Methyl Jasmonate Effect on Betulinic Acid Content and Biological Properties of Extract from Senna obtusifolia Transgenic Hairy Roots.

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity.

Endophytic fungal extracts: evaluation as photosynthesis and weed growth inhibitors.

A central pillar of modern weed control is the discovery of new herbicides which are nontoxic to humans and the environment and which have low application dosage. The natural products found in plants and microorganisms are well suited in this context because they are generally nontoxic and have a wide variety of biological activities. In this work, Diaporthe phaseolorum (Dp), Penicillium simplicissimum (Ps) and Trichoderma spirale (Ts) (methanolic extracts) were evaluated as photosynthesis and plant growth inhibitors in Senna occidentalis and Ipomoea grandifolia. The most significant results were observed for Ts and Dp in S. occidentalis and I. grandifolia, respectively. Ts reduced PI(abs), ET0/CS0, PHI(E0) and PSI0 parameters by 64, 28, 40 and 38%, respectively, indicating a reduction on electron transport efficiency. Additionally, Ts decreased shoot length by 9%, affecting the plant growth. Dp reduced PI(abs), ET0/CS0 and PHI(E0) parameters by 50, 20, 26 and 22%, respectively, revealing the inhibition competency on PSII acceptor site. Furthermore, Dp decreased by 50% the shoot length on germination assay. Thus, the phytotoxic behaviors based on endophytic fungal extracts may serve as a valuable tool in the further development of a bioherbicide since natural products represent an interesting alternative to replace commercial herbicides.

Does the sequence of addition of herbicide and adjuvants to the spray solution influence sicklepod control? / A sequência de adição de herbicida e adjuvantes à calda influencia no controle de fedegoso?

The use of adjuvants associated with herbicides aims at improving the performance of application and the consequent increase in the biological effect of the treatment. However, the sequence of product added to the sprayer tank can influence the phytosanitary spray solution. Thus, this study aimed to evaluate the control of Senna obtusifolia as a function of the sequence of addition of the herbicide aminopyralid + fluroxypyr and adjuvants in the preparation of spray solution. Two herbicide doses (1 and 2 L c.p. ha-1) associated with the adjuvants mineral oil (MO), silicone polyether copolymer (SIL), and a mixture of phosphatidylcholine and propionic acid (LEC), all in the proportion of 0.3% v v-1, with alternate addition to the spray solution to evaluate the effects of the preparation sequence. The spray solution volume considered was 150 L ha-1. Evaluations of spray solution stability were performed from the visual evaluation of homogeneity, electrical conductivity, and pH. The effect of treatment on S. obtusifolia control was measured using a scoring scale and dry matter. Correlation coefficients between the evaluations were also determined. No difference of the preparation sequence of spray solution was observed for stability, pH, and electrical conductivity, but an influence was observed on S. obtusifolia control, without changing dry matter accumulation. The treatment with the adjuvant LEC previously added to the herbicide provided a higher control rate at the highest dose, while the adjuvant SIL had the opposite effect.(AU)

O uso de adjuvantes associados a herbicidas visa melhorar o desempenho da aplicação e o consequente aumento do efeito biológico do tratamento. Porém, a ordem de adição dos produtos ao tanque do pulverizador pode trazer importantes influências à calda fitossanitária. Assim, o objetivo desta pesquisa foi avaliar o controle de Senna obtusifolia em função da sequência de adição do herbicida aminopiralide + fluroxipir e de adjuvantes no preparo das caldas. Foram utilizadas duas dosagens de herbicida (1 e 2 L p.c. ha-1), associadas aos adjuvantes óleo mineral (OM); copolímero de poliéter e silicone (SIL); mistura de fosfatidicolina e ácido propiônico (LEC), todos na proporção de 0,3% v v-1, com adição alternada à calda para avaliar os efeitos da sequência de preparo. O volume de calda considerado foi de 150 L ha-1. Foram realizadas avaliações da estabilidade da calda a partir da avaliação visual de homogeneidade, condutividade elétrica e pH. O efeito do tratamento no controle de S. obtusifolia foi mensurado por meio de uma escala de pontuação e pela massa seca. Também foram determinados os coeficientes de correlação entre as avaliações. Verificou-se que não houve diferença da sequência de preparo da calda para a estabilidade, o pH e a condutividade elétrica. Porém, a sequência de preparo influenciou o controle inicial de S. obtusifolia, sem efeito sobre a massa seca. O tratamento com o adjuvante LEC adicionado ao herbicida proporcionou maior taxa de controle na maior dosagem, enquanto o adjuvante de SIL teve o efeito oposto.(AU)

Piriformospora indica, an excellent system for heavy metal sequestration and amelioration of oxidative stress and DNA damage in Cassia angustifolia Vahl under copper stress.

