Robert M. Zablotowicz

Pesticide metabolism in plants and microorganisms

Abstract Understanding pesticide metabolism in plants and microorganisms is necessary for pesticide development, for safe and efficient use, as well as for developing pesticide bioremediation strategies for contaminated soil and water. Pesticide biotransformation may occur via multistep processes known as metabolism or cometabolism. Cometabolism is the biotransformation of an organic compound that is not used as an energy source or as a constitutive element of the organism. Individual reactions of degradation–detoxification pathways include oxidation, reduction, hydrolysis, and conjugation. Metabolic pathway diversity depends on the chemical structure of the xenobiotic compound, the organism, environmental conditions, metabolic factors, and the regulating expression of these biochemical pathways. Knowledge of these enzymatic processes, especially concepts related to pesticide mechanism of action, resistance, selectivity, tolerance, and environmental fate, has advanced our understanding of pesticide science, and of plant and microbial biochemistry and physiology. There are some fundamental similarities and differences between plant and microbial pesticide metabolism. In this review, directed to researchers in weed science, we present concepts that were discussed at a symposium of the American Chemical Society (ACS) in 1999 and in the subsequent book Pesticide Biotransformation in Plants and Microorganism: Similarities and Divergences, edited by J. C. Hall, R. E. Hoagland, and R. M. Zablotowicz, and published by Oxford University Press, 2001. Nomenclature: American Chemical Society; fenchlorazole-ethyl; glutathione; glutathione-S-transferase; naphthalic anhydride; polyaromatic hydrocarbons; polychlorinated biphenyls; reductive dehalogenation; trichloroethylene.

Glyphosate-resistant soybean response to various salts of glyphosate and glyphosate accumulation in soybean nodules

Abstract A field study was conducted during 2000 and 2001 at Stoneville, MS, to determine the effects of isopropylamine, trimethylsulfonium (Tms), diammonium, and aminomethanamide dihydrogen tetraoxosulfate (Adt) salt formulations of glyphosate on weed control, growth, chlorophyll content, nodulation, nitrogen content, and grain yield in glyphosate-resistant soybean and to assess potential glyphosate accumulation in soybean nodules. Glyphosate-Tms and glyphosate-Adt injured soybean, and visible injury ranged from 29 to 38% 2 d after late postemergence (LPOST) application; however, soybean recovered by 14 d. Glyphosate formulations had no effect on chlorophyll content, root and shoot dry weight, or nodule number but reduced nodule biomass by 21 to 28% 14 d LPOST. Glyphosate levels in nodules from treated plants ranged from 39 to 147 ng g−1 (dry weight), and leghemoglobin content was reduced by as much as 10%. Control of five predominant weed species 14 d after LPOST was > 83% with one application and > 96% with two applications regardless of the glyphosate salts used. Soybean yields were generally higher with two applications than with one application regardless of glyphosate formulation. These results indicate that soybean injury and inhibition of nodule development with certain glyphosate formulations can occur, but soybean has the potential to recover from glyphosate stress. Nomenclature: Glyphosate; soybean, Glycine max (L.) Merr. ‘DP 5806 RR’.

Biocontrol of aflatoxin in corn by inoculation with non-aflatoxigenic Aspergillus flavus isolates

Abstract The ability of two non-aflatoxigenic Aspergillus flavus Link isolates (CT3 and K49) to reduce aflatoxin contamination of corn was assessed in a 4-year field study (2001–2004). Soil was treated with six wheat inoculant treatments: aflatoxigenic isolate F3W4; two non-aflatoxigenic isolates (CT3 and K49); two mixtures of CT3 or K49 with F3W4; and an autoclaved wheat control, applied at 20 kg ha−1. In 2001, inoculation with the aflatoxigenic isolate increased corn grain aflatoxin levels by 188% compared to the non-inoculated control, while CT3 and K49 inoculation reduced aflatoxin levels in corn grain by 86 and 60%, respectively. In 2002, the non-toxigenic CT3 and K49 reduced aflatoxin levels by 61 and 76% compared to non-inoculated controls, respectively. In 2001, mixtures of aflatoxigenic and non-aflatoxigenic isolates had little effect on aflatoxin levels, but in 2002, inoculation with mixtures of K49 and CT3 reduced aflatoxin levels 68 and 37% compared to non-inoculated controls, respectively. In 2003 and 2004, a low level of natural aflatoxin contamination was observed (8 ng g−1). However, inoculation with mixtures of K49 + F3W4 and CT3 + F3W4, reduced levels of aflatoxin 65–94% compared to the aflatoxigenic strain alone. Compared to the non-sclerotia producing CT3, strain K49 produces large sclerotia, has more rapid in vitro radial growth, and a greater ability to colonize corn when artificially inoculated, perhaps indicating greater ecological competence. Results indicate that non-aflatoxigenic, indigenous A. flavus isolates, such as strain K49, have potential use for biocontrol of aflatoxin contamination in southern US corn.

