Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.     

"Inert" formulation ingredients with activity: toxicity of trisiloxane surfactant solutions to twospotted spider mites (Acari: Tetranychidae)

Organosilicone molecules are important surfactant ingredients used in formulating pesticides. These methylated silicones are considered inert ingredients, but their superior surfactant properties allow them to wet, and either suffocate or disrupt important physiological processes in mites and insects. Aqueous solutions of the tri-siloxane surfactants Silwet L-77, Silwet 408, and Silwet 806 were bioassayed against adult female two-spotted spider mites, Tetranychus urticae Koch, with leaf dip methods to compare their toxicity with organosilicone molecules containing bulkier hydrophobic components. All three tri-siloxanes in aqueous solutions were equivalently toxic (LC50 = 5.5-8.9 ppm), whereas Silwet L-7607 solutions were less toxic (LC50 = 4,800 ppm) and Silwet L-7200 was nontoxic to mites. In another experiment, the toxicity of Silwet L-77 was affected by the wettability of leaf surfaces. The LC50 shifted from 22 to 84 ppm when mites were tested on bean and strawberry leaf disks, respectively. Droplet spreading on paraffin and surface tension were both related to the toxicity of surfactant solutions. Surface tensions of solutions below 23 mN/m caused > 90% mite mortality in leaf dip bioassays. A field test of Conserve SC and its formulation blank, with and without Dyne-Amic adjuvant (a vegetable oil-organosilicone surfactant mixture) revealed that Dyne-Amic had the greatest miticidal contribution, reducing mite populations by 70%, followed by formulation inactive ingredients. Spinosad, the listed active ingredient in Conserve, only contributed miticidal activity when synergized by Dyne-Amic. Researchers should include appropriate surfactant or formulation blank controls when testing insecticides or miticides, especially when using high spray volumes.     

Adjuvants enhance the biological control potential of an isolate of Colletotrichum gloeosporioides for biological control of sicklepod (Senna obtusifolia)

In greenhouse experiments, unrefined corn oil, Silwet L-77, and an invert emulsion were tested as adjuvants with the mycoherbicidal fungus Colletotrichum gloeosporioides, a weakly virulent pathogen of sicklepod (Senna obtusifolia). A 1:1 (v/v) fungus/corn oil tank mixture containing 0.2% (v/v) Silwet L-77 surfactant reduced the dew period requirements for maximum weed infection and mortality from 16 to 8 h, and delayed the need for free moisture for greater than 48 h. This formulation also resulted in the ability of the pathogen to infect and kill weeds in larger (>5 leaf) growth stages. The invert emulsion resulted in similar effects upon these parameters. These results suggest that invert emulsions, unrefined corn oil and Silwet L-77 surfactant greatly improve the bioherbicidal potential of this pathogen for control of sicklepod, a serious weed pest in the southeastern US.     

Influence of surfactants on the sorption of two chloroacetanilide in an Romanian chernozem soil

Pesticides have been extensively used in modern agriculture. Due to the prevalent use, there have been serious problems generated by pesticides wastes which could eventually endanger water resources and human health. The development of technologies for the decontamination of soils and waters polluted by hydrophobic organic compounds has encouraged research into the use of non-ionic surfactants as potential agents for the enhanced solubilization and removal of contaminants from soils and sediments. Sorption of two chloroacetanilide herbicides, acetochlor and metolachlor was studied on a representative chernozem soil of the Main Agricultural Research Station Ezareni belonging to the "Ion Ionescu de la Brad" University of Agriculture and Veterinary Medicine lasi, Romania, in the presence and absence of surfactants. Three different non-ionic surfactants were selected: Tween-20, Synperonic 91/5 and Silwet L-77, to verify the influence of their presence on herbicide sorption at different concentrations. Our results showed that the sorption of the studied herbicides within the soil-water-non-ionic surfactant system was influenced by the presence of non-ionic surfactants. The n values obtained were lower than 1 for all pesticide-surfactant combinations, which indicates that the amount of acetochor and metolachlor sorbed decreased with an increase in pesticide concentration. The sorption of acetochlor increased in the following order: Acetochlor+Synperonic 91/5 < Acetochlor < Acetochlor+Tween-20 < Acetochlor+Silwet L-77. In the case of metolachlor+Synperonic and metolachlor+Silwet L-77, the Kf values were significantly higher than the Kf value of metolachlor+Tween-20 on soil, where a lower Kf value could be observed with however a higher n value which indicate a higher sorption capacity at higher concentrations.     

