Characterization of Strigolactones, Germination Stimulants for the Root Parasitic Plants Striga and Orobanche, Produced by Maize, Millet and Sorghum

The germination stimulants for root parasitic plants Striga and Orobanche produced by sorghum (Sorghum bicolor (L.) Moench), maize (Zea mays L.), and pearl millet (Pennisetum typhoideum Rich.) were examined. Characterization of strigolactones in the root exudates from the plants grown hydroponically was conducted by comparing retention times of germination stimulants on reverse phase high performance liquid chromatography (HPLC) with those of synthetic standards, and by using HPLC linked with tandem mass spectrometry (LC/MS/MS). All the plants tested, except for a sorghum cultivar Swarna, were found to exude two major stimulants, 5-deoxy-strigol, which is known as a branching factor for arbuscular mycorrhizal (AM) fungi, and an isomer of strigol, tentatively named sorghumol. Swarna was found to exude 5-deoxy-strigol and strigol. These results imply that 5-deoxy-strigol is one of major germination stimulants of gramineous plants and that major stimulants may differ even among cultivars within the same species.     

PHYTOTOXIC EXUDATES FROM VELVETLEAF (ABUTILON THEOPHRASTI) GLANDULAR TRICHOMES

Nonvolatile exudates from velvetleaf glandular trichomes inhibited root and shoot growth of several weed and crop species in petri plate bioassays, but had no effect on seed germination per se. The exudate was efficiently collected by wiping both the stems and petioles with cotton swabs or by leaching with water, but was absent on the leaves of velvetleaf plants. Cress (Lepidium sativum L.) was the most sensitive indicator species. Four types of trichomes appeared on the stem surface as revealed by scanning electron microscopy. Water soluble globules on the apices of 12- to 15-celled glandular trichomes recurred and demonstrated their original potency within eight days after removal with cotton swabs. Both the quantity and phytotoxicity of the exudates from velvetleaf plants cultured under varying environmental conditions were determined. While total exudate production was not affected at 16, 24, or 36 C, the exudates from plants cultured at 24 and 36 C were about twice as toxic as the exudate collected from plants grown at the lower temperature. Water stress decreased the amount of exudate collected, but the phytotoxic activity was increased by approximately the same factor.     

Ethylene biosynthesis and strigol-induced germination of Striga asiatica

Germination of witchweed [ Striga asiatica (L.) Kuntze], an important parasite on cereal crops, is stimulated by several natural and synthetic compounds. In the present study the role of ethylene in germination of Striga asiatica in response to strigol was examined. Unconditioned seeds and those conditioned for 3 days produced negligible amounts of ethylene in response to strigol. However, extending the conditioning period to 5 and 8 days increased ethylene evolution by more than 10-fold. Ethylene production preceded radicle protrusion and was detectable within 3h after treatment. No germination was observed in the first 6 h of exposure to strigol. Germination and ethylene production increased with strigol concentration. Strigol-induced germination was considerably reduced by the ethylene action inhibitors. 2. 5-norbornadiene, silver thiosulphate and CO₂. The ethylene precursor 1-aminocyclopropane-1-carboxyac acid (ACC) at 5 to 200 μ M elicited neither germination nor ethylene production. However, a combination of strigol and ACC resulted in a high germination rate and copious ethylene production. Both germination and ethylene production were reduced by CoCl₂ and cyclobeximide, inhibitors of the ethylene-forming enzyme and of protein synthesis, respectively. The results are consistent with a model in which conditioning and strigol are required to remove a restriction on the ethylene biosynthetic pathway and in which the ethylene-forming enzyme is rate limiting.     

Comparison of the seed germination effects of synthetic analogs of strigol,gibberellic acid,cytokinins, and other plant growth regulators

Four synthetic multiring analogs of strigol, a naturally occurring sesquiterpene lactone that promotes germination of dormant seeds ofStriga (witchweed), were found to stimulate germination of dormantLactuca (lettuce) seeds. The effects on light-sensitive and light-insensitive lettuce seeds were concentration-dependent and exceeded those produced by equimolar (0.1 mM) solutions of gibberellic acid. Strigol and epistrigol promoted lettuce seed germination to a lesser degree than did the synthetic analogs. The strigol group compounds had minimal effect on the germination of monocot seeds. The results indicate that the synthetic strigol analogs have plant growth regulatory activity in dormant seeds of genera beyondStriga in which germination stimulation by strigol and the synthetic analogs was first demonstrated.Names of companies of commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

