In situ assessment of pesticide genotoxicity in an integrated pest management program I--Tradescantia micronucleus assay.

The genotoxicity induced by pesticides applied in an integrated pest management (IPM) program was evaluated with the Tradescantia micronucleus assay (Trad-MCN). Three pesticide application rates were prescribed as follows: (a) Low, no field pesticide spray; (b) Medium, IPM test rate: banded cyanazine plus metolachlor (2.7 kg a.i. and 2.3 l a.i./ha of herbicides, respectively); and (c) High, a preventative pesticide application program: broadcast cyanazine plus metolachlor (same application rates as above) plus chlorpyrifos (1 kg a.i./ha of insecticide). The Trad-MCN was employed for the assessment of (a) the formulated compounds, singly and in combinations; (b) pesticide residues extracted from soils sampled before and after application, and (c) in situ exposures (14-h exposure to pesticide-sprayed field). All pesticides showed clastogenic potency at doses between 10 and 50 ppm. Aqueous extracts of the two pesticide-sprayed soils were clastogenic, but the unsprayed soil extracts were not. Plants exposed in situ to pesticide-sprayed soils (inside a chamber receiving vapors from the soil) also showed significant increases in micronuclei frequency in relation to controls exposed to unsprayed soil. In general, there was no significant reduction in the genotoxic effects from the High to the Medium treatment levels of the IPM program. This suggests that the reduction in pesticide application rates attained with the implementation of the proposed IPM program was not sufficient to abate the genotoxicity of the pesticides, as perceived with the sensitive assays employed. The results indicate that replacing genotoxic compounds may be the only effective remediation measure to eliminate the risks imposed by mutagenic compounds in the agricultural environment.

In situ assessment of pesticide genotoxicity in an integrated pest management program: II. Maize waxy mutation assay.

The mutagenicity induced by pesticides applied in an integrated pest management (IPM) program was evaluated in situ with the maize forward waxy mutation bioassay. Three pesticide application rates were prescribed as follows: (1) Low--no field pesticide spray; (2) Medium--IPM test rate: banded cyanazine plus metolachlor (2.7 kg a.i. and 2.3 l a.i./ha of herbicides, respectively); and (3) High--a preventative pesticide application program: broadcast cyanazine plus metolachlor (same application rates as above) plus chlorpyrifos (1 kg a.i./ha of insecticide). In general, there was no significant reduction in the genotoxic effects from the high to the medium treatment levels of the IPM program. This suggests that the reduction in pesticide application rates attained with the implementation of the proposed IPM program was not sufficient to abate the genotoxicity of the pesticides. The results indicate that replacing genotoxic compounds may be the only effective remediation measure if concern about environmental mutagenesis were to result in changes in agricultural management.

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue.

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

A review of physical, chemical, and biological properties of fly ash and effects on agricultural ecosystems.

Fly ash is the solid material which is carried away from the power plant boiler in the flue gas during coal combustion. The properties of fly ash may vary considerably according to several factors such as the geographical origin of the source coal, conditions during combustion, and sampling position within the power plant. A typical aggregate of fly ash from the combustion of eastern U.S. coals consists of spherical particles embedded in an amorphous matrix. Most fly ash particles are in the silt-sized range of 2-50 microns. The three major mineralogical matrices identified in fly ash are glass, mullite-quartz, and magnetic spinel. The major elemental constituents of fly ash are Si, Al, Fe, Ca, C, Mg, K, Na, S, Ti, P, and Mn. Nearly all naturally occurring elements can be found in fly ash in trace quantities. Certain trace elements, including As, Mo, Se, Cd, and Zn, are primarily associated with particle surfaces. The solubility of fly ash has been extensively investigated. Results of these investigations are largely dependent on factors specific to the extraction procedure. The most abundant species in fly ash extracts are inorganic ions derived from Ca, Na, Mg, K, Fe, S, and C. Boron is much more soluble than other trace elements in fly ash. The forms of some elements in fly ash extracts have been determined, but the species of most trace elements remain unidentified. Long-term leaching studies predict that fly ash will lose substantial amounts of soluble salts over time, but simulation models predict that the loss of trace elements from fly ash deposits through leaching will be very slow. The constituents of coal fly ash include small amounts of radioisotopes which do not appear to be hazardous. A complex mixture of organic compounds is also associated with fly ash particles. The organic compounds identified in fly ash extracts include known mutagens and carcinogens. Better methods for the extraction of organic compounds from fly ash particles must be developed before these compounds can be fully identified and quantified.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitation of polyamines using thin-layer chromatography and image analysis.

Efficient separation of dansylated polyamines can be achieved by thin-layer chromatography (TLC). Quantitation, however, can be laborious because it requires removal of the silica gel and the fluorescing derivative from the glass plates, elution in a suitable solvent, and estimation with a fluorescence spectrophotometer. We report here a relatively simple and rapid method for the quantitation of dansylated polyamines that employs an image analyzer without removal from the glass TLC plates.

Protection of wheat against leaf and stem rust and powdery mildew diseases by inhibition of polyamine metabolism.

