Distribution and Accumulation of Thiophenes in Plants and Calli of Different Tagetes Species

The diversity of thiophenes (natural biocides) and the differencesbetween the concentrations of these compounds in the leavesand roots of Tagetes erecta L., T. patula L. cv. Nana furia,and T. minuta L. (marigolds) indicated the presence of at leasttwo different sites of accumulation: leaves and roots. Leafexplants of Tagetes, however, are used by preference to obtaincallus cultures. Once subcultured, secondary (C₂) calli of T.patula obtained from leaves of 4 to 7-week-old plants, containedhigher amounts of accumulated thiophenes (up to 80% of the amountsin the leaves) than original (C₁) or twice subcultured calli(C₃). The concentrations of thiophenes in C₂ calli of T. minutawere about half those of C₁ calli, while the concentrationsof thiophenes of C₁ calli amounted to 1-2% of the leaf values.Most of the C₃ calli of T. minuta did not contain thiophenesat all. Although C₁ calli of T. erecta also contained considerableamounts of thiophenes, the C₂ calli died, most likely owingto high levels of accumulated polyphenolic compounds. The combinationof species effects and the physiological state of plants andcalli provides adequate information to decide whether Tagetescalli are able to produce thiophenes or not. It is concludedthat the ability to produce thiophenes does not depend on theorgan used, but on the genetic information present in the species,and on the physiological state of plants and calli, particularlytheir age. Key words: Callus, explant selection, Tagetes erecta, Tagetes minuta, Tagetes patula, thiophenes     

Thiophene Content in Normal and Transformed Root Cultures of Tagetes erecta: A Comparison with Thiophene Content in Roots of Intact Plants

Transformed roots of Tagetes erecta were obtained followinginfection of stems of sterile plantlets with Agrobacterium rhizogenesstrain TR105. The thiophenes detected were 5-(4-hydroxy-l-butenyl)-2,2'-bithienyl,5-(4-acetoxy-l-butenyl)-2,2'-bithienyl, 5-(3-buten-l-enyl)-2,2',-bithienyl and 2.2': 5', 2'-terthienyl. The thiophene patternwas the same in normal root cultures and roots of the intactplant. Transformed roots showed a higher growth rate and a higherbiomass yield than normal root cultures on a hormone-free media. Key words: Transformed roots normal roots, Tagetes erecta, thiophenes, Agrobacterium rhizogenes     

Elicitation of thiophene production by cultured hairy roots of Tagetes patula

Hairy roots of Tagetes patula were initiated by infecting the seedlings with Agrobacterium rhizogenes. The hairy roots were grown in liquid medium MS (Murashige and Skoog 1962) supplemented with sucrose. The roots were treated with three different elicitors obtained from mycelial culture of Aspergillus niger, Rhizopus oligosporus and Pencillium notatum. Accumulation of biomass and the production of thiophenes were studied over a period of six weeks in culture. The HPLC separation profile of the thiophenes indicated the presence of several structurally different thiophenes. α-terthienyl being predominent. Maximum production of thiophene was recorded at the end of the fourth week in culture with a content of 0.138 % (w/w on dry weight basis). Treatment of hairy roots with mycelial extract of Aspergillus niger (1.5 % v/v) elicited an increase in thiophene content by 1.6 folds over the control.     

Quantitative relationships between boron and mannitol concentrations in phloem exudates of Olea europaea leaves under contrasting boron supply conditions

Root exudates are implicated in the chemical defense of plants, but testing such hypotheses has been hindered by the difficulties of quantifying allelochemical concentrations in soil. Here we describe a new, simple method to quantify the dynamics of non-polar root exudates in soil. Novel soil probes were constructed using stainless steel wire inserted into polydimethylsiloxane (PDMS) tubing. Probes were inserted into soil for 24 h, removed and extracted, and analyzed by HPLC. Lipophilic thiophenes produced by roots of Tagetes and Rudbeckia species were chosen as candidate compounds to test the method. Probes recovered microgram quantities of the highly phytotoxic thiophenes 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT) and α-terthienyl per probe per day from the root zone of Tagetes patula, and distribution of thiophenes beneath plants was spatially and temporally heterogeneous. Flux-proportional sampling of soil provides a means to test hypotheses about the role of root exudates in plant–plant and other interactions.     

