Frank A. Einhellig

Phytotoxicity of sorgoleone found in grain sorghum root exudates

Root exudates ofSorghum bicolor consist primarily of a dihydroquinone that is quickly oxidized to ap-benzoquinone named sorgoleone. The aim of this investigation was to determine the potential activity of sorgoleone as an inhibitor of weed growth. Bioassays showed 125μM sorgoleone reduced radicle elongation ofEragrostis tef. In liquid culture, 50-μM sorgoleone treatments stunted the growth ofLemna minor. Over a 10-day treatment period, 10μM sorgoleone in the nutrient medium reduced the growth of all weed seedlings tested:Abutilon theophrasti, Datura stramonium, Amaranthus retroflexus, Setaria viridis, Digitaria sanguinalis, andEchinochloa crusgalli. These data show sorgoleone has biological activity at extremely low concentrations, suggesting a strong contribution toSorghum allelopathy.

Potentials for exploiting allelopathy to enhance crop production

Strategies for utilizing allelopathy as an aid in crop production include both avoidance and application protocols. There are immediate opportunities for management of weed and crop residues, tillage practices, and crop sequences to minimize crop losses from allelopathy and also to use allelopathic crops for weed control. Varieties of grain and forage sorghums (Sorghum Spp.), sunflower (Helianthus annuus L.), oats (Avena sativa L.), wheat (Triticum sativum L.),rye (Secale cereale L.), and others may provide weed control and in some instances crop stimulation from their residues. Our four-year field study with cultivated sunflower resulted in no differences in weed biomass between plots with and without herbicide (EPTC) applications. Strip cropping that included sorghum showed that in the subsequent year weed density and biomass were significantly lower in the previous-year sorghum than in soybean strips. Possibilities exist for modification of crop plant metabolism to alter production of allelochemicals. Allelochemical-environmental interactions must be considered in efforts to benefit from allelopathy. Under greenhouse conditions, joint application of low levels of atrazine, trifluralin, alachlor, or cinmethylin with a phenolic allelochemical showed that these two categories of inhibitors acted in concert to reduce plant growth. Allelochemicals may also be adapted as yield stimulants or environmentally sound herbicides, such as cinmethylin and methoxyphenone. Isolation of bialophos, tentoxin, and others shows that bacteria and fungi are good sources of biologically active compounds.

Effects of salicylic acid on plant-water relationships

Soybean seedlings [Glycine max (L.) Merr.] were used as the test species to study the allelopathic influence of salicylic acid (SA) on short- and long-term plant water status. Plants were grown in greenhouse conditions in nutrient culture medium amended with SA. Treatments were initiated 10 days after germination and continued for either 14 or 28 days. The threshold for inhibition of seedling growth over a 28-day treatment was 0.15 mM SA. Seedlings grown with 0.3 mM SA consistently had higher leaf diffusive resistance and lower transpiration and water potentials than control plants. The stable carbon isotope ratio (13C:12C) in tissue from both the 0.15 and 0.30 mM SA-treated plants was significantly higher than control seedlings, indicating SA caused a chronic water stress during the 28-day treatment. These data show that an interference with plant-water relationships is one mechanism whereby this allelochemical inhibits plant growth.

Effects of root exudate sorgoleone on photosynthesis

The aim of this investigation was to determine if sorgoleone (SGL), ap-benzoquinone inSorghum bicolor root exudate, is a photosynthesis inhibitor. Assays usingGlycine max leaf disks showed concentrations as low as 10μM SGL inhibited oxygen evolution more than 50%. Tests conducted on chloroplasts isolated fromPisum sativum showed that SGL is a powerful inhibitor of CO2-dependent oxygen evolution. Using a chloroplast suspension equivalent to 80–100μg chlorophyll, the I50 was approximately 0.2μM SGL. These data indicate inhibition of photosynthesis is part of the explanation for growth reduction caused by this allelochemical.

Prior cropping with grain sorghum inhibits weeds

Three years of field data in northeastern Nebraska demonstrate that a grain sorghum crop reduces weediness in the following crop year. Weed growth was consistently lower in sorghum areas the year after strip-cropping fields with sequences of four-row bands of grain sorghum, soybeans, and corn. Percentage weed cover was significantly lower early in the year, and midsummer weed biomass was well below that found after corn and soybeans. Weed biomass in June and July following corn was two to four times that of grain sorghum strips. Inhibitory effects of grain sorghum were primarily on broadleaf weeds, often showing no action on grass weeds. No obvious differences were noted in the weed species present after the three crops. Allelopathy provides a logical explanation for the sorghum-mediated weed inhibition found in this study. The data have implications for weed management strategies in agriculture.

