Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita.

More than 70% of vascular plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. In addition to other effects this symbiosis is known for its significance for plant nutrition including iron. Fungal iron mobilization from soil is commonly dependent on siderophores. This study reports on a search for such iron-chelators in root tissue of Tagetes patula nana var. plena colonized by Gigaspora margarita. The AM colonized plants and uninoculated controls were grown under strictly axenic conditions. HPLC analyses of aqueous extracts from plant roots have provided clear evidence for the presence of a rhizoferrin type siderophore, named glomuferrin, in root tissue of mycorrhizal seedlings. Results from HPLC analytical work are seconded by molecular biological data: A BLASTp search revealed that the AM fungal species Gigaspora rosea, Rhizophagus irregularis (formerly Glomus intraradices), Glomus cerebriformis and Diversispora epigea encode a non-ribosomal peptide synthetase (NRPS)-independent siderophore synthase (NIS), which is homologous to the rhizoferrin synthetase of Rhizopus delemar. Thus this study indicates that the biosynthesis of rhizoferrin type siderophores such as glomuferrin (= bis-imidorhizoferrin) may be widespread in the AM symbiosis.

Soil lead pollution modifies the structure of arbuscular mycorrhizal fungal communities.

The impact of lead (Pb) pollution on native communities of arbuscular mycorrhizal fungi (AMF) was assessed in soil samples from the surroundings of an abandoned Pb smelting factory. To consider the influence of host identity, bulk soil surrounding plant roots soil samples of predominant plant species (Sorghum halepense, Bidens pilosa, and Tagetes minuta) growing in Pb-polluted soils and in an uncontaminated site were selected. Molecular diversity was assessed by sequencing the 18S rDNA region with primers specific to AMF (AMV4.5NF/AMDGR) using Illumina MiSeq. A total of 115 virtual taxa (VT) of AMF were identified in this survey. Plant species did not affect AMF diversity patterns. However, soil Pb content was negatively correlated with VT richness per sample. Paraglomeraceae and Glomeraceae were the predominant families while Acaulosporaceae, Ambisporaceae, Archaeosporaceae, Claroideoglomeraceae, Diversisporaceae, and Gigasporaceae were less abundant. Acaulosporaceae and Glomeraceae were negatively affected by soil Pb, but Paraglomeraceae relative abundance increased under increasing soil Pb content. Overall, 26 indicator taxa were identified; four of them were previously reported in Pb-polluted soils (VT060; VT222; VT004; VT380); and five corresponded to cultured spores of Scutellospora castaneae (VT041), Diversispora spp. and Tricispora nevadensis (VT060), Diversispora epigaea (VT061), Glomus proliferum (VT099), and Gl. indicum (VT222). Even though AMF were present in Pb-polluted soils, community structure was strongly altered via the differential responses of taxonomic groups of AMF to Pb pollution. These taxon-specific differences in tolerance to soil Pb content should be considered for future phytoremediation strategies based on the selection and utilization of native Glomeromycota.

Arbuscular mycorrhizal fungi enhance the copper tolerance of Tagetes patula through the sorption and barrier mechanisms of intraradical hyphae.

Arbuscular mycorrhizal fungi (AMF) are widespread soil fungi that can form endosymbiotic structures with the root systems of most plants and can improve the tolerance of host plants to heavy metals. In the present study, we investigated the effects of AMF (Glomus coronatum) inoculation on the tolerance of Tagetes patula L. to Cu. Almost all of the non-mycorrhizal plants exposed to 100 µM Cu died after 3 d, whereas phytotoxicity was only observed in mycorrhizal plants that were exposed to Cu concentrations greater than 100 µM. Analysing the dynamic accumulation of Cu indicated that, after 7 d of Cu exposure, less Cu was absorbed or accumulated by mycorrhizal plants than by control plants, and significantly less Cu was translocated to the shoots. Meanwhile, analysing the root morphology, the integrity of the root plasma membranes, the photosynthesis rate, and the content of essential elements of plants growing in cultures with 50 µM Cu revealed that AMF inoculation markedly alleviated the toxic effects of Cu stress on root system activity, photosynthesis rate, and mineral nutrient accumulation. In addition, to understand the Cu allocation, an energy spectrum analysis of Cu content at the transverse section of root tips was conducted and subsequently provided direct evidence that intraradical hyphae at the root endodermis could selectively immobilise large amounts of Cu. Indeed, the sorption and barrier mechanisms of AMF hyphae reduce Cu toxicity in the roots of T. patula and eventually enhance the plants' Cu tolerance.

A search for glomuferrin: a potential siderophore of arbuscular mycorrhizal fungi of the genus Glomus.

