Effects of rice seed surface sterilization with hypochlorite on inoculated Burkholderia vietnamiensis.

When a combination of hydrogen peroxide and hypochlorite was used to surface sterilize rice seeds, a 10(2)- to 10(4)-fold decrease in CFU was observed during the first 15 h after inoculation of the rice rhizosphere organism Burkholderia vietnamiensis TVV75. This artifact could not be eliminated simply by rinsing the seeds, even thoroughly, with sterile distilled water. When growth resumed, a significant increase in the frequency of rifampin- and nalidixic acid-resistant mutants in the population was observed compared to the control without seeds. This phenomenon was a specific effect of hypochlorite; it was not observed with hydrogen peroxide alone. It was also not observed when the effect of hypochlorite was counteracted by sodium thiosulfate. We hypothesized that the hypochlorite used for disinfection reacted with the rice seed surface, forming a chlorine cover which was not removed by rinsing and generated mutagenic chloramines. We studied a set of rifampin- and nalidixic acid-resistant mutants obtained after seed surface sterilization. The corresponding rpoB and gyrA genes were amplified and sequenced to characterize the induced mutations. The mutations in five of seven nalidixic acid-resistant mutants and all of the rifampin-resistant mutants studied were found to correspond to single amino acid substitutions. Hypochlorite surface sterilization can thus be a source of artifacts when the initial bacterial colonization of a plant is studied.     

Contamination control of microbe Ziziphus spina [christti] seed in vitro culture

Ziziphus spina [christti] is a naturally distributed tree in subtropical, arid and semi-arid parts of Iran. It is ecologically and economically important due to its tolerance to drought and salinity. Most tree seeds are infected with parasitic and saprophytic microorganisms which decrease the seed germination and seedling establishment. The goal of this paper was to evaluate the ability of selected chemical solutions to inhibit the growth of variety of microbial contaminants in Z. spina [christti] seeds and to enhance the seed germination. Different chemical treatments were used in surface sterilization of seeds: (Treatment 1) sodium hypochlorite (NaOCl) in concentrations of 1, 2 and 4% for 20 min. (Treatment 2) hydrogen peroxide (H₂O₂) in concentration of 4, 8, 12% treated for 10 min. (Treatment 3) 1% mercuric chloride (HgCl₂) at duration 10, 15, 20, 25 min. Seeds were scarified and aseptically, planted on agar Murashige and Skoog (MS) medium. Contaminants were identified according to their morphological and cultural characteristics. Bacterial contaminants included Xanthomonas sp. While Fungal isolates were Fusarium, Penicillium, Alternaria, Rhizopus, and Aspergillus. Our experiment reveals that 4% NaOCl followed by benomyl is the best sterilization treatment for Z. spina [christti] seeds, since the highest number of germination and highest number of sterilized seeds was observed after this treatment.     

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.     

Role of nalidixic acid in isolation ofSalmonella typhimurium strains capable of growth at 48°C

Salmonella typhimurium thermotolerant mutants dependent on the presence of nalidixic acid for growth at 48°C were isolated and designated nalidixic acid-dependent, thermotolerant mutants,nal ^d ttl. Genetic mapping revealed thatnal ^d ttl alleles map within thegyrA gene. WhenS. typhimurium strain Q was plated in the dark on nutrient agar containing nalidixic acid (20 g/ml) as a photosensitizer and briefly exposed to white light or near VU light prior to incubation at 42°C, nalidixic acid-resistant mutants arose in about 16 h at frequencies of 5×10^–8 for white light and 1×10^–6 for near UV light. About 10% of these nalidixic acid-resistant mutants derived from photodynamic mutagenesis exhibited the thermotolerant characteristic.     