Thiophene accumulation in hairy roots ofTagetes patula in response to fungal elicitors

Summary Hairy roots ofTagetes patula were treated with fungal extracts from 3 different fungi. Treated roots were found to accumulate thiophenes at a higher level than untreated control cultures. The kinetics of thiophene formation varied with the fungus from which the elicitor was prepared, elicitor concentration and duration of exposure.     

Greenhouse Studies on the Effect of Marigolds (Tagetes spp.) on Four Meloidogyne Species

The effects of preplanted marigold on tomato root galling and multiplication of Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla were studied. Marigold cultivars of Tagetes patula, T. erecta, T. signata, and a Tagetes hybrid all reduced galling and numbers of second-stage juveniles in subsequent tomato compared to the tomato-tomato control. All four Meloidogyne spp. reproduced on T. signata ''Tangerine Gem''. Several cultivars of T. patula and T. erecta suppressed galling and reproduction of Meloidogyne spp. on tomato to levels lower than or comparable to a fallow control. Phytotoxic effects of marigold on tomato were not observed. Several of the tested marigold cultivars are ready for full-scale field evaluation against Meloidogyne spp.     

Influence of polyamines on growth and formation of secondary metabolites in hairy root cultures of Beta vulgaris and Tagetes patula

Growth of hairy roots of Beta vulgaris, which produces betalaines, and of Tagetes patula, which produces thiophenes, was studied under the influence of externally treated polyamines. Of the three polyamines, viz. putrescine, spermidine and spermine, administered singly at 1.5 mM concentration, putrescine and spermidine at 0.75 mM concentration influenced increase in the accumulation of biomass of B. vulgaris and T. patula hairy roots by 1.42 and 1.30 fold over the control. Whereas, the treatment of spermine (1.5 mM) alone resulted in decrease in the biomass in both the systems. Combined administration of putrescine (0.75 mM) and spermidine (0.75 mM) enhanced growth in both B. vulgaris and T. patula than that observed in individual treatments. Polyamines administered alone or in combination did alter production of betalaine and thiophene content. Dose response experiments showed that, when putrescine and spermidine was administered at 0.75 mM concentration, it resulted in maximum biomass and production of beta-laine and thiophene in B. vulgaris and T. patula respectively as compared to the control and the media treated with double and triple strength of nitrates and in combination with putrescine and spermidine at equimolar concentration. In B. vulgaris and T. patula hairy root cultures, endogenous spermine titers were maximum in putrescine and spermidine 0.75 mM each treated, cultures, which was 1.63 and 2.0 fold higher than in control on 28^th and 35^th days respectively.     

Thiophene accumulation in relation to morphology in roots of Tagetes patula

Roots of marigold (Tagetes patula L.) accumulate thiophenes, heterocyclic sulfurous compounds with strong biocidal activity. In detached roots cultured in vitro, the thiophene content was 5 mol·(g fresh weight)^-1 which is 25-times higher than in roots attached to the plant. In roots derived from tissues transformed by Agrobacterium tumefaciens and A. rhizogenes, the morphology and thiophene content varied with the bacterial strain used. Transformation stimulated the elongation of the root tips and the formation of lateral roots but lowered the thiophene level to 20–50% relative to the concentration in untransformed detached roots. A negative correlation was found between the number of laterals in a root system and the thiophene content. Extensive branching and a decrease in thiophene accumulation was evoked in untransformed roots by indole-3-acetic acid (1–10 mol·l^-1) added to the medium. Within the roots, the highest thiophene concentrations were found in the tips. The results indicate that auxin directly or indirectly plays a role in the regulation of the thiophene level in root tips.Abbreviations B5 Gamborg's B5 medium - IAA Indole-3-acetic acid     

Postharvest changes in endogenous ABA levels and ABA metabolism in relation to dormancy in potato tubers

In this paper the effects of indole-3-acetic acid (IAA) on growth of Tagetes patula hairy root cultures and secondary product formation are presented. The biosynthesis of thiophenes, sulfurous compounds with nematicidal activity, was inhibited by IAA application, as was evident from a decrease of [³⁵S] sulfur incorporation. The inhibition only occurred after the roots had developed numerous laterals as a result of auxin action. However, in roots cultured in the absence of IAA, there was no significant correlation between branching and thiophene accumulation. Therefore, development of lateral roots is not a sufficient condition for a low capacity to synthesize thiophenes. The highest rate of thiophene accumulation in the roots culture is at its maximum. Hence, growth and the production of thiophenes appear to be compatible in T. Patula hair roots.     

