The Effect of 2,4-Dichlorophenoxyacetic Acid and Related Compounds on the Fine Structure of the Primary Leaves of Phaseolus vulgaris

The epidermal cuticle of the abaxial surface of the primaryleaves of Phaseolus vulgaris used for these experiments is notmore than 0.15 µm thick except on the walls of basal cellsof epidermal hairs. The cuticle apparently has no internal structure;neither channels or canals can be seen passing through it. An examination of the fine structure of leaf tissue 24 h afterthe application of 1.6 x 10^–3 M solutions of 2-chlorophenoxyaceticacid (2-CPA), 4-chlorophenoxyacetic acid (4-CPA), 2,6-dichlorophenoxyaceticacid (2,6-D), and 2,4-dichlorophenoxyacetic acid (2,4-D) hasshown that 2,4-D alone, and then only in the light, induceschanges in the morphology and internal structure of the chloroplast.As early as 4 h after application, there is an apparent breakdownin the structure of the membrane systems of the cells of theepidermis, palisade, and mesophyll. After 8 h the chloroplastsare distorted and contain electron-dense granules, and the cellsappear to be plasmolysed. Subsequently these changes becomemore pronounced. It is possible that the specific disturbancesbrought about by 2,4-D reflect its phytotoxic properties.     

Growth Rates of a Pseudomonad on 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol

The growth of a pseudomonad on 2,4-D (2,4-dichlorophenoxyacetic acid) and 2,4-DCP (2,4-dichlorophenol) was studied in batch and continuous culture. The optimum growth rate using 2,4-D was 0.14/h at 25 C in a pH range from 6.2 to 6.9. Highest specific growth rate using 2,4-DCP was 0.12/h at 25 C in a pH range from 7.1 to 7.8. Growth was strongly inhibited by 2,4-DCP above a concentration of 25 mg/liter whereas no appreciable inhibition was observed with 2,4-D at concentrations up to 2,000 mg per liter. Growth on 2,4-DCP was described by Monod kinetics at subinhibitory concentrations but the inhibition by 2,4-DCP exhibited an unusual linear response to substrate concentration, and did not fit a model based on noncompetitive inhibition. The lag phase of batch cultures was found to depend on both 2,4-DCP concentration and prior adaptation of the inoculum. A study such as this on the kinetics of growth on related substrates may be useful as a method of finding the rate-limiting step in a metabolic sequence.     

Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D) by a Hypersaline Microbial Mat and Related Functional Changes in the Mat Community

Microbial mats possibly possess degradation capacities for haloorganic pollutants because of their wide range of different functional groups of microorganisms combined with extreme diurnal changes in pH, oxygen, and sulfide gradients. In this study, 20 mg/l of the chlorinated herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to a pristine hypersaline cyanobacterial mat from Guerrero Negro, Mexico, under a light regime of 12 h dark/12 h light (600 mol photons/m²s). The loss of 2,4-D was followed by chemical GC analysis; functional changes within the mat were determined with microelectrodes for oxygen, photosynthesis, pH, and sulfide. The depletion of 2,4-D due to photooxidation or sorption processes was checked in control experiments. Within 13 days, the light/dark incubated mats degraded 97% of the herbicide, while in permanent darkness only 35% were degraded. Adsorption of 2,4-D to the mat material, agar, or glass walls was negligible (4.6%), whereas 21% of the herbicide was degraded photochemically. The 2,4-D removal rate in the light/dark incubations was comparable to values reported for soils. The phototrophic community of the mat was permanently inhibited by the 2,4-D addition by 17% on average. The sulfate reduction in the entire mat and the respiration in the photic zone were inhibited more strongly but returned to original levels. Since at the end of the experiment the photosynthetic and respiratory activity of the mats were almost as high as in the beginning and 2,4-D almost completely disappeared, we conclude that the examined mats represent a robust and effective system for the degradation of the herbicide where probably the aerobic heterotrophic population is a major player in the degradation process.This revised version was published online in November 2004 with corrections to Volume 48.     

