The Allelochemical Sorgoleone Inhibits Root H+-ATPase and Water Uptake

Sorghum plants inhibit the growth of some adjacent species. Root exudates from grain sorghum (Sorghum bicolor), consisting primarily of the quinone sorgoleone, are phytotoxic to several plant species, yet the mechanisms of growth inhibition remain to be fully explained. Disruption of electron transport functions in isolated mitochondria and chloroplasts has been reported as one explanation for growth inhibition. In the studies reported here, however, soybean seedlings grown in nutrient solution with 10, 50, or 100 microM sorgoleone showed no disruption of photosynthesis, as measured by leaf fluorescence and oxygen evolution, yet their mean leaf surface area was less when grown in 100 microM sorgoleone. Furthermore, in the presence of these same concentrations of sorgoleone, decreased nutrient solution use by soybean seedlings and decreased H+-ATPase activity in corn root microsomal membranes were observed. This suggests that impairment of essential plant processes, such as solute and water uptake, driven by proton-pumping across the root cell plasmalemma should also be considered as a mechanism contributing to observed plant growth inhibition by sorgoleone.     

Effects of juglone on growth,photosynthesis, and respiration

The impacts of juglone on plant growth and several other physiological functions were evaluated in this study. Juglone inhibitedLemna minor growth, chlorophyll content, and net photosynthesis at treatments between 10 and 40M. Soybean leaf disks vacuum infiltrated with as little as 10M juglone had reduced photosynthesis. Oxygen evolution by chloroplasts isolated fromPisum sativum was inhibited by juglone with an I₅₀ of 2M. Micromolar treatments of juglone stimulated oxygen uptake in mitochondria isolated fromGlycine max. These data suggest perturbations of chloroplast and mitochondrial functions may contribute to plant growth reductions observed in juglone-mediated allelopathy.This study was supported by National Science Foundation grant STI-8902066.     

Synergism of Polygodial and trans-Cinnamic Acid on Inhibition of Root Elongation in Lettuce Seedling Growth Bioassays

A bicyclic sesquiterpene dialdehyde, polygodial did not inhibit root elongation up to a concentration of 12.5 g/ml in a lettuce seedling assay: trans-Cinnamic acid inhibited the elongation by 50% at 1.2 g/ml (8.1 M). The inhibitory activity of trans-cinnamic acid was enhanced 17-fold when used in combination with 6.25 g/ml (26.5 M) of polygodial. A decrease in the pH of the medium was observed during normal seedling growth, indicating transport of protons from the cells by a plasma membrane H⁺-ATPase. The inhibitory effect of trans-cinnamic acid on the elongation was reduced to some extent in 2 mM phosphate buffer (pH 7.0) during seedling growth. Although polygodial did not inhibit the activity of H⁺-ATPase in the plasma membrane fraction of roots in normally growing seedlings, a decrease in activity was found in the fraction obtained from seedlings incubated with 20 g/ml of polygodial. These results suggest that polygodial functions synergistically with trans-cinnamic acid in the inhibition of root elongation via restriction of proton transport from the cytoplasm of germinated cells.     

Milieu-Dependent Pro- and Antioxidant Activity of Juglone May Explain Linear and Nonlinear Effects on Seedling Development

Juglone, 5-hydroxy-1,4-naphthoquinone, is known for its wide range of biological activities. It has been suggested that juglone’s excellent redox cycling properties contribute to this reputation. Many biological activities are nonlinear with low concentrations exerting stimulating effects, whereas only higher concentrations cause inhibition. Here, we corroborate studies on the nematode Caenorhabditis elegans that point out hormetic effects by showing that juglone may cause a nonlinear effect on postgerminative shoot and root growth of Sinapis alba. This effect was only significantly visible, however, when seedlings were stressed with methanol. Classic and modified versions of the deoxyribose assay were applied successfully to characterize antioxidative (purposeful generation of hydroxyl radicals) and prooxidative (no purposeful generation of hydroxyl radicals) activities. Variants of the assay with and without the addition of the iron chelator EDTA showed that the antioxidant activity is independent on chelation of iron ions by juglone; by contrast, the strength of the prooxidative activity depended on the chelation of iron ions by juglone. The hormetic effects of lower concentrations on germination of Sinapis alba, thus, may be caused by the antioxidant activities of this compound, which are especially effective when the test organism is subjected to higher oxidative challenge. The present study suggests that pronounced prooxidative activities, which are considerably accelerated by chelation of iron ions, may contribute to the toxic effects of juglone at higher concentrations.     