Present investigation reveals copper induced phytotoxicity, oxidative stress and DNA damage in Cassia angustifolia Vahl and its amelioration by employing a symbiotic fungus, Piriformospora indica. Seeds were germinated on Knop's medium containing five Cu levels (0, 1, 10, 50, 100 and 200 mg L-1), with and without P. indica. Colonization with P. indica significantly (P < 0.05) ameliorated Cu induced oxidative stress. However, maximum amelioration was observed at 50 mg L-1 Cu with P. indica. Atomic absorption spectroscopy revealed that P. indica colonization significantly inhibited Cu accumulation in shoots. Maximum decline in Cu accumulation in shoots was observed at 50 mg L-1 (27.27%) with P. indica over Cu alone. Besides, P. indica colonized seedlings stored 16.86% higher Cu in roots as compared to Cu alone at 200 mg L-1. Similarly, maximum proline accumulation increased up to 19.32% over Cu alone at 50 mg L-1 Cu with P. indica. Significant elevation in antioxidant enzyme levels of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase and glutathione reductase was seen with P. indica. Contrary to increase in antioxidant level, toxic parameters such as lipid peroxidation and hydrogen peroxide decreased significantly with P. indica. Maximum decline in lipid peroxidation (13.76%) and hydrogen peroxide (18.58%) was observed at 50 mg L-1 with P. indica over Cu alone. P. indica significantly reduced DNA damage as well as changed the protein profile in C. angustifolia seedlings. Thus, P. indica proved to be an excellent system to alleviate Cu induced oxidative stress and might be useful as a phytostabilization tool.

Croton argenteus preparation inhibits initial growth, mitochondrial respiration and increase the oxidative stress from Senna occidentalis seedlings.

Senna ocidentalis is a weed, native to Brazil, considered to infest crops and plantations, and is responsible for yield losses of several crops, particularly soybean. The aim of this work was to evaluate if the Croton argenteus extract and fractions possess phytotoxic activity on S. ocidentalis. The crude ethanolic extract (CEE) and its hexanic (HF), chloroformic (CLF) and ethyl acetate (EAF) fractions were tested in germination, growth, oxidative stress increase, Adenosine triphosphate, L-malate and succinate synthesis. The crude extract and its fractions slowed down the germination of S. ocidentalis and decreased the final percentage of germination. Oxidative stress was also increased in the seedlings, by an increase of catalase, peroxidase, superoxide dismutase, glutathione reductase and lipid peroxidation; and it became clear that the ethyl acetate fraction was more phytotoxic. The results indicate that the crude extract and fractions of C. argenteus compromise the mitochondrial energy metabolism, by the inhibition of mitochondrial ATP production, with a decrease in the production of L-malate and succinate. The ethyl acetate fraction of C. argenteus showed high activity on germination and growth, and these effects take place by means of mitochondrial metabolism alterations and increase the oxidative stress, leading the seedling death.

Synthetic seed production and physio-biochemical studies in Cassia angustifolia Vahl. - a medicinal plant.

Synthetic seed technology is an alternative to traditional micropropagation for production and delivery of cloned plantlets. Synthetic seeds were produced by encapsulating nodal segments of C. angustifolia in calcium alginate gel. 3% (w/v) sodium alginate and 100 mM CaCl2 · 2H2O were found most suitable for encapsulation of nodal segments. Synthetic seeds cultured on half strength Murashige and Skoog medium supplemented with thidiazuron (5.0 µM) + indole-3-acetic acid (1.0 µM) produced maximum number of shoots (10.9 ± 0.78) after 8 weeks of culture exhibiting (78%) in vitro conversion response. Encapsulated nodal segments demonstrated successful regeneration after different period (1-6 weeks) of cold storage at 4 °C. The synthetic seeds stored at 4 °C for a period of 4 weeks resulted in maximum conversion frequency (93%) after 8 weeks when placed back to regeneration medium. The isolated shoots when cultured on half strength Murashige and Skoog medium supplemented with 1.0 µM indole-3-butyric acid (IBA), produced healthy roots and plantlets with well-developed shoot and roots were successfully hardened off in plastic pots containing sterile soilrite inside the growth chamber and gradually transferred to greenhouse where they grew well with 85% survival rate. Growth performance of 2 months old in vitro-raised plant was compared with in vivo seedlings of the same age. Changes in the content of photosynthetic pigments, net photosynthetic rate (PN), superoxide dismutase and catalase activity in C. angustifolia indicated the adaptation of micropropagated plants to ex vitro conditions.