The effect of varying rates of glyphosate and an organosilicone surfactant on the control of gorse

The herbicidal effect of glyphosate applied to gorse (Ulex europaeus L.) was improved by the addition of increasing amounts (0.5–20 g/litre) of Silwet L-77, an organosilicone surfactant. Increasing the rate of herbicide also enhanced control. There was a highly significant interaction between surfactant rate and herbicide dosage; as the amount of Silwet L-77 was increased the rate of glyphosate could be reduced without loss of herbicide efficacy. However, without any added organosilicone surfactant, glyphosate did not provide more than 73% control of gorse at any rate up to 6.5 kg a.i./ha. With the addition of Silwet L-77, complete mortality of all plants could be achieved with 2.2 kg glyphosate/ ha.     

Adsorption of surfactants on a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain and the effect on cell surface lypohydrophilic property

The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton X-100, Tween 80, and rhamnolipid.     

Extending the host range of the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene

Spore formulations of the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene (ATCC No. 20358) (CGA) were evaluated for control of three weed species: northern jointvetch (Aeschynomene virginica), Indian jointvetch (A. indica), and hemp sesbania (Sesbania exaltata) in greenhouse experiments. Mortality, dry weight reduction, and plant height reduction of A. virginica seedlings ranged from 98% to 100%, 15 days after inoculation with CGA in water, in an invert emulsion, or in Silwet L-77 surfactant. However, CGA in water caused no effects of these parameters on S. exaltata, and only slight effects on A. indica. A. indica and S. exaltata were also severely injured (mortality, dry weight and plant height reduction, 98–100%) by CGA in the invert emulsion or in Silwet L-77. The CGA in Silwet formulation incited severe disease development more rapidly than the invert emulsion or water formulations of CGA in all species. These results suggest that the host range of CGA can be expanded though formulation modification to enable this bioherbicide to control multiple weeds, thus making this product more economically acceptable.     

Improvement of Agrobacterium-mediated transformation of embryogenic calluses from maize elite inbred lines

Summary This study was carried out to evaluate the effects of purine synthesis inhibitor mizoribine, purine and pyrimidine synthesis inhibitors azaserine and acivicin, and surfactant Silwet L-77 on Agrobacterium-mediated transformation efficiency of embryogenic calluses from maize elite inbred lines Qi 319 and Ye 515. After transformation and three rounds of selection on 2.8 μM chlorsulfuron, resistant calluses were obtained subsequently, and morphologically normal plantlets were regenerated from 80 to 90% of the resistant calluses treated with the compounds. There were no obvious discrepancies between the frequencies of plantlet regeneration and the ratio of PCR positive plantlets of calluses treated with different compounds. Results of PCR assay with primers for betA showed that 40.2% (103/256) of the regenerated plantlets were positive. The percentage of resistant calluses was 2–3-fold higher than the control after being treated with 0.19–0.27 mM mizoribine. The most suitable concentration of azaserin was 0.36 mM, which gave a 4-fold increase in the percentage of resistant calluses. Acivicin at 0.28–0.84 mM yielded a 3–5-fold increase in the percentage of resistant calluses, which is significantly better than the control. When the calluses were treated with 0.01 or 0.02% Silwet L-77, the percentages of resistant calluses were 34.89 and 25.60%, respectively. We concluded that purine synthesis inhibitors, purine and pyrimidine synthesis inhibitor and the surfactant Silwet L-77 at optimal concentrations significantly improved the Agrobacterium-mediated transformation of maize calluses.     

Potential of a combination of entomopathogenic fungal strains and a non-ionic surfactant to control the fall armyworm (Spodoptera frugiperda)

The fall armyworm, Spodoptera frugiperda, is an emerging invasive pest in Taiwan that feeds on a wide range of crops and causes serious damage. Herein, an entomopathogenic fungal library (EFLib) was constructed to identify potential microbes to control FAW. Twenty-eight indigenous entomopathogenic fungi (EPF) were isolated and investigated for their potential pathogenicity, with Metarhizium pinghaense (Mp-NCHU-124) and Beauveria bassiana (Bb-NCHU-157) exerting dose-dependent effects on the 4th instar FAW larvae. The non-ionic surfactant Silwet L-77 rapidly killed FAW larvae after spraying at a concentration of 300 mg/kg and the toxic effect of Silwet L-77 on FAW larvae was dose-dependent. When the EPF isolates (10⁶ conidia/mL) were applied to FAW larvae in combination with the non-ionic surfactant Silwet L-77 (30–90 mg/kg), the mortality rate dramatically increased and the LT₅₀ reduced, with increased fungal mycosis (Mp-NCHU-124: 38% to 72% and Bb-NCHU-157: 20 to 62%), indicating the high compatibility of EPF with the non-ionic surfactant. Thus, the Silwet L-77+EPF combined formulation has potential for practical field application for FAW pest control and sustainable agriculture in the future.     