Allelopathic effect of parthenium (Parthenium hysterophorus L.) extract and residue on some agronomic crops and weeds

Allelopathic effects of entire shoot extract, plant part extracts, and shoot residue of parthenium (Parthenium hysterophorus L.) on corn (Zea mays L.), ryegrass (Lolium multiflorum Lam.), wheat (Triticum aestivum L.), velvetleaf (Abutilon theophrasti Medik.), and soybean [Glycine max (L) Merr.] growth were examined. Parthenium shoot contained water-soluble materials that were toxic to root growth of velvetleaf and wheat. At 4% (w/ v) concentration, root growth of velvetleaf and wheat were reduced by 60 and 75%, respectively. The order of increasing sensitivity to parthenium was ryegrass, corn, wheat, and velvetleaf. There was a strong correlation between extract concentration and increased toxicity to test species. The toxicity of plant part extracts was also concentration dependent. At 1 and 2% (w/v), the inflorescence and leaves caused more root inhibition than stem extract. Parthenium shoot incorporated in soil at 1% (w/w) caused significantly more root inhibition of wheat than soybean, corn, and ryegrass. At 4% (w/w), root growth of all the test species was inhibited compared to the control. Toxicity of parthenium residue to wheat diminished with increasing periods of decomposition. Residue decomposed for four weeks was less toxic than the undecomposed residue.Florida Agricultural Experiment Stations Journal Series No. 7506.     

Inhibition of salvinia (Salvinia molesta Mitchell) by parthenium (Parthenium hysterophorus L.). II. Relative effect of flower,leaf, stem,and Root residue on Salvinia and Paddy

The relative effect of parthenium (Parthenium hysterophorus L.) plant residue on growth of salvinia and paddy seedlings was studied. The inhibitory activity of the residue as shown by its effect on the number of healthy fronds (HFN) and biomass was in the order: flower and leaf > stem and root. The flower (FP) and leaf (LP) residue was lethal at and above 0.75% (w/v, the convention used throughout), and inhibitory at lower doses. The stem (SP) and root (RP) residue supported growth of salvinia at lower doses and were slightly inhibitory at higher (1.25%) dose. All the above residue types supported the growth of paddy seedlings except at 1.25%, the highest concentration tested, which was slightly inhibitory. The amounts of chlorophylla, b, total chlorophyll, and carotenoid pigments in the leaves of the paddy seedlings grown in the medium were comparable to the amounts in the leaves of seedlings grown in distilled water. This demonstrates beneficial effects of the treatments. The study shows that salvinia is more sensitive to allelochemicals released by FP and LP into the aqueous medium. Both salvinia and paddy responded similarly to SP and RP by supporting growth at lower doses, probably due to lower levels of inhibitors. The results are discussed with reference to the possible role of allelopathy by parthenium on the population dynamics of aquatic weeds in natural ecosystems.     

Consumption and utilization of experimentally altered corn by southern armyworm: Iron,nitrogen, and cyclic hydroxamates

The effects of differential leaf water, leaf nitrogen and cyclic hydroxamate (DIMBOA) concentrations in corn seedlings were analyzed for a polyphagous insect, the southern armyworm (Spodoptera eridania Cram.). Six different combinations of nutrients and allelochemicals [DIMBOA = 2,4-dihydroxy-7-methoxy(2H)-benzoxazin-3(4H)-one] were generated using two corn genotypes (WF9 and CI3IA) and three fertility regimes (complete nutrient, Fe-deficient, and N-deficient solutions) in the University Biotron. Poorest larval growth was observed in the low-nitrogen treatments (1.2% and 1.7% leaf N) and was the result of both low consumption rates and high metabolic costs (low efficiency of conversion of digested food, ECD). Fastest growth rates were observed forthe larvae fed leaves from the high-nitrogen treatments (4.6% and 4.4% leaf N). It is noteworthy that these treatments also contained the highest concentration of cyclic hydroxamates, which are generally believed to be the primary defensive chemicals mediating resistance against the European corn borer,Ostrinia nubilalis (Hubner). If these hydroxamates do have any deleterious or costly effects (perhaps accounting for a large portion of metabolic expenditures), the high digestibility of the leaf tissue and the increased consumption rates more than compensate, resulting in rapid growth (growth rate = consumption rate × approximate digestibility × efficiency of conversion of the digested food). These studies illustrate that variation in key nutrients and allelochemicals within a single plant species (Zea mays L.) may have significantly different effects upon various potential leaf-chewing caterpillars, such as these armyworms versus corn borers (which cannot handle the cyclic hydroxamates, even if provided with young nutritious leaf tissues).     