Variation in Phenolic Profile, β‐Carotene and Flavonoid Contents,Biological Activities of Two Tagetes Species from Pakistani Flora

The objective of present study was to evaluate the variation in phenolic profile, β‐carotene, flavonoid contents, antioxidant and antimicrobial properties of Tagetes erecta and Tagetes patula (T. erecta and T. patula) through different in vitro assays. Antioxidant activity was determined through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical scavenging and inhibition of linoleic acid peroxidation assays and antibacterial and antifungal activities studied using the disc diffusion and resazurin microtiter‐plate assays against bacterial and fungal strains. Moreover, total phenolics (TP), total carotenoids (TC) and total flavonoids (TF) were also determined. Highest (TP 35.8 mg GAE/g) and TF (16.9 mg CE/g) contents were found in MeOH extract of T. patula. T. erecta extract showed higher TC contents (6.45 mg/g) than T. patula extract (6.32 mg/g). T. erecta exhibited the highest DPPH radical‐scavenging activity (IC₅₀) (5.73 μg/mL) and inhibition of linoleic acid peroxidation (80.1%). RP‐HPLC revealed the presence of caffeic acid, sinapic acid and ferulic acid in Tagetes extracts, m‐coumaric acid in T. erecta whereas chlorogenic acid in T. patula extract only. Both extracts possessed promising antimicrobial activity compared to the ciprofloxacin and flumequine (+ve controls) against Bacillus subtilis and Alternaria alternate. Both extract were rich source of polyphenols exhibiting excellent biological activities.     

Investigation of P and S Distributions in the Roots of Tagetes patula L. using Micro-PIXE

A proton microprobe for energy-dispersive PIXE-analysis hasbeen used to measure elemental distributions of sulphur andother elements in roots of Tagetes patula L. by making a line-scanalong the diameter of the cross-section. Higher concentrationsof phosphorus and sulphur were found in or near the phloem.In addition the endodermis contained an increased sulphur concentration.The results obtained and future possibilities for this kindof investigation are discussed. Key words: Tagetes patula L., micro-PIXE, sulphur, thiophenes, endodermis, Pratylenchus penctrans (Cobb)     

Quantitative profiling of polyacetylenes in tissue cultures and plant parts of three species of the Asteraceae

Polyacetylenes are a group of fatty-acid derived specialized metabolites with several C–C-triple bonds and derived compounds which are widely distributed in the plant kingdom, but are especially abundant and structurally diverse in the Asteraceae family. Despite their interesting structural and biological properties, the biosynthesis of polyacetylenes is only poorly understood. We have used three species of the Asteraceae (Carthamus tinctorius, Tagetes patula, and Arctium lappa) to compare their suitability for studies of polyacetylene biosynthesis when used after cultivation on soil or as tissue culture. The polyacetylene profiles detected in different plant parts together with information from the literature indicate that C. tinctorius seedlings and flowers as well as T. patula roots and flower buds are major sites of polyacetylene biosynthesis. Highest levels of polyacetylenes were detected in T. patula [about 30 µmol/g dry weight (d.w.) thiophenes in roots] while A. lappa contained less than 1 µmol/g d.w.. Methyljasmonate (MeJ)-induced T. patula hairy root cultures proved to be an excellent source of butenynyl-bithiophene (200 µmol/g d.w., 43 mg/g d.w.) while T. patula flower buds could serve as a source of pentenynyl-bithiophene and α-terthienyl (5–10 µmol/g d.w.) and C. tinctorius flowers or seedlings as a source of polyacetylenic C₁₃ hydrocarbons, the biosynthetic precursors of thiophenes (5–10 µmol/g d.w.). Upon addition of elicitors to tissue cultures, highest elicitation factors (between four and seven) were reached for 1,11-tridecadiene-3,5,7,9-tetrayne in C. tinctorius cell suspension cultures with 40 µM MeJ and α-terthienyl in T. patula hairy root cultures with 100 µM MeJ.     