The pathway of betalain biosynthesis: Effect of cytokinin on enzymic oxidation and hydroxylation of tyrosine in Amaranthus tricolor seedlings

The effect of exogenously added tyrosine or l -3-(3,4-dihydroxyphenyl) alanine on the accumulation of betacyanin in response to cytokinin in Amaranthus tricolor (L.) var. tricolor half-seedlings depends on the age of the seedlings and the treatment of the seedlings prior to induction of pigment by benzyladenine. Neither extracted polyphenol oxidase, peroxidase or tyrosine hydroxylase activity, nor in vivo tyrosine hydroxylation is increased in response to exposure of seedlings to cytokinin. However a small percentage of the polyphenol oxidase activated or unmasked by Triton X-100 treatment of membrane fractions is increased after cytokinin treatment of half-seedlings for 22 h. It is concluded that cytokinin control may be on a multi-enzyme membrane-located complex involving part of the polyphenol oxidase activity of the tissue (possibly an isoenzyme), and that the majority of the polyphenol oxidase activity in Amaranthus is a constitutive membrane enzyme which is not involved in betacyanin synthesis. Although cytokinins do not affect in vivo tyrosine hydroxylation, this activity follows closely the accumulation of betacyanin which is first detectable about 6.5 h after the application of cytokinin. Only a very low level of in vivo hydroxylation can be demonstrated in half-seedlings treated for 6 h either with or without cytokinin but it begins to increase shortly after this time. A large increase in this activity by 16 h (independent of cytokinin) can be almost completely (79%) prevented by chloramphenicol (300 μM) although the drug increases accumulation of betacyanin. At this concentration about 30% of the protein synthesis in inhibited. In vitro tyrosine hydroxylation is, however, not reduced in half-seedlings treated with chloramphenicol during 16 h induction nor is extractable polyphenol oxidase reduced. It is concluded that chloramphenicol is inhibiting the synthesis of some protein essential for in vivo hydroxylation other than the activity measured during in vitro hydroxylation and that the inhibition of synthesis of 79% in vivo hydroxylation still leaves enough activity for maximum betacyanin synthesis.     

Efficacy of Silwet L-77 against several arthropod pests of table grape

Silwet L-77, an organosilicone surfactant, was applied to several arthropod pests of California table grapes. Eggs of grape mealybug, Pseudococcus maritimus (Ehrhorn), and omnivorous leafroller, Platynota stultana Walsingham, were tolerant to 0.1, 0.25, and 0.5% treatment solutions; however, eggs of Pacific spider mite, Tetranychus pacificus McGregor, were highly susceptible with mortality >99.4% (0.1% Silwet L-77). Mortality of immature and adult stages of cotton aphid (Aphis gossypii Glover), Western flower thrips (Frankliniella occidentalis Pergande), and Pacific spider mite (Tetranychus pacificus McGregor) was > or = 93.8, > or = 98.5, and > or = 99.4% for 0.1, 0.25, and 0.5% Silwet L-77, respectively. Grape mealybug crawlers had 100% mortality when treated with 0.5 and 1.0% Silwet L-77 solutions; however, mortality was only 6.7% when 0.1% Silwet L-77 was applied. 'Thompson Seedless' table grapes were not damaged when treated with up to 1% Silwet L-77; however, grapes treated with the 0.5 and 1.0% solutions appeared wet after removal from cold storage because of the effect of the surfactant spreading the water condensation. Grapes dried with the normal bloom on the berries when they reached room temperature.     