Phytotoxic Effects of Leukamenin E (an ent-kaurene diterpenoid) on Root Growth and Root Hair Development in Lactuca sativa L. seedlings

Leukamenin E, an ent-kaurene diterpenoid isolated from Isodon racemosa (Hemsl) Hara, showed phytotoxic effects on root growth and root hair development of lettuce seedlings (Lactuca sativa L.). Lower concentrations (10 μM) of leukamenin E did not affect root growth, but at concentrations higher than 50 μM, the rate was inhibited. The influence of leukamenin E on root growth rate was closely correlated with alterations in the mitotic index. A low incidence of aberrant mitosis image was observed when lettuce roots were treated with higher concentrations (100 and 200 μM) of leukamenin E. This suggests that inhibition of root growth may be due to inhibition of cell division. All tested concentrations of the diterpenoid (10 μM or more) inhibited root hair development in a dose-dependent manner. At a concentration of 80 μM, leukamenin E completely blocked root hair initiation. Application of Ag⁺—an ethylene action inhibitor—to lettuce seedlings inhibited root hair elongation similar to the diterpenoid. Enhanced root hair length was stimulated by exogenous ethephon—an ethylene-releasing agent—and could be reversed by addition of leukamenin E. This suggests that leukamenin E may act as a potential ethylene action antagonist in the inhibition of lettuce root hair development. We conclude that leukamenin E may curb root hair development by interfering with ethylene action at concentrations above 10 μM and inhibits root growth via inhibition of cell division at concentrations above 50 μM. Lan Ding and Linlin Qi contributed equally to this work.     

Inhibitory effect of parthenium (Parthenium hysterophorus L.) residue on growth of water hyacinth (Eichhornia crassipes Mart Solms.) II. Relative effect of flower,leaf, stem,and root residue

The relative effect of residue of leaf, flower, stem, and root of parthenium (Parthenium hysterophorus L.) on growth of water hyacinth was studied. The inhibitory activity of the residue as shown by its effect on biomass and healthy leaf number (HLN) of treated plants was in the order: leaf and flower >stem >root. Total phenolic acids in the medium after 72 hr of suspending the plant part residue were maximum in flower followed by leaf, root, and stem, successively. The dry leaf powder (DLP) and dry flower powder (DFP) at and above 0.50% (w/v) and dry stem powder (DSP) at 1.00% (w/v) killed water hyacinth in about one month. Dry root powder (DRP) at the highest dose (1.25% w/v) reduced the growth of the treated plants drastically, but the plants recovered after about one month. The DSP at 0.50% (w/v) and DRP at 0.25–0.75% (w/v) supported growth of treated plants, probably due to lower levels of inhibitors, allowing utilization of constituents of the residue as nutrients. Using wheat seedlings as a reference material, it was observed that in aquaculture at different levels of parthenium plant parts residue, water hyacinth plants were much more sensitive to inhibitory activity. Thus, water hyacinth is suggested as a material for bioassay of inhibitory activity of the parthenium plant residue.     