The effect of surface sterilization and the type of sterilizer on the genus composition of lichen-inhabiting fungi with notes on some frequently isolated genera

Surface sterilization is generally used for isolating lichen-inhabiting fungi as well as endophytic fungi, and ethanol and sodium hypochlorite are commonly used as the sterilizer. However, there are few studies on whether the type of chemicals used for surface sterilization affects the isolation results of lichen-inhabiting fungi. In this study, the genus composition of the lichen-inhabiting fungi of two lichen species (Flavoparmelia caperata and Peltigera dilacerata) were investigated 1) to reveal how the isolation result changes before and after surface sterilization and 2) to examine the effect of the sterilizer (ethanol, sodium hypochlorite, or hydrogen peroxide) on the composition of the isolated fungi. We isolated 652 non-lichenized fungal isolates from the two-lichen species and identified 84 genera. It was found that 1) every sterilizer effectively removed the fungi on the lichen surface and that 2) the composition of isolated fungi varied depending on the type of surface sterilizer. It was also shown that, such as the genus Sarea, there were some lichen-inhabiting fungi which could not be isolated at all by surface sterilization with ethanol or sodium hypochlorite, which are commonly used. In addition, the genus Virgaria was detected as lichen-inhabiting fungi for the first time. Our results suggest that single surface sterilization alone may underestimate the genus composition of lichen-inhabiting fungi.     

Evaluation of chemical seed coat sterilants

Summary Three seed coat sterilants were evaluated for effectiveness as surface sterilants and for effects on seed germination. Several exposure times in sodium hypochlorite, hydrogen peroxide and peracetic acid were evaluated. Seeds of the following crop plants were used: wheat (Triticum aestivum L.); sorghum [Sorghum bicolor (L.) Moench]; soybean [Glycine max (L.) Merrill]. All treatments reduced germination of wheat and soybean seed, but only the most severe peracetic acid treatments substantially reduced germination of sorghum seed. Considering both sterilization effectiveness and seed germination, a treatment with 5% sodium hypochlorite for 45 min appeared to be the most satisfactory of the treatments used.     

Asymbiotic seed germination and in vitro seedling development of the threatened orchid Hoffmannseggella cinnabarina

Hoffmannseggella cinnabarina has not been found in the wild for the last 70?yr in the State of S?o Paulo and, therefore, wild populations of this native orchid are thought to be extinct. This investigation studied seed storage at a low temperature, in vitro germination, and seedling development of H. cinnabarina in order to establish an optimized protocol for propagation, and thus assure species conservation. Seeds of different ages were incubated on Knudson C (KC), Murashige and Skoog, and Vacin and Went media with or without 1???M of N⁶-benzyladenine (BA) and exposed to either 12 or 16?h of light (30???mol?m^?2?s^?1 at 26?±?2°C). Seed surface sterilization was deleterious to 3-mo-old seeds and severely reduced the viability of the 4-mo-old seeds. More mature seeds were not affected by the sterilization procedure. In general, the germination of 4-, 5-, 6-, and 7-mo-old seeds increased when BA was added to the culture medium especially under 16?h of light. Germination rates were highest with 8- and 9-mo-old seeds, and application of BA failed to enhance germination rates further. Developmental studies revealed that this cytokinin reduced seed and protocorm mortality rates; however, protocorm development was negatively affected in its presence. Seedling development was more pronounced when KC medium with 16?h of light was used. Long-term seed storage at 4°C did not provide promising results. The protocol described in this study proved to be efficient and relevant to in vitro seed germination and initial development of H. cinnabarina, and thus will contribute to conservation of this orchid species.     

Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8–71.9 % in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8 % reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.     

Verticillium dahliae (Kleb.) Infecting Pumpkin Seed

This study investigated the natural occurrence of Verticillium dahliae (Kleb.) infection in pumpkin (Cucurbita pepo L.) seed. The mean incidence of infection was found to be 21.0%. Isolates recovered from seeds were pathogenic to pumpkin (cultivar ‘Jamaican squash’). Surface sterilization by immersion in 0.6% sodium hypochlorite for 20 min eradicated V. dahliae from infected pumpkin seeds without affecting germinability. Plating of seed components revealed that the fungus was present in the seed coat but not in the embryo or cotyledons. In a growing‐on test, 25% of 6‐week‐old plants grown from untreated seeds were infected. Germination and production of normal seedlings were unaffected by V. dahliae infection of seeds. Verticillium dahliae in pumpkin seed was found to be external and transmissible to plants. The findings of this study are important in devising disease control strategies.     

