtain a clean, decontaminated prosthesis by removing the oral microbial contamination.[5] There are many studies, which showed more efficacy of chemical than physical methods.[6] The 2% and 1% sodium hypochlorite showed the highest cleaning effect followed by 0.2% chlorhexidine in the current study as also reported by Montagner et al.,[7] Furthermore 0.5% sodium hypochlorite, deconex (1%) and 4% benzalconium chloride showed a lower effect in disinfection of denture in the current study. Yilmaz et al. used 5.25% and 2% sodium hypochlorite as well as deconex and salvex and showed the same results;[8] however 5% sodium hypochlorite can damage dentures[9] and was not used in the present study.

This research aimed to findout efficient sterilization method and MS media to germinate dragonfruit seeds in vitro , conducting at Biotechnology Laboratory, Agriculture Faculty, Tadulako University, using factorial completely randomized design. The first factor was sterilization technique (S), i.e : S1 (The sliced fruit was washed with sterile distilled water three times, then rinsed in Bayclin (household bleach containing 5.25% NaOCl) 5% for 15 minutes, followed by washing in sterile distilled water three times. S2 (The seeds were taken from the fruit meat, then rinsed in 5% Bayclin for 15 minutes, then washed with sterile distilled water three times. S3 (The seeds were taken from the fruit meat, then rinsed in 15% Bayclin for 15 minutes, then washed with sterile distilled water three times. The second factor was the strength of MS media (M), i.e full strength MS (M1) and half strength MS for macro and micro nutrients (M2). Each treatment combination consisted of 35 seeds, and was replicated three times. Germination responses were observed as time to germinate, germination percentage and percentage of opened cotyledone seedlings. All datas were subjected to Analysis of Variance and the mean differences among the treatments were analyzed using Honest Significant Difference (HSD) at the level of 1%. The results showed that removing seed pulp, prior to rinsing the seeds in 15% Bayclin for 15 minutes followed by washing in sterile aquadest three times, and cultured in half MS produce the fastest and highest seed germination of 99.05% with 92.38% opened cotyledon seedlings after 2 weeks in culture. Key Words: Dragonfruit, germination, in vitro, MS strength, sterilization.

This study aimed to evaluate the effect of sulfinic acid sodium salt interposition after acid and sodium hypochlorite treatment (NC treatment) on dentin bonding durability using a mild type one-step self-etching adhesive. Fifteen human third molars were randomly assigned into three experimental groups according to dentin pretreatment before applying the one-step self-etching adhesive: Cont group, without pretreatment; NC group, pretreatment with phosphoric acid and sodium hypochlorite gel; and NC+AC group, additional treatment with sulfinic acid sodium salt followed by the same pretreatment of the NC group. Microtensile bond strength was measured and the pre-treated dentin surface, fracture modes, and bonding interface were observed. The bond strength of the NC+AC group was significantly higher than that of the other groups (p<0.001). The dentin-adhesive interface was degraded after 1 year only in the Cont group. Our results demonstrated NC treatment improves bonding durability and application of sodium sulfinic acid salt after NC treatment improves bonding effectiveness.

The influence of ultrasound combined with sodium hypochlorite (US-NaClO) treatment on microorganisms and quality of fresh-cut cucumber during storage were investigated. Ultrasound (400 W, 40 kHz, US: 5, 10 and 15 min) and sodium hypochlorite (NaClO: 50, 75, 100 ppm) were used to treat fresh-cut cucumber in a single or combined treatment and stored at 4 °C for 8 days and analyzed for texture, color and flavor. The results showed that US-NaClO treatment had a synergistic effect on the inhibition of microorganisms during storage. It could significantly reduce (p < 0.05) the number of microorganisms by 1.73 to 2.17 log CFU/g. In addition, US-NaClO treatment reduced the accumulation of malondialdehyde (MDA) during storage (4.42 nmol/g) and water mobility, and maintained the integrity of the cell membrane, delayed the increase of weight loss (3.21%), reduced water loss, thus slowing down the decline of firmness (9.20%) of fresh-cut cucumber during storage. The degradation of chlorophyll (6.41%) was reduced to maintain the color of freshly cut cucumbers. At the same time, US-NaClO could maintain the content of aldehydes, the main aromatic substance of cucumber, and reduced the content of alcohols and ketones during storage. Combined with the electronic nose results, it could maintain the cucumber flavor at the end of the storage period and reduce the odor produced by microorganisms. Overall, US-NaClO was helpful to inhibit the growth of microorganisms during storage, improve the quality of fresh-cut cucumber.