Role of allelopathy as a possible factor associated with the rising dominance ofBunias orientalis L. (Brassicaceae) in some native plant assemblages

Leaf extracts ofBunias orientalis were shown to inhibit seed germination of a variety of cultivar plant species and of species cooccurring withB. orientalis in the field. Root exudate solutions and leaf litter leachates ofB. orientalis were tested for their allelopathic activity using seedling growth assays. Additionally, in comparative seedling growth assays soil cores removed from denseB. orientalis stands were tested bimonthly for elevated allelopathic effects. The impact of root exudates on seedling growth was generally weak and varied between species. Similar results were obtained for the effect ofB. orientalis leaf litter leachates on seedlings grown in sand culture relative to the effect of leaf litter leachates of a plant species mixture. When soil as a growth substrate was used, no consistent differences in seedling growth were obtained between the two litter leachate treatments. In the soil core experiment seedlings grown in soil cores collected from a denseB. orientalis stand unexpectedly showed better performance than seedlings grown in soil cores collected from a nearby mixed plant stand withoutB. orientalis, at least in early spring and late autumn. Predominating nutrient effects are, therefore, assumed to conceal a potentially increased allelopathic effect of soil beneath denseB. orientalis stands. It is concluded that other factors than allelopathy must be investigated to explain the rapid establishment of dense stands of this alien plant species.     

Root growth and phosphorus and potassium uptake by two corn genotypes in the field

In a field study P and K uptake by two corn (Zea mays L.) genotypes which differed in root growth was investigated. The effect of differences in root growth on P and K uptake was assessed using a mechanistic-mathematical model which describes nutrient uptake by growing plant roots in soil. Nitrogen was applied at 0 and 227 kg ha^–1 to Pioneer 3732 and B73xMo17 corn grown on Raub silt loam (fine-silty, mixed, mesic Aquic Argiudoll) and at 227 kg N ha^–1 to these two genotypes on Chalmers silt loam (finesilty, mixed, mesic, Typic Haplaquoll). Root growth and P and K uptake by the two corn genotypes was measured 31, 47, 75 and 91 d after planting on the Raub and 31, 47, 61 and 75 d after planting on the Chalmers soil.Root growth and P and K uptake by B73xMo17 was greater than that of Pioneer 3732 on N-fertilized Raub soil. On Chalmers soil the difference in root growth between the two genotypes resulted in an increase in K but not P uptake. The higher soil P level of the Chalmers appears to have offset possible differences in P uptake due to root size. There were no differences between the two genotypes in either the percentage of roots with root hairs, or the density or length of root hairs. Phosphorus and K uptake calculated with the simulation models for both corn genotypes on both soils over each of three growth periods agreed with observed P (Y = 0.68X + 1.71; r = 0.944^**) and K (Y = 0.88X + 15.52; r = 0.928^**) uptake. Differences in P and K uptake between B73xMo17 and Pioneer 3732 resulted primarily from the difference in root growth in the topsoil. A high correlation was found between root surface area and P (r = 0.893^**) and K (r = 0.928^**) uptake by both corn genotypes on both the soils.Journal paper No. 10,316 Purdue Univ. Agric. Exp. Stn., W. Lafayette, In 47906. Contribution from the Dep. of Agron. This paper was supported in part by a grant from the Tennessee Valley Authority.     

Effects of benzoic and cinnamic acids on growth,mineral composition,and chlorophyll content of soybean

Organic acids are major water-soluble allelochemicals found in soil infested with quackgrass and are involved in several processes that are important in plant growth and development. This study was carried out to gain more information on the effects of benzoic acid (BEN) andtrans-cinnamic acid (CIN) on growth, mineral composition, and chlorophyll content of soybean [Glycine max (L.) Merr. cv. Maple Bell] grown in nutrient solution. The two allelochemicals reduced root and shoot dry biomass of soybean. Treated plants had fewer lateral roots and tended to grow more horizontally compared to the untreated plants. Lateral roots were stunted and less flexible. The amounts of P, K, Mg, Mn, Cl^–, and SO ₄ ^2– were lower, and Zn and Fe contents were higher in roots of plants grown with BEN or CIN as compared to untreated plants. Shoots of plants grown with the allelochemical showed greater accumulation of Ca, Mg, and Zn, whereas P and Fe contents were reduced. The BEN and CIN also caused reductions in leaf chlorophyll content. The BEN and CIN may be responsible for negative allelopathic effects of quackgrass on soybean by inhibiting root growth, by altering ion uptake and transport, and by reducing chlorophyll content.Contribution 493 of the Soils and Crop Research Center.     