Efficacy of Ciprofloxacin for Treatment of Cholera Associated with Diminished Susceptibility to Ciprofloxacin to Vibrio cholerae O1

Objective

We identified a poor clinical response to treatment of cholera with a single 1 g dose of ciprofloxacin, a standard treatment for cholera.

Methods

To determine reasons for the poor response and better therapeutic approaches we examined the minimal inhibitor concentration (MIC, n = 275) and disc-diffusion zone sizes (n = 205) for ciprofloxacin and nalidixic acid of V. cholerae O1 strains isolated in Bangladesh from 1994 to 2012, and reexamined data from 161patients infected with Vibrio cholerae O1 recruited in four clinical trials who received single- or multiple-dose ciprofloxacin for treatment of cholera and compared their clinical response to the V. cholerae O1 susceptibility.

Results

Although all 275 isolates of V. cholerae O1 remained susceptible to ciprofloxacin using standard MIC and disc-diffusion thresholds, the MIC⁹⁰ to ciprofloxacin increased from 0.010 in 1994 to 0.475 μgm/ml in 2012. Isolates became frankly resistant to nalidixic with the MIC⁹⁰ increasing from 21 μgm/ml in 1994 to >256 μgm/ml and 166 of 205 isolates from 1994 to 2005 being frankly resistant using disc-diffusion testing. Isolates resistant to nalidixic acid by disc-diffusion testing had a median ciprofloxacin MIC of 0.190 μgm/ml (10^th-90^th centiles 0.022 to 0.380); nalidixic acid-susceptible isolates had a median ciprofloxacin MIC of 0.002 (0.002 to 0.012).The rate of clinical success with single-dose ciprofloxacin treatment for nalidixic acid-susceptible strains was 94% (61 of 65 patients) and bacteriologic success 97% (63/65) compared to 18% (12/67) and 8% (5/67) respectively with nalidixic acid-resistant strains (P<0.001 for both comparisons). Multiple-dose treatment with ciprofloxacin had 86% and 100% clinical and bacteriologic success rates respectively in patients infected with nalidixic acid-susceptible strains of V. cholerae O1 compared to clinical success 67% and bacteriologic success 60% with nalidixic acid-resistant strains.

Conclusions

Single-dose ciprofloxacin is not effective for treating cholera caused by V. cholerae O1 with diminished susceptibility to ciprofloxacin, and nalidixic acid disc-diffusion testing effectively screens for such isolates.     

Antimicrobial solid media for screening non-sterile Arabidopsis thaliana seeds

Stable genetic transformation of plants is a low-efficiency process, and identification of positive transformants usually relies on screening for expression of a co-transformed marker gene. Often this involves germinating seeds on solid media containing a selection reagent. Germination on solid media requires surface sterilization of seeds and careful aseptic technique to prevent microbial contamination, but surface sterilization techniques are time consuming and can cause seed mortality if not performed carefully. We developed an antimicrobial cocktail that can be added to solid media to inhibit bacterial and fungal growth without impairing germination, allowing us to bypass the surface sterilization step. Adding a combination of terbinafine (1 μM) and timentin (200 mg l⁻¹) to Murashige and Skoog agar delayed the onset of observable microbial growth and did not affect germination of non-sterile seeds from 10 different wild-type and mutant Arabidopsis thaliana accessions. We named this antimicrobial solid medium “MSTT agar”. Seedlings sown in non-sterile conditions could be maintained on MSTT agar for up to a week without observable contamination. This medium was compatible with rapid screening methods for hygromycin B, phosphinothricin (BASTA) and nourseothricin resistance genes, meaning that positive transformants can be identified from non-sterile seeds in as little as 4 days after stratification, and transferred to soil before the onset of visible microbial contamination. By using MSTT agar we were able to select genetic transformants on solid media without seed surface sterilization, eliminating a tedious and time-consuming step.     