Effect of Glyphosate on Growth, Chlorophyll, and Nodulation in Glyphosate-Resistant and Susceptible Soybean (Glycine max) Varieties

ABSTRACT Greenhouse and growth chamber experiments were conducted to examine glyphosate [isopropylamine salt of N-(phosphono-methyl) glycine] effects on growth, chlorophyll content, nodulation, and nodule leghemoglobin content of glyphosate-resistant and susceptible soybean (Glycine max[L.] Merr.) varieties. In susceptible soybean, a single application of 0.28 kg/ha reduced chlorophyll content (49%), and shoot and root dry weight (50 and 57%, respectively) at 2 wk after treatment. In glyphosate-resistant soybean, there were no significant effects on these parameters by single application up to 1.12 kg/ha, but 2.24 kg/ha reduced shoot and root dry weight by 25 to 30%. Application of glyphosate 1.12 kg/ha, followed by sequential applications at 0.56 or 1.12 kg/ha, did not affect plant growth and chlorophyll content, but application of 2.24 kg/ha followed by sequential application of 2.24 kg/ha reduced root growth. In glyphosate-resistant soybean, an application of 1.12 kg/ha 3 wk after planting did not affect nodule number or mass, but 2.24 kg/ha reduced these parameters by 30 and 39%, respectively, compared to untreated. Leghemoglobin content of nodules was reduced (6 to 18%) by both glyphosate rates, but effects were inconsistent with rate. At post-treatment temperatures of 18/13_C (day/night), glyphosate at 1.12 kg/ha or 2.24 kg/ha did not affect chlorophyll and growth of glyphosate-resistant soybean. However, at 25/20 and 32/27_C (day/night), glyphosate at 2.24 kg/ha reduced both chlorophyll content and growth of glyphosate-resistant soybean. Overall, treatment of gly-phosate-resistant soybean with glyphosate at 1.12 had little or no effect on chlorophyll content and dry weight of shoots and roots in five of five trials. But treatment of glyphosate at 2.24 kg/ha reduced these parameters in three of five trials, suggesting potential for soybean injury at higher rates. Results showed subtle reductions of nodulation in glypho-sate-resistant soybean using label rates of glyphosate, but these effects may be of minimal consequence due to the potential of soybean to compensate after short durations of stress.

Ecology of Aspergillus flavus, regulation of aflatoxin production, and management strategies to reduce aflatoxin contamination of corn

The contamination of corn (maize) by fungi and the accumulation of mycotoxins are a serious agricultural problem for human and animal health. One particular devastating group of mycotoxins, called aflatoxins, has been intensely studied since the 1960s. Studies of Aspergillus flavus, the agriculturally relevant producer of aflatoxins, have led to a well-characterized biosynthetic pathway for aflatoxin production, as well as a basic understanding of the organism’s life cycle. Unfortunately, these efforts have not resulted in corn production practices that substantially reduce aflatoxin contamination. Similarly, the use of agrochemicals (e.g., fungicides) results in very limited reduction of the fungus or the toxin. Thus, cultural management (fertility and irrigation) coupled with aggressive insect management is current recommendation for integrated aflatoxin management. The development of resistant hybrids appears to be a very promising technology, but commercial hybrids are still not available. Thus, biocontrol appears to be the most promising available avenue of reducing aflatoxin accumulation. Biocontrol utilizes nontoxigenic strains of Aspergillus to reduce the incidence of toxin-producing isolates through competitive displacement. To maximize the effectiveness of biocontrol, a thorough knowledge of the environmental factors influencing colonization and growth of Aspergillus is needed. A. flavus not only colonizes living plant tissue, but it also grows saprophytically on plant tissue in the soil. These residues serve as a reservoir for the fungus, allowing it to overwinter, and under favorable conditions it will resume growth and release new conidia. The conidia can be transmitted by air or insects to serve as new inoculum on host plants or debris in the field. This complex ecology of Aspergilli has been studied, but our understanding lags behind what is known about biosynthesis of the toxin itself. Our limited understanding of Aspergilli soil ecology is in part due to limitations in evaluating Aspergilli, aflatoxin, and the biosynthetic genes in the varying aspects of the environment. Current methods for assessing Aspergillus and aflatoxin accumulation rely heavily on cultural and analytical methods that are low throughput and technically challenging. Thus to understand Aspergillus ecology and environmental effects in contamination to maximize biocontrol efforts, it is necessary to understand current treatment effects and to develop methodologies capable of assessing the fungal populations present. In this manuscript we discuss the current knowledge of A. flavus ecology, the application of selected molecular techniques to field assessments, and crop practices used to reduce aflatoxin contamination, focusing on chemical treatments (fungicides and herbicides), insect management, and crop management.

