David A. Danehower

CHANGES OVER TIME IN THE ALLELOCHEMICAL CONTENT OF TEN CULTIVARS OF RYE (Secale cereale L.)

Published studies focused on characterizing the allelopathy-based weed suppression by rye cover crop mulch have provided varying and inconsistent estimates of weed suppression. Studies were initiated to examine several factors that could influence the weed suppressiveness of rye: kill date, cultivar, and soil fertility. Ten cultivars of rye were planted with four rates of nitrogen fertilization, and tissue from each of these treatment combinations was harvested three times during the growing season. Concentrations of a known rye allelochemical DIBOA (2,4-dihydroxy-1,4-(2H)benzoxazine-3-one) were quantified from the harvested rye tissue using high performance liquid chromatography (HPLC). Phytotoxicity observed from aqueous extracts of the harvested rye tissue correlated with the levels of DIBOA recovered in harvested tissue. The amount of DIBOA in rye tissue varied depending on harvest date and rye cultivar, but was generally lower with all cultivars when rye was harvested later in the season. However, the late maturing variety ‘Wheeler’ retained greater concentrations of DIBOA in comparison to other rye cultivars when harvested later in the season. The decline in DIBOA concentrations as rye matures, and the fact that many rye cultivars mature at different rates may help explain why estimates of weed suppression from allelopathic agents in rye have varied so widely in the literature.

Vitamer Levels, Stress Response, Enzyme Activity, and Gene Regulation of Arabidopsis Lines Mutant in the Pyridoxine/Pyridoxamine 5′-Phosphate Oxidase (PDX3) and the Pyridoxal Kinase (SOS4) Genes Involved in the Vitamin B6 Salvage Pathway

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5′-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5′-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B6 pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5′-phosphate than the wild type, reflected in an increase in total vitamin B6 observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B6 de novo pathway enzyme PDX1, correlating with increased total B6 levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B6 observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.

Influence of tobacco leaf surface chemicals on germination ofPeronospora tabacina adam sporangia.

Chromatographic procedures were utilized to isolate and purify components of tobacco cuticular extracts and leaf surface chemicals.In vitro microbial bioassays determined the influence of these leaf surface compounds on germination and germ tube morphology ofP. tabacina sporangia, the tobacco blue mold pathogen, and to a lesser extentAlternaria alternata, the tobacco brown spot pathogen. Exposure to 10 μg/cm2 of α- and β-duvatrienemonols, sucrose esters, or hydrocarbons did not inhibit germination, whereas germination was significantly decreased bycis-abienol.cis-Abienol did not inhibit sporangial germination when combined with sucrose esters or hydrocarbons at a combined 10 μg/cm2. Germination of sporangia was completely inhibited by α- and β-duvatrienediols. In contrast to a previous report, α-DVT-diol was more inhibitory than the β isomer. Toxic effects of the DVT-diols were not altered by pH. Diluting the DVT-diols to less than 0.1 μg/cm2 resulted in a small but significant stimulation of germination. Previously, the DVT-diols had been identified only as inhibitory toP. tabacina. None of the leaf surface chemicals affected germination ofA. alternata conidia.

Physiological basis for cotton tolerance to flumioxazin applied postemergence directed

Abstract Previous research has shown that flumioxazin, a herbicide being developed as a postemergence-directed spray (PDS) in cotton, has the potential to injure cotton less than 30 cm tall if the herbicide contacts green stem tissue by rain splash or misapplication. In response to this concern, five-leaf cotton plants with chlorophyllous stems and older cotton, 16-leaf cotton plants, with bark on the lower stem were treated with a PDS containing flumioxazin plus crop oil concentrate (COC) or nonionic surfactant (NIS). Stems of treated plants and untreated plants at the respective growth stage were cross-sectioned and then magnified and photographed using bright-field microscopy techniques. More visible injury consisting of necrosis and desiccation was evident in younger cotton. Also, there was a decrease in treated-stem diameter and an increase in visible injury with COC compared with NIS in younger cotton. The effects of plant growth stage and harvest time on absorption, translocation, and metabolism of 14C-flumioxazin in cotton were also investigated. Total 14C absorbed at 72 h after treatment (HAT) was 77, 76, and 94% of applied at 4-, 8-, and 12-leaf growth stages, respectively. Cotton at the 12-leaf stage absorbed more 14C within 48 HAT than was absorbed by four- or eight-leaf cotton by 72 HAT. A majority (31 to 57%) of applied 14C remained in the treated stem for all growth stages and harvest times. Treated cotton stems at all growth stages and harvest times contained higher concentrations (Bq g−1) of 14C than any other tissues. Flumioxazin metabolites made up less than 5% of the radioactivity found in the treated stem. Because of the undetectable levels of metabolites in other tissues when flumioxazin was applied PDS, flumioxazin was foliar applied to determine whether flumioxazin transported to the leaves may have been metabolized. In foliar-treated cotton, flumioxazin metabolites in the treated leaf of four-leaf cotton totaled 4% of the recovered 14C 72 HAT. Flumioxazin metabolites in the treated leaf of 12-leaf cotton totaled 35% of the recovered 14C 48 HAT. These data suggest that differential absorption, translocation, and metabolism at various growth stages, as well as the development of a bark layer, are the bases for differential tolerances of cotton to flumioxazin applied PDS. Nomenclature: Flumioxazin; cotton, Gossypium hirsutum L.