Juglone reduces growth,nitrogenase activity,and root respiration of actinorhizal black alder seedlings

European black alder trees [Alnus glutinosa (L.) Gaertn.] fix nitrogen with nodular symbionts and are interplanted with valuable black walnut trees (Juglans nigra L.) to increase soil nitrogen fertility. However, on some soils interplanted alder can be killed by black walnut's allelochemical juglone. In order to better understand the effects of juglone directly on the growth, nitrogen fixation, and root respiration of black alder, we grew nodulated alders hydroponically in a nitrogen-free nutrient solution at juglone levels of 2 × 10^–6, 2 × 10^–5, and 0 molar (M). Results indicate that nitrogenase activity (acetylene reduction) of alders growing in 2 × 10^–3 M juglone was reduced relative to alders without added juglone after one day, and in 2 × 10^–6 M juglone after five days. Root respiration (CO₂ evolution) and the relative increase of plant fresh weight were reduced in the 2 × 10^–5 M juglone treatment. In a related experiment, black alder germinants were grown in Flanagan silt loam soil dosed with 10^–3, 10^–4, and 0 M juglone. The inhibitory effects of 10^–3 M juglone on radicle elongation ceased 22–37 days after juglone treatments were started, suggesting that this soil can readily detoxify juglone.     

Growth Inhibition and Root Ultrastructure of Cucumber Seedlings Exposed to Allelochemicals from Rye (Secale cereale)

Inhibition of "Calypso" cucumber seedling growth by rye allelochemicals, 2(3H)-benzoxazolinone BOA and 2,4-dihydroxy-1,4(2H)-benzoxazin-3-one DIBOA, was studied by analyzing the growth of seedling tissues and organs. Light and electron microscopy of seedling root cells were also carried out to investigate the mechanism(s) of root growth inhibition and mode of action of these compounds. BOA inhibited root elongation and reduced the number of cucumber lateral roots by 77 and 100% at 0.1 and 0.43 mg BOA/ml deionized (DI) water, respectively. DIBOA also inhibited root growth, but did not affect the number of lateral roots. BOA increased size of cucumber cortical root cells fivefold, but DIBOA had no effect. Both compounds reduced the regeneration of root cap cells and increased the width of cortical cells resulting in increased root diameter. BOA and DIBOA caused increased cytoplasmic vacuolation, reduced ribosome density and dictyosomes, reduced number of mitochondria, and reduced lipid catabolism. Starch granules in amyloplasts of seedling roots treated with BOA and DIBOA were also greatly reduced compared to the control. Changes in cellular ultrastructure indicated that BOA and DIBOA reduced root growth by disrupting lipid metabolism, reducing protein synthesis, and reducing transport or secretory capabilities.     

Phytotoxicity of <Emphasis Type="Italic">Phytolacca americana</Emphasis> Leaf Extracts on the Growth,and Physiological Response of <Emphasis Type="Italic">Cassia mimosoides</Emphasis>

We examined the allelochemical effects of control soil, native soil (treated soil), and leaf extracts of Phytolacca americana (pokeweed) on the germination rate and seedling growth of Cassia mimosoides var. nomame. We also studied the resulting changes in root-tip ultrastructure and peroxidase isozyme biochemistry. P. americana leaf extract inhibited seed germination, seedling growth, and biomass when compared to control and treated soil. Root and shoot growth in treated soil was stimulated relative to control soil, but root growth was inhibited by 50% in the leaf extract treatment. Biomass of C. mimosoides seedlings grown on leaf extract was reduced sevenfold when compared to the control seedlings. The amounts of total phenolic compounds in the leaf extract, treated soil, and control soil were 0.77, 0.14, and 0.03 mg l⁻¹, respectively. The root tips of C. mimsoides treated with leaf extracts of P. americana showed amyloplasts and large central vacuoles with electron-dense deposits inside them when compared to control root tips. The activity of guaiacol peroxidase (GuPOX) in whole plant, roots, and shoots of C. mimosoides increased as leaf extract increased; maximum activity was observed in extract concentrations of 75% and higher. Root GuPOX activity was three times higher than in shoots. Therefore, we conclude that inhibition of C. mimosoides growth is related to the phenolic compounds in the P. americana leaf extract and the ultrastructure changes in root-tip cells and increased GuPOX activity is a response to these allelochemicals.     