Agronomic and environmental implications of enhanced s-triazine degradation.

Novel catabolic pathways enabling rapid detoxification of s-triazine herbicides have been elucidated and detected at a growing number of locations. The genes responsible for s-triazine mineralization, i.e. atzABCDEF and trzNDF, occur in at least four bacterial phyla and are implicated in the development of enhanced degradation in agricultural soils from all continents except Antarctica. Enhanced degradation occurs in at least nine crops and six crop rotation systems that rely on s-triazine herbicides for weed control, and, with the exception of acidic soil conditions and s-triazine application frequency, adaptation of the microbial population is independent of soil physiochemical properties and cultural management practices. From an agronomic perspective, residual weed control could be reduced tenfold in s-triazine-adapted relative to non-adapted soils. From an environmental standpoint, the off-site loss of total s-triazine residues could be overestimated 13-fold in adapted soils if altered persistence estimates and metabolic pathways are not reflected in fate and transport models. Empirical models requiring soil pH and s-triazine use history as input parameters predict atrazine persistence more accurately than historical estimates, thereby allowing practitioners to adjust weed control strategies and model input values when warranted.

Microbial adaptation for accelerated atrazine mineralization/ degradation in Mississippi Delta soils

Abstract Most well-drained Mississippi Delta soils have been used for cotton production, but corn has recently become a desirable alternative crop, and subsequently, atrazine use has increased. Between 2000 and 2001, 21 surface soils (0 to 5 cm depth) with known management histories were collected from various sites in Leflore, Sunflower, and Washington counties of Mississippi. Atrazine degradation was assessed in 30-d laboratory studies using 14C-ring–labeled herbicide. Mineralization was extensive in all soils with a history of one to three atrazine applications with cumulative mineralization over 30 d ranging from 45 to 72%. In contrast, cumulative mineralization of atrazine from three soils with no atrazine history was only 5 to 10%. However, one soil with no history of atrazine application mineralized 54 and 29% of the atrazine in soils collected in 2000 and 2001, respectively. Methanol extracted 15 to 23% of the 14C-atrazine 7 d after treatment in soils having two applications within the past 6 yr, whereas 65 to 70% was extracted from no-history soils. First-order kinetic models indicated soil with 2 yr of atrazine exposure exhibited a half-life of less than 6 d. Most probable number (MPN) estimates of atrazine-ring mineralizing-microorganisms ranged from 450 to 7,200 propagules g−1 in atrazine-exposed soils, and none were detected in soils with no history of atrazine use. Although most soils exhibited rapid atrazine mineralization, analysis of DNA isolated from these soils by direct or nested polymerase chain reaction (PCR) failed to amplify DNA sequences with primers for the atzA atrazine chlorohydrolase gene. These results indicate that microbial populations capable of accelerated atrazine degradation have developed in Mississippi Delta soils. This may reduce the weed control efficacy of atrazine but also reduce the potential for off-site movement. Studies are continuing to identify the genetic basis of atrazine degradation in these soils. Nomenclature: Atrazine; cyanazine; DEA, de-ethyl atrazine; DIA, de-isopropyl atrazine; corn, Zea mays L.; cotton, Gossypium hirsutum L.