Effects of Temperature and dl-Strigol on Seed Conditioning and Germination of Witchweed (Striga asiatica)

Seed conditioning and germination in witchweed (Striga asiatica(L.) Kuntze) were temperature-dependent. With higher conditioningtemperatures, shorter conditioning time was required for germinationwith terminal dl-strigol (strigol) treatment at 30 °C. Maximumgermination (80–100%) was obtained by conditioning inwater at 20, 25, 30 and 35 °C for 14, 7, 5 and 3 d, respectively,and terminally treating with 10^–6 M strigol at 30 °C.Seeds conditioned in 10^–8 M strigol instead of water germinatedmuch less with the same terminal strigol treatment. Generally,conditioning was slower when seeds were conditioned in strigolrather than water. The reduction in germination rate by pretreatmentin strigol or pretreatment at low temperatures could be overcomeby increasing the terminal strigol concentration in the germinationtest. Conditioned seeds did not germinate at 10 and 15 °Cwith a terminal 10^–6 M strigol treatment but yielded closeto maximum germination at 25, 30 and 35 °C with the sameterminal strigol treatment. To obtain maximum germination, boththe minimum conditioning temperature and the minimum germinationtemperature for conditioned seeds were 20 °C. Factors suchas conditioning time, and strigol concentration and temperatureduring conditioning and/or germination determine whether seedsremain in the conditioning phase or shift to a germination phase. dl-Strigol, germination stimulation, parasitic plants, seed conditioning, seed germination, Striga asiatica, temperature, weed control     

Influence of pea-root exudates on germination of conidia and chlamydospores of physiologic races of Fusarium oxysporum f. pisi

Sterile root exudates from wilt susceptible and wilt resistant pea cultivars showed no differential effects on spore germination of Fusarium oxysporum Schl. f.pisi (Linf.) Snyd. & Hans, races 1 and 2 which could be correlated with the pathogenicity of a particular isolate to a given cultivar. Uniformly high percentages of germination were obtained with conidia of the two races in aseptic shake culture with exudates collected from resistant or susceptible plants of various ages. Chlamydospores of the two races incubated with exudates under sterile conditions germinated to uniformly high levels irrespective of exudate origin. Conidia and chlamydospores of Fusarium solani (Mart.) Sacc. f. pisi (Jones) Snyd. & Hans., used for comparative purposes, also germinated to high levels in the presence of exudate solutions of all cultivars. Non-specific germination of the two races of F. oxysporum f. pisi occurred in soil when the exudates were supplied to populations of chlamydospores via diffusion units. Germination was lower than that recorded under sterile conditions and was rapidly followed by germling lysis.     

A standardized bioassay for evaluation of potential germination stimulants for seeds of parasitic weeds

A standard bioassay for the evaluation of the biological activity of potential germination stimulants for seeds of parasitic weeds has been designed by carefully reconsidering all steps of a literature procedure. Addition of a surfactant to the solution for sterilization of the seeds gave a considerable reduction of contamination during the conditioning and stimulation process. Furthermore, it was shown that the water/seed ratio during conditioning affects the germination results. For the seed conditioning and stimulation process, the sandwich technique was introduced, whereby the seeds were placed between two layers of glass fiber filter paper discs. The standardized bioassay has been used for the evaluation of the stimulatory activity of numerous (new) synthetic analogues of strigol (the natural germination stimulant). It is essential to include a reference compound—GR24, an analogue of strigol, is recommended—in every test series, because it was observed that, although a standard bioassay was used, germination percentages obtained with GR24 solutions vary from test to test. For seeds of Striga hermonthica, seasonal effects were found in the germination percentages obtained with GR24 as stimulant. Test results with strigol analogues having modifications in ring D reveal that structural variations in this part of the molecule have dramatic effects on the biological activity of strigol analogues.     

Bleach-induced Germination and Breakage of Dormancy of Seeds of Alectra vogelii

The effects of solutions of sodium hypochlorite and calcium hypochlorite (bleach) on dormancy and germination of Alectra vogelii seeds were investigated. Dry (non-pretreated) and 10-day water-pretreated seeds were exposed to various bleach concentrations, as well as to the host (Vigna unguiculata) root exudate. The 15-month-old Alectra seeds used were partially dormant in that only 40% or less of the 10-day pretreated seeds could be stimulated to germinate by the standard host root exudate. Comparable percentage germination of nonpretreated seeds was achieved with certain concentrations of bleach and the halogens, chlorine and bromine. Bleach and the halogens served not only as germination stimulant for pretreated Alectra seeds but appropriate concentrations induced also high percentage (70–90%) germination indicating a breakage of seed dormancy as well. The activity of the bleach in stimulating high percentage germination could be significantly reduced when the optimally bleach-treated seeds were rinsed daily with water during the germination period. Vigna root exudate, but not bleach, was shown to act as root stimulant for Alectra seedlings. The possible mechanism of the bleach effect on germination and dormancy of Alectra seeds is discussed.     