Mineralization of 2,4-Dichlorophenoxyacetic Acid (2,4-D) and Mixtures of 2,4-D and 2,4,5-Trichlorophenoxyacetic Acid by Phanerochaete chrysosporium

Evidence is presented for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in nutrient-rich media (high-nitrogen and malt extract media) by wild-type Phanerochaete chrysosporium and by a peroxidase-negative mutant of this organism. Mass balance analysis of [U-ring-¹⁴C]2,4-D mineralization in malt extract cultures showed 82.7% recovery of radioactivity. Of this, 38.6% was released as ¹⁴CO₂ and 27.0, 11.2, and 5.9% were present in the aqueous, methylene chloride, and mycelial fractions, respectively. 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were simultaneously mineralized when presented as a mixture, and mutual inhibition of degradation was not observed. In contrast, a relatively higher rate of mineralization of 2,4-D and 2,4,5-T was observed when these compounds were tested as mixtures than when they were tested alone.     

Control of Morphogenesis in Sorghum by 2,4-Dichlorophenoxyacetic Acid and Cytokinins

2, 4-Dichlorophenoxyacetic acid caused a shortening of rootsand shoots when mature seeds of Sorghum bicolor (L.) Moenchcv. BT3197 were germinated on Murashige and Skoog (MS) mediumthat contained 2,4-D. Shoot growth was restored with cytokinins.A callus formed at the nodal region, the further differentiationof which was determined by the ratio of 2,4-D and cytokininsin the initial culture medium. A high auxin to cytokinin ratiopromoted primarily root differentiation while a high cytokininto auxin ratio promoted multiple bud development. Isolated shootapical meristem with the subtending node produced embryogeniccallus at low cytokinin levels and green buds on high cytokininlevels when cultured in the presence of 2,4-D. It is concludedthat cells potentially capable of differentiation into roots,somatic embryos or axillary buds are present in the first nodalregion. Sorghum bicolor, organogenesis, embryogenesis, 2, 4-D: cytokinin ratios, tissue culture     

Interaction between 2,4-Dichlorophenoxyacetic Acid and Vitamin K in Datura stramonium

Plants of the cross Datura stramonium L. var. stramonium×Datura stramonium L. var. inermis (Juss. ex Jacq.) Timm. were sprayed either with a water solution of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D) or with a combination of 2,4-D (sodium salt) and the vitamin K compound menadione sodium bisulfite in water solution. The plants were found to differ in one of their responses to the treatments, namely the development of necrotic spots on the leaf blades. Many necroses appeared on the plants treated with 2,4-D plus menadione sodium bisulfite while only a few necroses or none at all developed on the plants treated with 2,4-D alone. Furthermore, 2,4-D in combination with menadione sodium bisulfite induced much larger necrotic spots than did 2,4-D by itself. Spraying with menadione sodium bisulfite in water solution did not produce any observable effects upon Datura plants. The great increase in necrotic lesion formation was probably due to an interaction between 2,4-D and menadione sodium bisulfite (or a metabolite of this compound) within leaf cells.     

A Bioluminescent Whole-Cell Reporter for Detection of 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol in Soil

A bioreporter was made containing a tfdRP_DII-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (R² = 0.9825) in the range of 2.0 μM to 1.1 × 10² μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 10² μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.     

Antifungal Activity of 2,4-Dihydroxyacylophenones and Related Compounds

The antifungal activity of 2,4-dihydroxyacylophenones and related compounds against Trichophyton spp and other fungi were investigated to determine their structure-activity relationships.

The activity of these compounds was found to be closely related to the length of the acyl and alkyl substituents attached to the 1,3-dihydroxybenzene moiety In addition, differences in activity were observed depending on the position of the alkyl substituents and on the number of substituents attached to the 1,3-dihydroxybenzene moiety. Some compounds tested showed potent antifungal activity against Trichophyton spp. and other fungi that was more active than amphotericin B.     

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10⁵ CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10⁵ CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.