Cover crop, tillage, and herbicide effects on weeds, soil properties, microbial populations, and soybean yield

Abstract A field study was conducted during 1997 to 2001 on a Dundee silt loam soil at Stoneville, MS, to examine the effects of rye and crimson clover residues on weeds, soil properties, soil microbial populations, and soybean yield in conventional tillage (CT) and no-tillage (NT) systems with preemergence (PRE)-only, postemergence (POST)-only, and PRE plus POST herbicide programs. Rye and crimson clover were planted in October, desiccated in April, and tilled (CT plots only) before planting soybean. Both cover-crop residues reduced density of barnyardgrass, broadleaf signalgrass, browntop millet, entireleaf morningglory, and hyssop spurge but did not affect yellow nutsedge at 7 wk after soybean planting (WAP) in the absence of herbicides. Densities of these weed species were generally lower with PRE-only, POST-only, and PRE plus POST applications than with no-herbicide treatment. Total weed dry biomass was lower when comparing CT (1,570 kg ha−1) with NT (1,970 kg ha−1), rye (1,520 kg ha−1) with crimson clover (2,050 kg ha−1), and PRE plus POST (640 kg ha−1) with PRE-only (1,870 kg ha−1) or POST-only (1,130 kg ha−1) treatments at 7 WAP. Soils with crimson clover had higher organic matter, NO3–N, SO4–S, and Mn, and lower pH compared with rye and no–cover crop soils. Total fungi and bacterial populations and fluorescein diacetate hydrolytic activity were higher in soil with crimson clover, followed by rye and no cover crop. Soybean yields were similar between CT (1,830 kg ha−1) and NT (1,960 kg ha−1), no cover crop (2,010 kg ha−1) and rye (1,900 kg ha−1), and rye and crimson clover (1,790 kg ha−1), but they were higher in PRE plus POST (2,260 kg ha−1) than in PRE-only (1,890 kg ha−1) or POST-only (1,970 kg ha−1) treatments. Nomenclature: Acifluorfen; bentazon; clethodim; flumetsulam; metolachlor; barnyardgrass, Echinochloa crus-galli (L.) Beauv. ECHCG; broadleaf signalgrass, Brachiaria platyphylla (Griseb.) Nash BRAPP; browntop millet, Brachiaria ramosa (L.) Stapf. PANRA; entireleaf morningglory, Ipomoea hederacea var. integriuscula Gray IPOHG; hyssop spurge, Euphorbia hyssopifolia L. EPHHS; yellow nutsedge, Cyperus esculentus L. CYPES; crimson clover, Trifolium incarnatum L. ‘Dixie’; rye, Secale cereale L. ‘Elbon’; soybean, Glycine max (L.) Merr.

Relationships between aflatoxin production and sclerotia formation among isolates of Aspergillus section Flavi from the Mississippi Delta

Aspergillus section Flavi isolates, predominately A. flavus, from different crops and soils differed significantly in production of aflatoxin and sclerotia. About 50% of the isolates from corn, soil and peanut produced large sclerotia, while only 20% of the rice isolates produced large sclerotia. There was a higher frequency of small sclerotia-producing isolates from rice compared to the other sources and isolates that did not produce sclerotia were significantly less likely to be toxigenic than strains that produced large sclerotia.

Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize

Biological control of toxigenic Aspergillus flavus in maize through competitive displacement by non-aflatoxigenic strains was evaluated in a series of field studies. Four sets of experiments were conducted between 2007 and 2009 to assess the competitiveness of non-aflatoxigenic strains when challenged against toxigenic strains using a pin-bar inoculation technique. In three sets of experiments the non-aflatoxigenic strain K49 effectively displaced toxigenic strains at various concentrations or combinations. The fourth study compared the relative competitiveness of three non-aflatoxigenic strains (K49, NRRL 21882 from Afla-Guard®, and AF36) when challenged on maize against two aflatoxin- and cyclopiazonic acid (CPA)-producing strains (K54 and F3W4). These studies indicate that K49 and NRRL 21882 are superior to AF36 in reducing total aflatoxin contamination. Neither K49 nor NRRL 21882 produce CPA and when challenged with K54 and F3W4, CPA and aflatoxins were reduced by 84–97% and 83–98%, respectively. In contrast, AF36 reduced aflatoxins by 20% with F3W4 and 93% with K54 and showed no reduction in CPA with F3W4 and only a 62% reduction in CPA with K54. Because AF36 produces CPA, high levels of CPA accumulate when maize is inoculated with AF36 alone or in combination with F3W4 or K54. These results indicate that K49 may be equally effective as NRRL 21882 in reducing both aflatoxins and CPA in maize.