Flavonoids in seed and root exudates of Lotus pedunculatus and their biotransformation by Mesorhizobium loti

Analysis of the flavonoid content of seed and root exudates of Lotus pedunculatus was undertaken using multiple coupled analytical techniques: capillary zone electrophoresis coupled to a UV spectral array detector (CZE-UV), high performance thin-layer chromatography with densitometry (HPTLC-UV) and gas chromatography with mass spectrometry (GC-MS). These procedures provided separation, identification and structural confirmation of the previously unidentified flavonoids in this plant's seed and root exudates and were particularly applicable to samples from a small seeded legume. Catechin, naringenin, kaempferol, quercetin aglycone and 3 different glycosides of quercetin were detected in seed exudate. Sterile root exudates contained catechin, naringenin and quercetin in addition to apigenin, kaempferol and other flavones and flavanones for which partial identifications were obtained. When sterile root exudate was incubated with Mesorhizobium loti , changes in its flavonoid content were detected. Analysis of bacterial cells after incubation revealed the presence of quercetin, kaempferol and one other flavone. The monocyclic aromatics protocatechuic acid and phloroglucinol were detected in both the incubated root exudate and its bacterial cells.     

Induction of chlorosis, ROs generation and cell death by a toxin isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.     

棉花根系分泌物对土壤速效养分和酶活性及微生物数量的影响

采用水培法收集棉花根系分泌物,在耕作1年的土壤中添加棉花根系分泌物,培养10 d后测定土壤中速效养分、酶活性及微生物数量.结果显示,(1)棉花根系分泌物能极显著提高土壤中速效K和速效P含量4.31%～15.03%和5.99%～24.31%(P<0.01);高浓度分泌物处理下速效N含量比对照显著提高11.39%(P<0.05),其它处理影响不显著;各浓度分泌物对土壤有机质含量均无显著影响.(2)各浓度棉花根系分泌物均使土壤中转化酶活性显著提高,且随分泌物浓度的增加而显著增强;低浓度分泌物能显著提高土壤中磷酸酶的活性,所有浓度处理对土壤脲酶活性均无显著影响.(3)中、高浓度的棉花根系分泌物能显著增加土壤中细菌的数量,低浓度的分泌物能显著增加土壤中真菌的数量,而不同浓度处理的土壤中放线菌的数量均无显著的变化.研究表明,棉花根系分泌物可通过促进土壤细菌及土壤真菌的繁殖来增强土壤转化酶和磷酸酶活性,提高土壤速效P、速效K及速效N含量,从而对棉花根际微环境产生深刻影响.     

Presence of trifoliin A,a rhizobium-binding lectin,in clover root exudate

Trifoliin A, a Rhizobium-binding glycoprotein from white clover, was detected in sterile clover root exudate by a sensitive immunofluorescence assay employing encapsulated cells of Rhizobium trifolii 0403 heat-fixed to microscope slides. Its presence in root exudate was further examined by immunoaffinity chromatography. The binding of trifoliin A to cells was specifically inhibited by the hapten, 2-deoxyglucose. Significantly higher quantities of trifoliin A were detected in root exudate of seedlings grown hydroponically in nitrogen-free medium than in rooting medium containing 15 mM NO, a concentration which completely suppressed root hair infection by the nitrogen-fixing symbiont. The presence of trifoliin A in root exudate may make it possible for recognition processes to occur before the microsymbiont attaches to its plant host.     