In higher plants, polyamines arise from arginine by one of two pathways: via ornithine and ornithine decarboxylase or via agmatine and arginine decarboxylase but in fungi, only the ornithine decarboxylase pathway is present. Since polyamines are required for normal growth of microorganisms and plants and since the ornithine pathway can be irreversibly blocked by alpha-difluoromethylornithine (DFMO) which has no effect on arginine decarboxylase, fungal infection of green plants might be controlled by the site-directed use of such a specific metabolic inhibitor. DFMO at relatively low concentrations provided effective control of the three biotrophic fungal pathogens studied, Puccinia recondita (leaf rust), P. graminis f. sp. tritici (stem rust), and Erysiphe graminis (powdery mildew) on wheat (Triticum aestivum L.) Effective control of infection by leaf or stem rust fungi was obtained with sprays of DFMO that ranged from about 0.01 to 0.20 mM in experiments where the inhibitor was applied after spore inoculation. The powdery mildew fungus was somewhat more tolerant of DFMO, but good control of the pathogen was obtained at less than 1.0 mM. In general, application of DFMO after spore inoculation was more effective than application before inoculation. Less control was obtained following treatment with alpha-difluoromethylarginine (DFMA) but the relatively high degree of control obtained raises the possibility of a DFMA to DFMO conversion by arginase.

Kinetic studies on the control of the bean rust fungus (Uromyces phaseoli L.) by an inhibitor of polyamine biosynthesis.

alpha-Difluoromethylornithine (DFMO), a specific and irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, effectively inhibits mycelial growth of several phytopathogenic fungi on defined media in vitro and provides systemic protection of bean plants against infection by Uromyces phaseoli L. race 0 (MV Rajam, AW Galston 1985 Plant Cell Physiol 26: 683-692; MV Rajam et al. 1985 Proc Natl Acad Sci USA 82: 6874-6878). We now find that application of 0.5 millimolar DFMO to unifoliolate leaves of Pinto beans up to 3 days after inoculation with uredospores of U. phaseoli completely inhibits the growth of the pathogen, while application 4 or 5 days after inoculation results in partial protection against the pathogen. Spores do not germinate on the surface of unifoliolate leaves treated with DFMO 1 day before infection, but addition of spermidine to the DFMO treatments partially reverses the inhibitory effect. The titer of polyamines in bean plants did not decline after DFMO treatment; rather, putrescine and spermidine contents actually rose, probably due to the known but paradoxical stimulation of arginine decarboxylase activity by DFMO.

Cadmium-induced accumulation of putrescine in oat and bean leaves.

The effects of Cd2+ on putrescine (Put), spermidine (Spd), and spermine (Spm) titers were studied in oat and bean leaves. Treatment with Cd2+ for up to 16 hours in the light or dark resulted in a large increase in Put titer, but had little or no effect on Spd or Spm. The activity of arginine decarboxylase (ADC) followed the pattern of Put accumulation, and experiments with alpha-difluoromethylarginine established that ADC was the enzyme responsible for Put increase. Concentrations of Cd2+ as low as 10 micromolar increased Put titer in oat segments. In bean leaves, there was a Cd(2+)-induced accumulation of Put in the free and soluble conjugated fractions, but not in the insoluble fraction. This suggests a rapid exchange between Put that exists in the free form and Put found in acid soluble conjugate forms. It is concluded that Cd2+ can act like certain other stresses (K+ and Mg2+ deficiency, excess NH4+, low pH, salinity, osmotic stress, wilting) to induce substantial increases in Put in plant cells.

Prevention of a plant disease by specific inhibition of fungal polyamine biosynthesis.

DL-alpha-Difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly retards the growth of several species of phytopathogenic fungi in vitro. Such inhibition can be completely reversed by putrescine or spermidine, confirming the essentiality of polyamines for growth of fungal hyphae. We now show that DFMO can protect bean plants (Phaseolus vulgaris Linnaeus cv. Pinto) against infection by uredospores of the bean rust fungus, Uromyces phaseoli Linnaeus, race O. Unifoliolate leaves of 10-day-old greenhouse-grown seedlings were sprayed with 400 microliter per leaf of DFMO at various concentrations in 0.01% Tween 20 at pH 7.0 before or after inoculation with uredospores of Uromyces. After 16 hr in darkness in dew chambers to facilitate spore germination, plants were transferred to the greenhouse, arranged randomly, and examined for local lesions 7 days later. All concentrations of DFMO 0.50 mM or higher gave complete protection against the pathogen; at lower concentrations, postinoculation treatments with DFMO were generally more effective than preinoculation. The appearance of lesions on plants treated with lower concentrations of DFMO was retarded 2-6 days. DFMO also confers protection on unsprayed parts of treated plants, indicating the translocation of some protective effect from sprayed areas. DL-alpha-Difluoromethylarginine, an analogous inhibitor of arginine decarboxylase (EC 4.1.1.19), which is the rate-limiting enzyme in an alternative pathway for polyamine biosynthesis in higher plants, confers no protection even at 5 mM. This emphasizes ornithine decarboxylase as the biochemical locus of choice for the prevention of plant diseases by inhibiting polyamine metabolism.