Surfactant inhibition of bacterial growth on solid anthracene

Surfactants have been proposed as a promising method to enhance bioremediation of hydrophobic compounds in contaminated soils. However, the results of effects of surfactants on bioremediation are not consistent. This study showed that Triton X-100 at low concentration (0.024 mM or 0.09 CMC) inhibited the rate of growth of either a Mycobacterium sp. or a Pseudomonas sp. on solid anthracene as sole carbon source. Recovery of microbial growth rate could be achieved by dilution of surfactants, while addition of more surfactant gave an immediate decrease in growth rate. No inhibition of growth by Triton X-100 was observed with growth on glucose. The surfactant sorbed onto the surfaces of both the cells and the anthracene particles, which could inhibit uptake of anthracene. The results were consistent with the hypothesis that inhibition of microbial adhesion of cells to anthracene was responsible for the inhibition of growth by Triton X-100.     

Alteration in cell surface properties of <Emphasis Type="Italic">Burkholderia</Emphasis> spp. during surfactant-aided biodegradation of petroleum hydrocarbons

Chemical surfactants may impact microbial cell surface properties, i.e., cell surface hydrophobicity (CSH) and cell surface charge, and may thus affect the uptake of components from non-aqueous phase liquids (NAPLs). This work explored the impact of Triton X-100, Igepal CA 630, and Tween 80 (at twice the critical micelle concentration, CMC) on the cell surface characteristics of Burkholderia cultures, Burkholderia cepacia (ES1, aliphatic degrader) and Burkholderia multivorans (NG1, aromatic degrader), when grown on a six-component model NAPL. In the presence of Triton X-100, NAPL biodegradation was enhanced from 21% to 60% in B. cepacia and from 18% to 53% in B. multivorans. CSH based on water contact angle (50–52°) was in the same range for both strains while zeta potential at neutral pH was −38 and −31 mV for B. cepacia and B. multivorans, respectively. In the presence of Triton X-100, their CSH increased to greater than 75° and the zeta potential decreased. This induced a change in the mode of uptake and initiated aliphatic hydrocarbon degradation by B. multivorans and increased the rate of aliphatic hydrocarbon degradation in B. cepacia. Igepal CA 630 and Tween 80 also altered the cell surface properties. For B. cepacia grown in the presence of Triton X-100 at two and five times its CMC, CSH increased significantly in the log growth phase. Growth in the presence of the chemical surfactants also affected the abundance of chemical functional groups on the cell surface. Cell surface changes had maximum impact on NAPL degradation in the presence of emulsifying surfactants, Triton X-100 and Igepal CA630.     

Stabilization of Crystalline Acid Carboxypeptidase from Penicillium janthinellum by Nonionic Surfactants,and Inhibition of Enzyme Activity by Anionic Compounds

The crystalline acid carboxypeptidase from Penicillium janthinellum IFO-8070 was stabilized by the addition of nonionic surfactants, such as Triton X-100, Brij 35, Span 40, and Tween 20. In the presence of these stabilizers, extremely diluted enzyme (0.3 μg/ml of 50 mm sodium acetate buffer, pH 3.7) was almost completely stable after 2 days incubation at 25°C. About 35% and 20% of the enzyme activities were activated by the addition of Triton X-100 and Brij 35, respectively. Triton X-100 completely retarded inactivation at freezing (?15°C). On the other hand, anionic surfactants of SLS and LBSA, and cationic surfactant of cetyltrimethylammonium bromide strongly inactivated the enzyme. The inhibition of the fatty acid series was roughly proportional to the molecular weight of the inhibitor. Di-, and Tri-carboxylic acids also inhibited the enzyme activity.     

Response of fluoranthene-degrading bacteria to surfactants

A prerequisite for surfactant-enhanced biodegradation is that the microorganisms survive, take up substrate and degrade it in the presence of the surfactant. Two Mycobacterium and two Sphingomonas strains, degrading fluoranthene, were investigated for their sensitivity towards non-ionic chemical surfactants. The effect of Triton X-100 and Tween 80 above their critical micelle concentration on mineralization of [¹⁴C]-glucose and [¹⁴C]-fluoranthene was measured in shaker cultures. Tween 80 had no toxic effect on any of the tested strains. The surfactant inhibited fluoranthene mineralization by the hydrophobic Mycobacterium spp. slightly, but more than doubled that by the two less hydrophobic Sphingomonas strains. Triton X-100 inhibited fluoranthene mineralization by all strains, yet this was more pronounced for the Sphingomonas spp. Both surfactants caused cell wall permeabilization, as shown by transient colouring of surfactant-containing media. Inhibition of glucose mineralization, indicating non-specific toxic effects of Triton X-100, was observed only for the Sphingomonas strains and the toxicity was caused by micelle-to-cell interactions. These strains, however, appeared to recover from initial Triton X-100 toxicity within 50–500 h of exposure. The ratio of surfactant concentration to initial cell density was found to determine critically the bacterial response to surfactants. For both Sphingomonas and Mycobacterium strains, this work indicates that fluoranthene solubilized in surfactant micelles is only partially available for mineralization by the bacteria tested. However, our results suggest that optimal conditions for polycyclic aromatic hydrocarbon mineralization can be developed by selection of the proper surfactant, bacterial strains, cell density and incubation conditions. Received: 6 February 1998 / Received revision: 19 June 1998 / Accepted: 19 June 1998     