转基因抗虫棉根系分泌物对棉花黄萎病菌生长的影响

转基因抗虫棉的抗病性下降已成为制约我国棉花生产的重要因素之一.以转基因抗虫棉及其亲本非转基因棉花对照为材料,研究转基因抗虫棉根系分泌物对棉花黄萎病菌孢子萌发和菌丝生长的影响,并对其根系分泌物中氨基酸及糖类的组成和含量进行了测定.结果表明：与亲本非转基因棉相比,2种转基因抗虫棉对棉花黄萎病菌的抗性下降,转基因抗虫棉的根系分泌物对黄萎病菌孢子萌发和菌丝生长均具有促进作用.与亲本常规棉中23相比,转基因双价抗虫棉中41根系分泌物中多了甲硫氨酸和赖氨酸,并且天冬氨酸、谷氨酸、丝氨酸、丙氨酸、缬氨酸、亮氨酸、酪氨酸的含量显著升高.泗棉三号与单价抗虫棉GK12的根系分泌物中虽然含有相同的氨基酸组分,但GK12的根系分泌物中酪氨酸、缬氨酸、亮氨酸的含量显著下降.中41根系分泌物中检测出4种糖类,而中23中仅检测出葡萄糖.抗虫棉GK12和泗棉三号根系分泌物中都检测出4种糖类,但其含量差异明显.     

Vibrational, 1H-NMR spectroscopic,and thermal characterization of gladiolus root exudates in relation to Fusarium oxysporum f. sp. gladioli resistance

Fourier transform Raman (FT Raman) and IR (FTIR) and (1)H-NMR spectroscopies coupled with differential scanning calorimetry (DSC) were applied to the characterization of root exudates from two cultivars of gladiolus (Spic Span and White Prosperity) with different degrees of resistance and susceptibility to Fusarium oxysporum gladioli, the main pathogen of gladiolus. This work was aimed at correlating the composition of root exudates with the varietal resistance to the pathogen. Spectroscopic analysis showed that White Prosperity root exudate differs from Spic Span root exudate by a higher relative amount of the aromatic-phenolic and sugarlike components and a lower relative amount of carbonylic and aliphatic compounds. DSC analysis confirmed the spectroscopic results and showed that White Prosperity root exudate is characterized by an aromatic component that is present in a higher amount than in the Spic Span root exudate. The results are discussed in relation to the spore germination tests showing that White Prosperity, which is characterized by a remarkable resistance toward F. oxysporum gladioli, exudes substances having a negative influence on microconidial germination of the pathogen; root exudates from Spic Span, one of the most susceptible cultivars to F. oxysporum gladioli, proved to have no effect. White Prosperity's ability to inhibit conidial germination of F. oxysporum gladioli can be mainly related to the presence of a higher relative amount of aromatic-phenolic compounds.     

Induction of Chlorosis,ROS Generation and Cell Death by a Toxin Isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.     

Characterization of strigolactones exuded by Asteraceae plants

Strigolactones (SLs), originally characterized as germination stimulants for root parasitic weeds, are now recognized as hyphal branching factors for symbiotic arbuscular mycorrhizal fungi and as a novel class of plant hormones inhibiting shoot branching. In the present study, SLs in root exudates of 13 Asteraceae plants including crops, a weed, and ornamental plants were characterized. High performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) analyses revealed that all the Asteraceae plants examined exuded known SLs and, except for sunflower (Helianthus annuus), high germination stimulant activities at retention times corresponding to these SLs were confirmed. The two major SLs exuded by these Asteraceae plants were orobanchyl acetate and orobanchol. 5-Deoxystrigol and 7-hydroxyorobanchyl acetate were detected in root exudates from several Asteraceae species examined in this study.     

Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation

* Strigolactones are rhizosphere signalling compounds that mediate host location in arbuscular mycorrhizal (AM) fungi and parasitic plants. Here, the regulation of the biosynthesis of strigolactones is studied in tomato (Solanum lycopersicum). * Strigolactone production under phosphate starvation, in the presence of the carotenoid biosynthesis inhibitor fluridone and in the abscisic acid (ABA) mutant notabilis were assessed using a germination bioassay with seeds of Orobanche ramosa; a hyphal branching assay with Gigaspora spp; and by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. * The root exudates of tomato cv. MoneyMaker induced O. ramosa seed germination and hyphal branching in AM fungi. Phosphate starvation markedly increased, and fluridone strongly decreased, this activity. Exudates of notabilis induced approx. 40% less germination than the wild-type. The LC-MS/MS analysis confirmed that the biological activity and changes therein were due to the presence of several strigolactones; orobanchol, solanacol and two or three didehydro-orobanchol isomers. * These results show that the AM branching factors and parasitic plant germination stimulants in tomato root exudate are strigolactones and that they are biosynthetically derived from carotenoids. The dual activity of these signalling compounds in attracting beneficial AM fungi and detrimental parasitic plants is further strengthened by environmental conditions such as phosphate availability.     