Analysis of a Nicotiana tabacum L. Genomic Region Controlling Two Leaf Surface Chemistry Traits

cis-Abienol and sucrose esters are Nicotiana tabacum leaf surface components that likely influence plant resistance to pests. Their breakdown products also contribute to flavor and aroma characteristics of certain tobacco types. Mapping of genes involved in the biosynthesis of these compounds could permit development of molecular-based tools for generating tobacco types with novel cured leaf chemistry profiles. A doubled haploid mapping population segregating for major genes (Abl and BMVSE) affecting the ability to accumulate cis-abienol and sucrose esters was generated and genotyped with a large set of microsatellite markers. The two genes were found to reside on chromosome A of the N. tabacum genome with a distance of 8.2 cM (centimorgans) between them. Seventeen microsatellite markers were also placed on this linkage group, several of which exhibited complete cosegregation with Abl and BMVSE. Results should aid breeding efforts focused on modification of this aspect of tobacco cured leaf chemistry.

Modulation of the Phenylacetic Acid Metabolic Complex by Quinic Acid Alters the Disease-causing Activity of Rhizoctonia solani on Tomato

The metabolic control of plant growth regulator production by the plant pathogenic fungus Rhizoctonia solani Kühn (teleomorph=Thanatephorus cucumeris (A.B. Frank) Donk) and consequences associated with the parasitic and saprobic activity of the fungus were investigated. Fourteen genetically distinct isolates of the fungus belonging to anastomosis groups (AG) AG-3, AG-4, and AG-1-IA were grown on Vogels minimal medium N with and without the addition of a 25 mM quinic acid (QA) source of carbon. The effect of QA on fungal biomass was determined by measuring the dry wt of mycelia produced under each growth condition. QA stimulated growth of 13 of 14 isolates of R. solani examined. The production of phenylacetic acid (PAA) and the chemically related derivatives 2-hydroxy-PAA, 3-hydroxy-PAA, 4-hydroxy-PAA, and 3-methoxy-PAA on the two different media was compared by gas chromatography coupled with mass spectrometry (GC-MS). The presence of QA in the growth medium of R. solani altered the PAA production profile, limiting the conversion of PAA to derivative forms. The effect of QA on the ability of R. solani to cause disease was examined by inoculating tomato (Solanum lycopersicum L.) plants with 11 isolates of R. solani AG-3 grown on media with and without the addition of 25 mM QA. Mean percent survival of tomato plants inoculated with R. solani was significantly higher when the fungal inoculum was generated on growth medium containing QA. The results of this study support the hypotheses that utilization of QA by R. solani leads to reduced production of the plant growth regulators belonging to the PAA metabolic complex which can suppress plant disease development.

Elucidating the role of the phenylacetic acid metabolic complex in the pathogenic activity of Rhizoctonia solani anastomosis group 3

The soil fungus Rhizoctonia solani produces phytotoxic phenylacetic acid (PAA) and hydroxy (OH-) and methoxy (MeO-) derivatives of PAA. However, limited information is available on the specific role that these compounds play in the development of Rhizoctonia disease symptoms and concentration(s) required to induce a host response. Reports that PAA inhibits the growth of R. solani conflict with the established ability of the fungus to produce and metabolize PAA. Experiments were conducted to clarify the role of the PAA metabolic complex in Rhizoctonia disease. In this study the concentration of PAA and derivatives required to induce tomato root necrosis and stem canker, in the absence of the fungus, and the concentration that inhibits mycelial growth of R. solani were determined. The effect of exogenous PAA and derivatives of PAA on tomato seedling growth also was investigated. Growth of tomato seedlings in medium containing 0.1–7.5 mM PAA and derivatives induced necrosis of up to 85% of root system. Canker development resulted from injection of tomato seedling stems with 7.5 mM PAA, 3-OH-PAA, or 3-MeO-PAA. PAA in the growth medium reduced R. solani biomass, with 50% reduction observed at 7.5 mM. PAA, and derivatives were quantified from the culture medium of 14 isolates of R. solani belonging to three distinct anastomosis groups by GC-MS. The quantities ranged from below the limit of detection to 678 nM, below the concentrations experimentally determined to be phytotoxic. Correlation analyses revealed that isolates of R. solani that produced high PAA and derivatives in vitro also caused high mortality on tomato seedlings. The results of this investigation add to the body of evidence that the PAA metabolic complex is involved in Rhizoctonia disease development but do not indicate that production of these compounds is the primary or the only determinant of pathogenicity.