Formation of natural thiophene derivatives from acetylenes by Tagetes patula

Six tritium-labelled thiophene derivatives were synthesized and their emulsions were applied to whole plants of Tagetes patula. From the root extra     

Degradation analysis of Reactive Red 198 by hairy roots of <Emphasis Type="Italic">Tagetes patula</Emphasis> L. (Marigold)

Tagetes patula L. (Marigold) hairy roots were selected among few hairy root cultures from other plants tested for the decolorization of Reactive Red 198. Hairy roots of Tagetes were able to remove dye concentrations up to 110 mg L^−l and could be successively used at least for five consecutive decolorization cycles. The hairy roots of Tagetes decolorized six different dyes, viz. Golden Yellow HER, Methyl Orange, Orange M2RL, Navy Blue HE2R, Reactive Red M5B and Reactive Red 198. Significant induction of the activity of biotransformation enzymes indicated their crucial role in the dye metabolism. UV–vis spectroscopy, HPLC and FTIR spectroscopy analyses confirmed the degradation of Reactive Red 198. A possible pathway for the biodegradation of Reactive Red 198 has been proposed with the help of GC–MS and metabolites identified as 2-aminonaphthol, p-aminovinylsulfone ethyl disulfate and 1-aminotriazine, 3-pyridine sulfonic acid. The phytotoxicity study demonstrated the non-toxic nature of the extracted metabolites. The use of such hairy root cultures with a high ability for bioremediation of dyes is discussed.     

Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta

Two mutants of Tagetes erecta displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M2 population using high-performance liquid chromatography analysis of root extracts. Both mutants, which may have originated from the same mutational event, contained high amounts of the C13 monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type. The mutant phenotype was also expressed in 21 Agrobacterium rhizogenes transformed root clones derived from both mutants. Feeding experiments with root cultures derived from one mutant and from the wild type indicated that the monothiophene accumulating in the mutant is the common precursor for all bithienyl thiophenes in wild-type and mutant Tagetes erecta. These experiments also showed that one mutant is deficient in demethylation of the monothiophene.     

Growth and Secondary Metabolite Production by Hairy Roots of Tagetes Patula in Aqueous Two-Phase Systems

Hairy roots of Tagetes patula have been grown in aqueous two-phase systems. After selecting suitable polymers from single-phase experiments (in which salt phases were unable to support growth in the desired concentrations) several two-phase systems were tested for their influence on cell growth and thiophene production. Cell growth occurred in all aqueous two-phase systems, but the highest growth rate was achieved in normal medium. There was no difference in thiophene production between medium and aqueous two-phase systems. The partition of thiophenes favoured slightly the more hydrophobic top phase in most cases, while the cells were confined to the bottom phase. One aqueous two-phase system (15% polyethyleneglycol 10,000 and 15% Reppal PES 200) was tested in a stirred tank reactor with normal medium as a control. The growth rate in medium was higher than in the aqueous two-phase system, while the thiophene production per unit cell weight was in the same range for both systems. The excretion of thiophenes in the reactor with the aqueous two-phase system was about ten times as high as in the control reactor. The amount excreted was however still not more than 3% of the total production.     

CYTOGENETIC STUDIES ON THE ORIGIN OF TAGETES PATULA. I. MEIOSIS AND MORPHOLOGY OF DIPLOID AND ALLOTETRAPLOID T. ERECTA × T. TENUIFOLIA

Towner , Joseph W. (U. California, Los Angeles.) Cytogenetic studies on the origin of Tagetes patula. I. Meiosis and morphology of diploid and allotetraploid T. erecta × T. tenuifolia. Amer. Jour. Bot. 48 (9): 743–751. Illus. 1961.—Cytological and morphological evidence is presented to show that Tagetes patula L. is an allotetraploid species (2n = 48) which probably originated by hybridization between the diploids T. erecta L. and T. tenuifolia Cav., or species closely related to them. The putative parent species are separated by strong reproductive isolation barriers, and controlled crosses between them yielded very few good seed. Most of the hybrid seedlings died before reproductive age, and the few survivors were sterile and extremely weak. At meiosis in the diploid hybrid, bivalent frequency per cell was 0–11 and averaged 4.4. Although 43.6% of the quartet stage consisted of restitution dyads, pollen was essentially all non-viable and seed set was less than 1%. A true-breeding, fertile, allotetraploid T. erecta-tenuifolia was synthesized by colchicine-doubling of the chromosome number of the F₁ hybrid. In the synthetic allotetraploid, average chromosomal association was 23.97 bivalents plus 0.05 univalents, and 97.4% of MI cells had 24 bivalents; there were no multivalents, bridges, or fragments. Tagetes erecta-tenuifolia showed a striking morphological similarity to T. patula. Both the natural and synthetic allotetraploids were intermediate between T. erecta and T. tenuifolia for nearly every character. Of 10 species of Tagetes whose chromosome numbers are known, 5 are tetraploid (2n = 48).     