Alteration of the Trifoliin A-Binding Capsule of Rhizobium trifolii 0403 by Enzymes Released from Clover Roots

The effect of white clover root exudate on capsules of Rhizobium trifolii 0403 was examined. The clover lectin trifoliin A was detected in root exudate of two clover varieties by indirect immunofluorescence with antibody against this lectin purified from clover seed. Trifoliin A bound uniformly to encapsulated, heat-fixed cells during 1 h of incubation with root exudate. After 4 to 8 h of incubation, trifoliin A was only bound to one pole of the cells. Transmission electron microscopy showed that the capsule itself was altered. The disorganization of the acidic polymers of the capsule began in the equatorial center of the rod-shaped cell and then progressed toward the poles at unequal rates. Trifoliin A could no longer be detected on heat-fixed cells after 12 h of incubation with root exudate. However, trifoliin A was detected in situ on one pole of cells grown for 4 days in the clover root environment of Fahraeus slide cultures. Inhibition studies with the hapten 2-deoxy-d-glucose showed that trifoliin A in root exudate had a higher affinity for one of the cell poles. Immunoelectrophoresis was used to monitor the alteration of the extracellular polysaccharides from R. trifolii 0403 by concentrated root exudate. These polysaccharides were converted into products which eventually lost their ability to immunoprecipitate with homologous antibody. This progressive loss of antigenic reactivity proceeded more rapidly with root exudate from seedlings grown under nitrogen-free conditions than with root exudate from plants grown with 15 mM KNO(3). The root exudate, depleted of trifoliin A by immunoaffinity chromatography, was still able to alter the capsule of R. trifolii 0403. Reconstitution experiments showed that the substance(s) in root exudate which induced this alteration of the capsule was of a high molecular weight, heat labile, trypsin sensitive, and antigenically unrelated to trifoliin A. A variety of glycosidase activities were also detected in the fraction depleted of trifoliin A. These results suggest that enzymes in clover root exudate alter the trifoliin A-binding capsule in a way which would favor polar attachment of R. trifolii to clover root hairs.     

泥胡菜等8种草本植物提取物除草活性的生物测定

采用顺序提取法制备了泥胡菜（Hemistepta lyrata Bunge）等8种草本植物全草的石油醚、乙酸乙酯和乙醇提取物,并以高粱（Sorghum vulgare Pers．）、黄瓜（Cucumis sativus L．）、小麦（Triticum aestivum L．）和油菜（Brassica campestris L．）为供试对象,用种子萌发法对提取物的除草活性进行了生物测定。结果表明,所有提取物对4种作物幼苗根和茎的生长均有一定的抑制作用,但抑制率有一定差异。泥胡菜和獯草[Humulus scandens（Lour．）Merr．]的乙酸乙酯提取物对作物幼苗根和茎的抑制作用最强,抑制率随提取物浓度的提高逐渐增高,且对作物幼苗根生长的抑制强度高于茎。在低浓度（12．5g·L^-1）条件下,泥胡菜乙酸乙酯提取物对小麦幼苗根和茎生长的抑制作用最强;蓓草乙酸乙酯提取物对高粱幼苗根的生长及油菜幼苗根和茎的生长抑制作用最强。研究结果显示,泥胡菜及蓰草的乙酸乙酯提取物具有潜在的除草活性。     

Germination stimulation in wild oats (Avena fatua L.) by synthetic strigol analogs and gibberellic acid

At concentrations of 0.01–1 mM, five synthetic multiring analogs of strigol were effective germination stimulants of intact and dehulled wild oat (Avena fatua L.) seeds. The effect was concentration-dependent and equaled or exceeded that produced by equimolar gibberellic acid (GA₃). The most effective strigol analog treatments induced 55–80% germination within 7 days in intact wild oat seeds and resulted in 63–86% germination and normal seedling growth over 14 days. Intact wild oat controls germinated 14% after 14 days. The stimulation of wild oat germination by these synthetic strigol analogs demonstrates that these compounds, initially developed as germination stimulants for the seeds of the parasitic weed, witchweed (Striga asiatica L. Kuntz.), have bioregulatory activity in dormant seeds of monocots, as well as dicots. None of the compounds tested significantly affected the germination of nondormant cultivated oat seeds (Avena sativa L.). The commonly used dispersal agent, Tween 20 (0.1%), was found to inhibit germination of cultivated oats, alone and in the presence of 2% acetone.