In January 2018, a large number of tuberous roots of sweet potato were lost in a production field in Jaiba, State of Minas Gerais, Brazil. Immediately after harvest, the roots showed dark spots on the surface, extending to the center, causing an internal, brown to black necrosis. Two symptomatic roots (cultivar Canadense) from different parts of the field were subjected to fungus isolation by removal of fragments between symptomatic and healthy tissue, followed by surface sterilization in 70% ethanol and then in sodium hypochlorite (1%) for 1 and 3 min, respectively, rinsing in sterile water, and incubation in PDA plates for 25°C in the dark. A gray to black, fast-growing fungus with abundant arthroconidia in aerial mycelia was isolated from all samples after 4 days of incubation. Hyphal tips were excised and transferred to new PDA plates, and two pure cultures (isolates) were obtained. Based on these characteristics, the fungus was identified as Neoscytalidium. For morphological analysis, culture sporulation was induced as described by Machado et al. (2014). Microscopic examination of the fungal structures identified arthroconidia with one to two septa, released by hyphal fragmentation, 4.2 to 9.5 × 3.2 to 5.3 μm, and a pycnidial synanamorph with hyaline, ellipsoid to nearly fusiform conidia, 7.4 to 12.7 × 4.2 to 6.3 μm. The morphological characteristics were similar to Neoscytalidium dimidiatum (Phillips et al. 2013). To confirm species identification, genomic DNA was extracted, and sequences of the internal transcribed spacer of the rDNA and translation elongation factor 1-α (TEF1-α) were obtained (Machado et al. 2014) and deposited in GenBank (accession nos. MH636327, MH636328, MH349427, and MH349428, respectively). According to a BLAST search, the TEF1-α sequences showed 99% identity with N. dimidiatum (accession no. KF553902). A combined phylogenetic tree obtained by Bayesian inference using MrBayes version 3.1.1 (Ronquist and Huelsenbeck 2003) grouped the isolates within the N. dimidiatum clade, confirming the species identification. One representative isolate (URM 7888) was deposited in the culture collection “Micoteca URM Profa. Maria Auxiliadora Cavalcanti” at the Universidade Federal de Pernambuco (Recife, Brazil). The pathogenicity test was performed by inoculating asymptomatic roots (cultivar Canadense), previously sterilized on the surface with 0.5% sodium hypochlorite and unwounded or wounded with a sterile scalpel at two equidistant points, with conidia suspension. Arthroconidia suspension (20 μl, 2.4 × 10⁶ conidia/ml) obtained from 7-day-old colonies was pipetted onto the inoculation sites. Sterile distilled water was used for the control instead of inoculum. Five roots were used for each treatment. The inoculated roots were maintained in plastic boxes containing moistened paper towels at approximately 25°C for 15 days. After 7 days, it was possible to observe initial symptoms of internal brown to black rot, which increased in size after 15 days. The fungus was reisolated, and pathogenicity was confirmed. The control and unwounded roots remained asymptomatic. In Brazil, N. dimidiatum has been reported causing collar and root rot in physic nut (Machado et al. 2012), black root rot in cassava (Machado et. al., 2014), and dieback in mango (Marques et al. 2013). To our knowledge, this is the first report of N. dimidiatum causing root rot of sweet potato in Brazil.

Introduction Sterilization methods adopted in Human Teeth Bank can promote structural changes in enamel and dentin. Thus, the ideal method should combine the antimicrobial efficacy and preservation of the substrate biomechanical properties. Aim Evaluated the morphology and physical properties of enamel and dentin of extracted human teeth, after being submitted to different sterilization methods. Method Sixteen extracted third molars were sectioned in the following tooth regions: mesio-distal, vestibular-lingual and at the cement enamel junction. Forty specimens were selected according to values of microhardness and roughness initials, and distributed in four experimental groups (n = 10/group): Group 1 = Autoclave 121 °C (30 minutes); Group 2 = 2.5% Sodium hypochlorite (07 days); Group 3 = 5.25% Sodium hypochlorite (07 days); Group 4= 30% Acetic Acid (7 days). After sterilization, the microhardness, roughness and morphology of enamel and dentin were evaluated. Data were analyzed by paired t-test, Wilcoxon, Kruskal-Wallis and Analysis of Variance. A significance level of 5% was adopted. Result All sterilization methods altered the microhardness and roughness of the dentine (p &lt;0.05). Regarding to enamel, the microhardness was affected only by Autoclave and 2.5% Sodium hypochlorite treatment (p &lt;0.05). A large aperture of the enamel pores and dentinal tubules was verified in the micrographs. Conclusion The sterilization methods adopted altered the morphology and/or physical properties of enamel and dentine.

A New Solution for the Removal of the Smear Layer

This study evaluated the efficacy of chlorinated water, sodium hypochlorite solution, sodium chloride solution and sterile distilled water in eliminating pathogenic bacteria on the surfaces of raw vegetables. Lettuce vegetables were dipped in different concentrations of chlorinated water, sodium hypochlorite solution, sodium chloride solution and sterile distilled water for 1,2,3,4 and 5 minutes and analyzed microbiologically. Treatments of the vegetables with chlorinated water, sodium hypochlorite solution, salt water and distilled water did not eliminate the pathogens, but reduced their populations. The bacterial loads were reduced by 38.18 – 69.83% (chlorinated water), 23.74 – 69.13% (sodium hypochlorite solution), 31.62 – 79.0% (salt water) and 14.95 – 42.59% (sterile distilled water). The coliform loads were reduced by 41.2 – 68.9% (chlorinated water), 22.2 – 55.6% (sodium hypochlorite solution), 36.0 – 90.0% (salt water) and 22.2% - 40.0% (sterile distilled water). This suggests that very high concentrations of salt water could be useful in reducing pathogenic microorganisms in fresh produce.