ß-Pinene inhibited germination and early growth involves membrane peroxidation.

ß-Pinene, an oxygenated monoterpene, is abundantly found in the environment and widely occurring in plants as a constituent of essential oils. We investigated the phytotoxicity of ß-pinene against two grassy (Phalaris minor, Echinochloa crus-galli) and one broad-leaved (Cassia occidentalis) weeds in terms of germination and root and shoot growth. ß-Pinene (0.02-0.80 mg/ml) inhibited the germination, root length, and shoot length of test weeds in a dose-response manner. The inhibitory effect of ß-pinene was greater in grassy weeds and on root growth than on shoot growth. ß-Pinene (0.04-0.80 mg/ml) reduced the root length in P. minor, E. crus-galli, and C. occidentalis over that in the control by 58-60, 44-92, and 26-85 %, respectively. In contrast, shoot length was reduced over the control by 45-97 % in P. minor, 48-78 % in E. crus-galli, and 11-75 % in C. occidentalis at similar concentrations. Further, we examined the impact of ß-pinene on membrane integrity in P. minor as one of the possible mechanisms of action. Membrane integrity was evaluated in terms of lipid peroxidation, conjugated diene content, electrolyte leakage, and the activity of lipoxygenases (LOX). ß-Pinene (≥0.04 mg/ml) enhanced electrolyte leakage by 23-80 %, malondialdehyde content by 15-67 %, hydrogen peroxide content by 9-39 %, and lipoxygenases activity by 38-383 % over that in the control. It indicated membrane peroxidation and loss of membrane integrity that could be the primary target of ß-pinene. Even the enhanced (9-62 %) activity of protecting enzymes, peroxidases (POX), was not able to protect the membranes from ß-pinene (0.04-0.20 mg/ml)-induced toxicity. In conclusion, our results show that ß-pinene inhibits root growth of the tested weed species through disruption of membrane integrity as indicated by enhanced peroxidation, electrolyte leakage, and LOX activity despite the upregulation of POX activity.

Inhibitory effects against pasture weeds in Brazilian Amazonia of natural products from the marine brown alga Dictyota menstrualis.

Fractions of the acetone extract and a mixture of two diterpenes from the marine brown alga Dictyota menstrualis were prepared with the aim of identifying potential effects on the germination of seeds and on elongation of the radicle and hypocotyl of the weeds Mimosa pudica and Senna obtusifolia. The bioassay on seed germination was performed in controlled conditions of 25 degreeC temperature and a 12 hour photoperiod, while the one concerning radicle and hypocotyl elongation was performed at the same temperature, though adopting a photoperiod of 24 hours. The results varied according to the receptor species, the fraction utilized, and its concentration. TLC analysis of the fractions and comparison with isolated products indicated that the diterpenes pachydictyol A and isopachydictyol A were the most abundant compounds in fraction HE, whereas the diterpene 6-hydroxy-dichotomano-2, 13-diene-16, 17-dial (3) was the most abundant compound in fractions DC and EA. Analysis of less polar fractions (in n-hexane, dichloromethane and ethyl acetate) revealed values of less than 30% inhibition. On the other hand, the ethanol/water fraction was the most active in all instances. The biological activity of these fractions must be due to the presence of known diterpenes and/or sulfated polysaccharides isolated in earlier studies.

Comparative study on elemental composition and DNA damage in leaves of a weedy plant species, Cassia occidentalis, growing wild on weathered fly ash and soil.

Open dumping of fly ash in fly ash basins has significant adverse environmental impacts due to its elevated trace element content. In situ biomonitoring of genotoxicity is of practical value in realistic hazard identification of fly ash. Genotoxicity of openly disposed fly ash to natural plant populations inhabiting fly ash basins has not been investigated. DNA damage, and concentrations of As, Co, Cr, Cu and Ni in the leaves of natural populations of Cassia occidentalis growing at two contrasting sites-one having weathered fly ash (fly ash basin) and the other having soil (reference site) as plant growth substrates-were assessed. The foliar concentrations of As, Ni and Cr were two to eight fold higher in plants growing on fly ash as compared to the plants growing on soil, whereas foliar concentrations of Cu and Co were similar. We report, for the first time, based upon comet assay results, higher levels of DNA damage in leaf tissues of Cassia occidentalis growing wild on fly ash basin compared to C. occidentalis growing on soil. Correlation analysis between foliar DNA damage and foliar concentrations of trace elements suggests that DNA damage may perhaps be associated with foliar concentrations of As and Ni. Our observations suggest that (1) fly ash triggers genotoxic responses in plants growing naturally on fly ash basins; and (2) plant comet assay is useful for in situ biomonitoring of genotoxicity of fly ash.