Correlation between seed longevity and RNA integrity in the embryos of rice seeds

The mature embryos of rice seeds contain translatable mRNAs required for the initial phase of germination. To clarify the relationship between seed longevity and RNA integrity in embryos, germinability and stability of embryonic RNAs were analyzed using the seeds of japonica rice cultivars subjected to controlled deterioration treatment (CDT) or long periods of storage. Degradation of RNA from embryos of a japonica rice cultivar “Nipponbare” was induced by CDT before the decline of the germination rate and we observed a positive relationship between seed germinability and integrity of embryonic RNAs. Moreover, this relationship was confirmed in the experiments using aged seeds from the “Nipponbare”, “Sasanishiki” and “Koshihikari” rice cultivars. In addition, the RNA integrity number (RIN) values, calculated using electrophoresis data and Agilent Bioanalyzer software, had a positive correlation with germinability (R²=0.75). Therefore, the stability of embryonic RNAs required for germination is involved in maintaining seed longevity over time and RIN values can serve as a quantitative indicator to evaluate germinability in rice.     

Differential Inactivation of Fungal Spores in Water and on Seeds by Ozone and Arc Discharge Plasma

Seed sterilization is essential for preventing seed borne fungal diseases. Sterilization tools based on physical technologies have recently received much attention. However, available information is very limited in terms of efficiency, safety, and mode of action. In this study, we have examined antifungal activity of ozone and arc discharge plasma, potential tools for seed sterilization. In our results, ozone and arc discharge plasma have shown differential antifungal effects, depending on the environment associated with fungal spores (freely submerged in water or infected seeds). Ozone inactivates Fusarium fujikuroi (fungus causing rice bakanae disease) spores submerged in water more efficiently than arc discharge plasma. However, fungal spores associated with or infecting rice seeds are more effectively deactivated by arc discharge plasma. ROS generated in water by ozone may function as a powerful fungicidal factor. On the other hand, shockwave generated from arc discharge plasma may have greatly contributed to antifungal effects on fungus associated with rice seeds. In support of this notion, addition of ultrasonic wave in ozone generating water has greatly increased the efficiency of seed disinfection.     

Effect of various sterilization procedures on the in vitro germination of cotton seeds

In vitro manipulations of cotton often require high-quality sterile seedlings as a source of hypocotyl and cotyledon explants for initiation of embryogenic cultures or embryo apexes for shoot production. Unfortunately, in vitro seed germination is often hindered if cotton seeds were collected from the open field and stored under improper conditions. In our case a limited supply of field-grown cotton seeds was received, which necessitated the development of a more effective surface sterilization protocol. Seeds from two accessions, designated as I and II, with very high contamination levels and lowered germination rate were used in this study. These seeds were treated with the most commonly used sterilizing agents, which included commercial bleach, chlorine gas and hydrogen peroxide. Additional steps such as soap-water washes (SW), 70 % ethanol (ETH) and plant preservative mixture rinses were also included in the sterilization procedures to improve the efficiency of tested protocols. Surface sterilized seeds were germinated on Linsmaier and Skoog medium and the percentage of contamination free and well-germinated seeds were recorded for each treatment. Seeds treated with hydrogen peroxide showed significant improvement in germination rate and level of contamination when compared to identical seeds treated with chlorine gas and commercial bleach. The most effective sterilization protocol for all genotypes tested consisted of SW wash followed by ETH rinse and H₂O₂ sterilization for 7 h. This protocol was successfully used to sterilize seeds of 55 cotton lines.     

RNAi mediated silencing of lipoxygenase gene to maintain rice grain quality and viability during storage

Lipoxygenase (LOX) is a common enzyme which catalyzes lipid peroxidation of seeds and consequently enhances seed quality deterioration and decreases seed viability. During seed storage, peroxidation of unsaturated fatty acids occur due to enhancement of LOX activity which directly leads to reduction in seed vigour and deterioration of grain nutritional quality. This study was undertaken to overcome these problem during rice seed storage by attenuating LOX activity using RNAi technology. To improve seed storage stability, we down regulated LOX gene activity by using a functional fragment of the LOX gene under the control of both constitutive (CaMV35S) and aleurone-specific (Oleosin-18) promoter separately. To understand the storage stability, RNAi–LOX seeds and non-transgenic control seeds were subjected to accelerated aging at 45 °C and 85 % relative humidity for 14 days. Our studies demonstrate that down regulation of LOX activity reduces the seed quality deterioration under storage condition. In addition GC–MS analysis revealed that reduction of fatty acid level in non-transgenic seeds during storage was higher when compared with that of transgenic rice seeds. Furthermore, the transgenic rice seeds with reduced LOX activity exhibited enhanced seed germination efficiency after storage than that of non-transgenic rice seeds. This study will have direct impact on nutritional stability of quality rice grains.     