GLC and GLC-mS analysis of thiophene derivatives in plants and in in vitro cultures of Tagetes patula L. (Asteraceae)

The occurrence of thiophenic compounds in diverse plant organs and in in vitro root-, callus- and cell suspension cultures of Tagetes patula cv. Carmen was investigated using capillary GLC and GLC-MS. The separation of thiophenes by capillary GLC and the group specific MS fragmentation with the typical sulfur isotope peaks allowed the unequivocal assignment of individual thiophenes in complex mixtures, even when occurring in traces and in the presence of different geometrical isomers. The extracts of Tagetes patula cv. Carmen contained the following 8 thiophene compounds: 5-(3-buten-1-ynyl)-2,2'-bithienyl (BBT), 5'-methyl-5-(3-buten-1-ynyl)-2,2'-bithienyl (MeBBT), 5-(1-pentynyl)-2,2'-bithienyl (PBT), 5-(4-hydroxy-1-butynyl)-2,2'-bithienyl (BBTOH), 2,2',5,2"-terthienyl (alpha-T), 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc), 5-methylaceto-5'-(3-buten-1-ynyl)-2,2'-bithienyl (AcOCH2BBT), and 5-(3,4-diacetoxy-1-butynyl)-2,2'-bithienyl (BBT(OAc)2). The most complex thiophene profile, including the less common PBT was detected in aerial parts of freshly harvested plant material. Under in vitro conditions only the root cultures, but not callus or cell suspension cultures produced substantial amounts of irregular thiophenes confirming that roots are the main site of thiophene biosynthesis.     

Endoroot bacteria derived from marigolds (Tagetes spp.) can decrease soil population densities of root-lesion nematodes in the potato root zone

Single isolates of bacterial endophytes, isolated from the nematode antagonistic plant species African (Tagetes erecta L.) and French (T. patula L.) marigold, were introduced into potatoes (Solanum tuberosum L.). Several bacterial species possessed activity against root-lesion nematodes (Pratylenchus penetrans) in soils around the root zone of potatoes, namely: Microbacterium esteraromaticum, Tsukamurella paurometabolum, isolate TP6, Pseudomonas chlororaphis, Kocuria varians and K. kristinae. Of these, M. esteraromaticum and K. varians depressed the population densities of root-lesion nematodes without incurring any yield penalty (tuber wet weight). No significant differences were found in the total numbers of P. penetrans nematodes, rhabditid nematodes or `other' parasitic nematode species within the root tissues of bacterized potato plants compared to the unbacterized check. Overall, tuber fresh weights and tuber number were equal to or significantly lower (P\le0.05) in bacterized plants than their unbacterized counterpart. We conclude that endoroot bacteria from Tagetes spp. can play a role in nematode suppression through the attenuation of nematode proliferation. We propose that these nematode control properties are capable of transfer to other crops in a rotation as a beneficial `residual' microflora – a form of beneficial microbial allelopathy.     

Thiophenes and benzofurans in the undisturbed rhizosphere ofTagetes patula L.

Summary Marigold (Tagetes patula L.) contains secondary metabolites toxic to various organisms. While these compounds may function as defensive agents in the plant, it is not clear whether they are exuded into the rhizosphere. Using a continuous root exudate trapping system and capillary column gas chromatography/mass spectrometry/data system, four thiophenes and two benzofurans were identified in the root exudates collected from the undisturbed rhizosphere of marigold. The importance of rhizospheric chemistry in the study of allelopathy is stressed.