Sorgoleone from root exudate inhibits mitochondrial functions

The aim of this investigation was to determine if sorgoleone (SGL), a hydrophobic compound inSorghum bicolor (L.) Moench root exudate, interferes with mitochondrial functions. Tests were conducted on mitochondria isolated from etiolated soybean [Glycine max (L.) Merr.] and corn (Zea mays L.) seedlings. The data show SGL is a potent inhibitor of state 3 and state 4 respiration rates in both soybean and corn. Using either NADH, succinate, or malate as substrate, the I₅₀ was about 0.5M SGL for state 3 and 5.0M for state 4 based on 0.3–0.5 mg mitochondrial protein. Absorption spectra indicate SGL blocks electron transport at theb-c ₁ complex. These data show that disruption of mitochondrial function may be a mechanism of SGL-mediated growth inhibition previously reported and demonstrate a probable role of SGL inSorghum allelopathy.This study was supported in part by National Science Foundation grant RII-8902066 (South Dakota EPSCOR).     

Manipulation of Root Hair Development and Sorgoleone Production in Sorghum Seedlings

Sorghum (Sorghum bicolor) roots exude a potent bioherbicide sorgoleone. Previous work indicates that sorgoleone is produced in living root hairs. We have developed a mist system that resulted in abundant production of root hairs exuding sorgoleone and a mat system that significantly inhibited root hair development and consequently sorgoleone production. Applying Ag+ (an ethylene action inhibitor) at 1.2 mM to the seedlings grown in the mist system also inhibited root hair formation and elongation. Hypoxic conditions in the mist system did not result in the inhibition of root hair growth as compared to the standard air atmosphere (20.8% O2). Applying ethephon (an ethylene-releasing agent) at 0.031 mM to the roots of seedlings grown in the mat system with water running at 1 ml/min reversed the inhibition of root hair development by water movement. These results indicate that either water movement or ethylene can be utilized to manipulate root hair development and sorgoleone production in sorghum seedlings. It is hypothesized that water movement reduced the local ethylene concentration on the root surface and consequently inhibited root hair development of sorghum seedlings grown in the mat system.     

Lignification and Related Enzymes in Glycine max Root Growth-Inhibition by Ferulic Acid

Changes in soluble and cell wall bound peroxidase (POD, EC 1.11.1.7) activity, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, and lignin content in roots of ferulic acid-stressed soybean (Glycine max (L.) Merr.) seedlings and their relationships with root growth were investigated. Three-day-old soybean seedlings were cultivated in half-strength Hoagland nutrient solution containing 1.0 mM ferulic acid for 24-72 hr. Length, fresh weight, and dry weight of roots decreased, while soluble and cell wall bound POD activity, PAL activity, and lignin content increased after ferulic acid treatment. These enzymes probably participate in root growth reduction in association with cell wall stiffening related to the formation of cross-linking among cell wall polymers and lignin production.     

Seasonal patterns of juglone in soil beneathJuglans nigra (black walnut) and influence ofJ. nigra on understory vegetation

The allelopathic nature ofJ. nigra L. was investigated in several planted mixed hardwood stands located near Syracuse, New York. Concentrations of chloroform-extracted juglone from soil collected beneathJ. nigra was determined by thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). Soil juglone concentrations were corrected based on recovery of synthetic juglone added to soil. Soil juglone levels were high in the spring, decreased during the summer, and were high again in the fall. The quantification of juglone from soil by HPLC was found to be more accurate than by TLC. Regression analysis indicated that individual tree variation in soil juglone levels could not be explained by differences in soil moisture, pH, organic matter content, and texture. The results of juglone recovery experiments suggest that chloroform-extractable juglone does not persist in soil. Juglone degradation by microorganisms could only explain a portion of the juglone decline. Ordinations revealed that the herbaceous and woody vegetation beneathJ. nigra, in comparison to vegetation beneathAcer saccharum andQuercus rubra, is distinct in only one of the four stands studied. This vegetational difference did not appear to be a consequence of any strong allelopathic influences ofJ. nigra (Scheffe's method of contrast, chi-square analysis). The allelopathic nature of juglone under these field conditions is questionable.