Enzyme production by growing cells of acinetobacter in presence of sophoroselipid and triton X-100

The effects of an extracellular microbial glycolipid, the interfacial active lactonic sophoroselipid, and of Triton X-100 on strains of Acinetobacter calcoaceticus are compared. Sophoroselipid diminished growth rates on n-heptadecane. Both surfactants led to the excretion of enzyme activities into the culture medium. Sophoroselipid increased the release of cytoplasmic malate dehydrogenase whereas in presence of Triton X-100 the quinoprotein glucose dehydrogenase was also excreted in large amounts.     

Relationships between nitrate level,nitrate reductase activity and anaerobic nitrite production inPisum sativum leaf tissue

Anaerobic nitrite production (thein vivo NO₃-R activity) in an incubation medium lacking exogenous nitrate but containing 0.5%n-propanol and 0.1% Triton X-100 showed higher correlation (y - ax ^b) with the level of endogenous nitrate inPisum sativum L. leaves than thein vitro nitrate reductase activity. Thein vivo NO₃-R activity correlated well with thein vitro activity up to the 50 ppm NO₃-N level of endogenous nitrate. The ratioin vivo: in vitro activity slightly decreased with increasing level of endogenous nitrate in leaf tissue.     

真空渗透法转化油菜及转化种子的筛选

真空渗透遗传转化法首先在拟南芥(Arabidopsis thaliana)中获得成功, 是一种简便、快速且无需经过组织培养阶段即可获得大量转化植株的基因转化方法。该研究以不同遗传背景的3个甘蓝型油菜(Brassica napus)品种(系)陕3B、L45和Mg23为材料, 对真空渗透遗传转化方法中真空渗透时间和Silwet L-77浓度与遗传转化效果的关系进行了比较, 同时对转化种子的筛选方法进行了优化。结果表明, 卡那霉素(Km)对油菜种子的萌发影响不显著, 但对其生长发育有明显的抑制作用。不同油菜品种对卡那霉素的敏感性不同, 各自的致死浓度也不一样。在0%-0.05%的浓度范围内, 随着Silwet L-77浓度的增加, 在相同的真空渗透时间内, 3个油菜品种的转化率逐渐升高。当Silwet L-77浓度为0.05%时, 10分钟的真空渗透时间可获得最高转化效率, 此时陕3B、L45和Mg23的转化率分别达到1.97%、2.09%和2.30%。     

Effect of abscisic acid on betacyanin leakage from plant tissues

Effect of abscisic acid on cell permeability in leaves ofIresine u allisi hort. and roots ofBeta vulgaris L. were examined. An increase of betacyanin leakage from leaf cells was shown by ABA at 10⁻⁴, 10⁻⁷ or 10⁻⁹ M concentrations in water solution at 25 °C. The efflux of batacyanin from tissues did not change during the joint action of ABA and PEG 1000. ABA could lower the betacyanin leakage fromIresine leaves and beet-root slices under severe osmotic stress, as was found by deplasmolysis. The results suggest that ABA elicits some alteration in density of tonoplast membranes under dehydration. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Calcium Ions Protect Beet Root Cell Membranes against Thermally Induced Changes

The initial heat induced increase in permeability of beet (Beta vulgaris L.) root cell membranes, as measured by betacyanin efflux, is reversible. The leakage of betacyanin stopped when tissue held for 90 min at 45°C was transferred to 27°C. Stronger heat treatments, 10 min at 60°C or 150 min at 45°C, resulted in a continuous release of betacyanin indicating an irreversible change in membrane structure. Calcium ions inhibited the heat induced betacyanin efflux only if this was due to a reversible increase in permeability.