Influence of Water Quality and Coapplied Agrochemicals on Efficacy of Glyphosate

Abstract Experiments were conducted in 2008, 2009, and 2010 to determine the influence of water source as carrier and other agrochemicals on glyphosate efficacy and physicochemical compatibility. Glyphosate efficacy was not affected by most water sources, when compared with deionized water, although response was not consistent across all weed species, including cereal rye, common lambsquarters, common ragweed, goosegrass, Italian ryegrass, large crabgrass, Palmer amaranth, tall morningglory, and wheat. Control by glyphosate was not negatively affected when coapplied with cloransulam-methyl, dicamba, flumioxazin, pyrithiobac-sodium, thifensulfuron-methyl plus tribenuron-methyl, trifloxysulfuron-sodium, and 2,4-D but was affected by acifluorfen and glufosinate. Calcium, manganese, and zinc solutions consistently reduced weed control by glyphosate, whereas boron seldom affected efficacy. Compared with deionized water, Italian ryegrass control was affected by water sources when applied at seedling and jointing stages more so than at tillering and heading growth stages. Calcium, manganese, and zinc reduced control regardless of growth stage. Precipitates were not produced when glyphosate was applied with the water sources or fertilizer solutions. However, transient precipitates developed when glyphosate was coapplied with cloransulam-methyl, flumioxazin, thifensulfuron-methyl plus tribenuron-methyl, and trifloxysulfuron-sodium but not when coapplied with acifluorfen, dicamba, glufosinate, pyrithiobac-sodium, and 2,4-D. Solution pH ranged from 4.11 to 5.60 after glyphosate was added, regardless of solution pH before glyphosate addition. Nomenclature: 2,4-D; acifluorfen; boron; calcium; cloransulam-methyl; dicamba; flumioxazin; glufosinate; glyphosate; manganese; pyrithiobac-sodium; thifensulfuron-methyl plus tribenuron-methyl; trifloxysulfuron-sodium; zinc; cereal rye, Secale cereale L.; common lambsquarters, Chenopodium album L.; common ragweed, Ambrosia artemisiifolia L.; goosegrass, Eleusine indica (L.) Gaertn.; Italian ryegrass, Lolium perenne L. ssp. multiflorum (Lam.) Husnot.; large crabgrass, Digitaria sanguinalis (L.) Scop.; Palmer amaranth, Amaranthus palmeri (L.) S. Wats.; tall morningglory, Ipomoea purpurea (L.) Roth; wheat, Triticum aestivum L.

Cuticular wax of Epilachna varivestis

Abstract Cuticular lipids of larvae, pupae and adults of the Mexican bean beetle (Epilachna varivestis) have been examined using gravimetric and thin layer densitometric techniques. The effects of rearing on different hostplants and of rearing temperature on lipid composition were studied. Total lipid varied with developmental stage as well as hosplant. The amount of lipid extracted ranged from 3.0 mg/g wet weight, in the case of larvae reared on snapbeans in the field, to 5.2 mg/g wet weight for pupae reared in limas under field conditions. Total lipid increased with increasing temperature for larvae reared under controlled climatic conditions. Thin layer densitometry was used to quantify lipid classes. Epicuticular lipids included hydrocarbons, wax esters, triacylglycerols, fatty alcohols, free fatty acids, sterols, three unidentified materials and alkaloid(s). Lipids of larval and pupal stages were composed primarily of wax esters, hydrocarbons and fatty alcohols in roughly equal proportions; free fatty acids, triacylglycerols, sterols, alkaloid(s) and two unknown materials made up of the remainder. Adult cuticular lipids consisted mainly of hydrocarbons (49–60% of total lipid).

Quantitative thin-layer chromatography of Nicotianatabacum leaf surface components

Abstract A thin-layer chromatographic (TLC) method and scanning densitometry technique have been developed for quantitation of major primary and secondary metabolites of Nicotiana tabacum L. leaf surface components. These components were resolved after a single development with isopropanol—chloroform—methylene chloride—hexane (7:8:6:79) using laned silica gel G TLC plates. R F values were reproducible to ± 0.01 units. Quantitation of all components of interest was accomplished by charring with 30% fuming sulfuric acid followed by densitometry using white light. Overall, charring results were semiquantitative (⩽ 10% relative error), but were quantitative (⩽ 6% relative error) for all major secondary metabolites except the sucrose esters. Quantitation of three secondary metabolites was also accomplished by scanning uncharred plates at a wavelength of 200 nm. In general, UV scanning provided semiquantitative results. For both charring and UV quantitation methods, highly correlated, curvilinear responses between mass and integration area were obtained. Advantages and limitations of these procedures versus an existing gas chromatographic procedure and their potential implementation are discussed.