Chemical basis of plant leaf movement.

Nyctinastic plants open and close leaves with a circadian rhythm. Here we discuss chemical aspects of the mechanism of nyctinastic leaf movement. Nyctinastic plants from five different genera are known to contain species-specific leaf-opening and leaf-closing factors. The relative concentrations of leaf-closing and leaf-opening factors of the nyctinastic plant Phyllanthus urinaria change circadianly, suggesting that nyctinastic movement is regulated by two classes of circadianly regulated factors with opposing functions. A closing and an opening factor of Albizzia, when linked to a fluorescent dye, both specifically labeled motor cells of pluvini. A membrane fraction of pluvini contains proteins of 210 and 180 kDa that bind to a leaf-opening factor of Cassia mimosoides. The molecular identification of these proteins is underway.

alpha-Pinene inhibits growth and induces oxidative stress in roots.

BACKGROUND AND AIMS: Determining the mode of action of allelochemicals is one of the challenging aspects in allelopathic studies. Recently, allelochemicals have been proposed to cause oxidative stress in target tissue and induce an antioxidant mechanism. alpha-Pinene, one of the common monoterpenoids emitted from several aromatic plants including forest trees, is known for its growth-inhibitory activity. However, its mechanism of action remains unexplored. The aim of the present study was to determine the inhibitory effect of alpha-pinene on root growth and generation of reactive oxygen species, as indicators of oxidative stress and changes in activities of antioxidant enzymes. METHODS: Effects of alpha-pinene on early root growth were studied in five test species, Cassia occidentalis, Amaranthus viridis, Triticum aestivum, Pisum sativum and Cicer arietinum. Electrolyte leakage, lipid peroxidation, hydrogen peroxide generation, proline accumulation, and activities of the enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR) were studied in roots of C. occidentalis. KEY RESULTS: alpha-Pinene inhibited the radicle growth of all the test species. Exposure of C. occidentalis roots to alpha-pinene enhanced solute leakage, and increased levels of malondialdehyde, proline and hydrogen peroxide, indicating lipid peroxidation and induction of oxidative stress. Activities of the antioxidant enzymes SOD, CAT, GPX, APX and GR were significantly elevated, thereby indicating the enhanced generation of reactive oxygen species (ROS) upon alpha-pinene exposure. Increased levels of scavenging enzymes indicates their induction as a secondary defence mechanism in response to alpha-pinene. CONCLUSIONS: It is concluded that alpha-pinene inhibits early root growth and causes oxidative damage in root tissue through enhanced generation of ROS, as indicated by increased lipid peroxidation, disruption of membrane integrity and elevated antioxidant enzyme levels.

Biofumigant compounds released by field pennycress (Thlaspi arvense) seedmeal.

Defatted field pennycress (Thlaspi arvense L.) seedmeal was found to completely inhibit seedling germination/emergence when added to a sandy loam soil containing wheat (Triticum aestivum L.) and arugula [Eruca vesicaria (L.) Cav. subsp. sativa (Mill.) Thell.] seeds at levels of 1.0% w/w or higher. Covering the pots with Petri dishes containing the soil-seedmeal mixture decreased germination of both species at the lowest application rate (0.5% w/w), suggesting that the some of the phytotoxins were volatile. CH2Cl2, MeOH, and water extracts of the wetted seedmeal were bioassayed against wheat and sicklepod (Senna obtusifolia (L.) H. S. Irwin & Barneby) radicle elongation. Only the CH2Cl2 extract was strongly inhibitory to both species. Fractionation of the CH2Cl2 extract yielded two major phytotoxins, identified by gas chromatography-mass spectrometry and NMR as 2-propen-1-yl (allyl) isothiocyanate (AITC) and allyl thiocyanate (ATC), which constituted 80.9 and 18.8%, respectively, of the active fraction. When seeds of wheat, arugula and sicklepod were exposed to volatilized AITC and ATC, germination of all three species was completely inhibited by both compounds at concentrations of 5 ppm or less. In field studies, where seedmeal was applied at 0.50, 1.25, and 2.50 kg/m2 and tarped with black plastic mulch, all of the treatments significantly reduced dry weight of bioassay plants compared to the tarped control, with the highest seedmeal rate decreasing dry matter to less than 10% of the control 30 d after seedmeal application. Field pennycress seedmeal appears to offer excellent potential as a biofumigant for high-value horticultural crops for both conventional and organic growers.