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 40μM. Soybean leaf disks vacuum infiltrated with as little as 10μM juglone had reduced photosynthesis. Oxygen evolution by chloroplasts isolated fromPisum sativum was inhibited by juglone with an I50 of 2μM. 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.

Effects of three phenolic acids on chlorophyll content and growth of soybean and grain sorghum seedlings

Experiments were designed to test the hypothesis that interference with chlorophyll metabolism may be one mechanism of inhibition of plant growth in allelopathic interactions. Effects of ferulic,p-coumaric, and vanillic acids on soybean and grain sorghum growth and chlorophyll content were quantified and compared after seedlings were treated with these compounds in a nutrient culture. Following a 6-day treatment cycle, dry weights of soybean seedlings were reduced by both 10−3 M and 5 × 10−4 M treatments of ferulic,p-coumaric and vanillic acids. Soybean weight reductions in each case were paralleled by a significant reduction in the concentration (μg Chl/mg dry wt) of chlorophylls a and b and total chlorophyll in the unifoliate leaves. Sorghum seedling growth was also reduced by each of the compounds at the 5 × 10−4 M level, but leaf chlorophyll concentration was not below that of control plants.

Synergistic inhibitory effects ofp-coumaric and ferulic acids on germination and growth of grain sorghum

The data support the hypothesis that there is a synergistic phytotoxic effect whenp-coumaric and ferulic acids are found together. Equimolar mixtures of both acids showed greater reduction in sorghum seed germination, shoot elongation, and total seedling growth than either phytotoxin caused when alone. Repeated experiments showed mixtures containing 5×10−3 Mp-coumaric and 5×10−3 M ferulic acids reduced germination to 34% of controls after 24 hr and 59% by 48 hr. The same concentration of either phenol-treated seeds alone showed 69 and 92% germination at comparable times. The phytotoxic action of the combination approximated the inhibitory effect on germination of 10−2 M ferulic acid and was a greater reduction than caused by 10−2 Mp-coumaric treatments. Sorghum seedling growth was more sensitive than germination, with an equimolar mixture of 2.5×10−4 Mp-coumaric and 2.5×10−4 M ferulic acids reducing seedling dry weight significantly below weights of seedlings treated separately with 2.5×10−4 Mp-coumaric or ferulic acids. Further dilutions showed a 1.25×10−4 M concentration of either phenol was stimulatory to seedling growth, whereas a mixture of these two produced inhibition.

Use ofLemna minor L. as a bioassay in allelopathy.

Investigations in allelopathy often require the use of a bioassay for evaluating limited quantities of potentially active growth regulators. A bioassay procedure was developed usingL. minor grown in 1.5-ml aliquots of nutrient medium with and without allelochemicals in wells of 24-well tissue culture cluster dishes with loose-fitting lids. Tests using six replications per treatment with several flavonoid compounds and derivatives of coumarin, benzoic acid, and cinnamic acid demonstrated that the bioassay was capable of measuring inhibition at levels of compound ranging from 50 to 1000 μmol. Strongly inhibitory treatments were visible after 1 or 2 days. After 7 days of growth, frond number, growth rate, and dry weight were used to evaluate effects. The bioassay system is relatively simple, very sensitive, reproducible, and can be used for testing small amounts and dilute concentrations of unknowns which have been separated by chromatography.

Synergistic inhibitory effects of vanillic andp-hydroxybenzoic acids on radish and grain sorghum

Radish and grain sorghum germination and sorghum growth were inhibited in a synergistic manner by combinations of vanillic andp-hydroxybenzoic acids. At threshold inhibition levels, 2.5 × 10−3 M vanillic acid-treated radish seeds had 71 % of control germination after 24 hr and 2.5 × 10−3 Mp-hydroxybenzoic acid-treated radish yielded 95% germination. A mixture of 2.5 × 10−3 M of each of these two phytotoxins showed 52% germination after 24 hr. Equimolar mixtures of 5 × 10−3 M vanillic andp-hydroxybenzoic acids allowed sorghum germination of 60% of untreated seeds after 24 hr, whereas separate treatments of individual phenols had 93% and 96% of control seed germination. Sorghum root and shoot elongation and total seedling growth were more sensitive than germination to vanillic andp-hydroxybenzoic acid treatments, and synergistic effects also were apparent. A combination of 5 × 10−3 M vanillic with 5 × 10−3 Mp-hydroxybenzoic reduced root length more than either did individually, and a mixture of 5 × 10−4 M vanillic with 5 × 10−4 Mp-hydroxybenzoic acid reduced sorghum seedling growth to approximately that resulting from a 10−3 M concentration of either phenol alone. Phytotoxin levels inhibitory to sorghum growth caused small increases in lower leaf surface diffusive resistance, but did not close stomates, and this effect was not judged to be the cause of reduced sorghum growth.