Dynamic analysis of Arabidopsis seed shape reveals differences in cellulose mutants

In a previous work, the shape of Arabidopsis seed was described as a cardioid modified by a factor of Phi. In addition, J index was defined as the similarity of the seed (in an orthogonal, bi-dimensional image) to a cardioid, thus allowing the quantitative comparison of seed shape in seeds of varieties and mutants at different stages of development. Here, J index is used for modeling changes in seed morphology during the dynamic process of seed imbibition before germination. The analysis was carried out by means of a general linear model with two fixed factors (genotype and time) applied to two Arabidopsis varieties: Columbia and Wassilewskija and two mutants in cellulose synthesis: prc1-1 (CESA6 in Columbia) and kor1-1 (in Wassilewskija). Equations representing the changes in seed form during imbibition are given. The analysis of changes in seed shape by this procedure provides (1) a quantitative method to record changes in seed shape and to compare between genotypes or treatments showing the time points with maximum differences, and (2) the observation of remarkable differences between wild-type seeds and mutants in cellulose biosynthesis, indicating new phenotypic characteristics previously unknown in the latter. While wild-type seeds increase their J index values during imbibition, in the cellulose mutants J index values decrease. In addition, shape comparisons were done with other mutants. Seeds of ga1-1 mutants behave like cellulose mutants, whereas different ethylene mutants present varied responses. Quantitative analysis of seed morphology is a new basis for the record of differences between wild-type and mutants as well as for phenotypic characterization.     

Effectiveness of certain sterilizing agents for seedling incubation in radiothymidine

Failure of seed sterilization methods and breakdown in sterile technique can result in catabolism of thymidine by microorganisms during seedling incubation in radioisotope. The effectiveness of some sterilizing agents was monitored by microbiological methods and by thin layer chromatography for the presence of thymidine degradation products. Sodium hypochlorite and ethylene oxide each sterilized pea and sunflower seeds. Sodium hypochlorite failed to sterilize onion seeds while ethylene oxide was only occasionally effective and often retarded germination. Captan, commonly used before germination to prevent fungal blight of seedlings, did not reduce the bacterial flora of onion seeds.     

Soil-borne seed pathogens: contributors to the naturalization gauntlet in Pacific Northwest (USA) forest and steppe communities?

Soil-borne seed pathogens are omnipresent but are often overlooked components of a community’s biotic resistance to plant naturalization and invasion. Using multi-year greenhouse experiments, we compared the seed mortality of single invasive, naturalized, and native grass species in sterilized and unsterilized soils collected from Pacific Northwest (USA) steppe and forest communities. Native Pseudoroegneria spicata displayed the greatest seed mortality, naturalized Secale cereale displayed intermediate seed mortality, and invasive Bromus tectorum was least affected by soil pathogens. Seed mortality across all three species was consistently greater in soils collected from steppe than soils collected from forest; seeds sown into sterilized steppe soil experienced half the overall seed mortality compared to seeds sown into unsterilized steppe soil. Soil sterilization did not affect grass seed mortality in forest soils. We conclude that (1) removing soil-borne pathogens with sterilization does increase native and non-native grass seed survival, and (2) soil-borne pathogens may influence whether an introduced species becomes invasive or naturalized within these Pacific Northwest communities as a result of differential seed survival. Soil-borne pathogens in these communities, however, have the greatest negative effect on the survival of native grass seeds, suggesting that the native microbial soil flora more effectively attack seeds of native plants than seeds of non-native species.