Most fungi are known to synthesize siderophores under iron limitation. However, arbuscular mycorrhizal fungi (AM fungi) have so far not been reported to produce siderophores, although their metabolism is iron-dependent. In an approach to isolate siderophores from AM fungi, we have grown plants of Tagetes patula nana in the presence of spores from AM fungi of the genus Glomus (G. etunicatum, G. mossae & unidentified Glomus sp.) symbiotically under iron limitation and sterile conditions. A siderophore was isolated from infected roots after 2-3 weeks of growth in pots containing low-iron sand with Hoagland solution. HPLC analysis of the root cell lysate revealed a peak at a retention time of 6.7 min which showed iron-binding properties in a chrome azurol S test. The compound was isolated by preparative HPLC and the structure was determined by high resolution electrospray FTICR-MS and GC/MS analysis of the hydrolysis products. From an observed absolute mass to charge ratio (m/z) of 401.11925 [M+H]+ with a relative mass error of ∆ = 0.47 ppm an elemental composition of C16H21N2O10 [M+H]+ was derived, suggesting a molecular weight of 400 Da for glomuferrin. Corresponnding ion masses of m/z 423.10 and m/z 439.06 were asigned to the Na-adduct and K-adduct respectively. A mass of 455.03836 confirmed an Fe- complex with an elemental composition of C16H19N2O10Fe (∆ = 0.15 ppm). GC/MS analysis of the HCl lysate (6 N HCL, 12 h) revealed 1,4 butanediamine. Thus the proposed structure of the isolated siderophore from Glomus species consisted of 1,4 butanediamine amidically linked to two dehydrated citrate residues, similar to the previously identified bis-amidorhizoferrin. Thus, the isolated siderophore (glomuferrin) is a member of the rhizoferrin family previously isolated from fungi of the Mucorales (Zygomycetes).

Catellatospora tagetis sp. nov., isolated from the root of a marigold (Tagetes erecta L.).

A Gram-stain positive, non-motile, mesophilic actinomycete, designated strain NEAU-YJC4(T) was isolated from the root of a marigold (Tagetes erecta L.) collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. Morphological and chemotaxonomic properties of strain NEAU-YJC4(T) were consistent with the description of the genus Catellatospora. 16S rRNA gene sequence similarity studies showed that strain NEAU-YJC4(T) belongs to the genus Catellatospora, being most closely related to Catellatospora bangladeshensis JCM 12949(T) (98.7 %). Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain NEAU-YJC4(T) formed a monophyletic clade with the closest relative. A combination of DNA-DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-YJC4(T) can be distinguished from C. bangladeshensis JCM 12949(T). Therefore, it is proposed that strain NEAU-YJC4(T) represents a novel species of the genus Catellatospora, for which the name Catellatospora tagetis sp. nov. is proposed. The type strain is NEAU-YJC4(T) (=CGMCC 4.7176(T) = JCM 30053(T)).

An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.

Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66 %) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5 mg/l BAP and 5 mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200 mg/l) for selection, standardization of cocultivation time (45 min) and acetosyringone concentration (150 µM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600 bp npt II fragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocol established during the study will open new vistas for genetic manipulation and introduction of desired genes for genetic improvement of T. erecta.

Streptosporangium subfuscum sp. nov., isolated from the rhizosphere of marigold (Tagetes erecta L.).

A Gram-stain positive, filamentous bacterial strain, designated strain NEAU-TWSJ13(T), was isolated from the rhizosphere of a marigold (Tagetes erecta L.) plant collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. The strain was observed to form abundant aerial hyphae differentiated into spherical sporangia. 16S rRNA gene sequence similarity studies showed that strain NEAU-TWSJ13(T) belongs to the genus Streptosporangium, being most closely related to Streptosporangium fragile DSM 43847(T) (98.6 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that it formed a phyletic line with S. fragile DSM 43847(T), Streptosporangium jomthongense NBRC 110047(T) (98.4 % 16S rRNA gene similarity) and Streptosporangium violaceochromogenes DSM 43849(T) (97.6 % 16S rRNA gene similarity). A combination of DNA-DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-TWSJ13(T) can be distinguished from S. fragile DSM 43847(T) and S. jomthongense NBRC 110047(T). Moreover, strain NEAU-TWSJ13(T) can also be differentiated from S. violaceochromogenes DSM 43849(T) and other Streptosporangium species showing high 16S rRNA gene sequence similarity (>98.0 %) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-TWSJ13(T) represents a novel species of the genus Streptosporangium, for which the name Streptosporangium subfuscum sp. nov. is proposed. The type strain is NEAU-TWSJ13(T) ( = CGMCC 4.7146(T) = DSM = 46724(T)).

Control de calidad de drogas vegetales: lavado y desinfección de Artemisia annua L y Tagetes lucida Cav / Quality control of vegetable drugs: washing and disinfection of Artemisia annua L and Tagetes lucida Cav

Introducción: utilizar el lavado y desinfección química como método que permita disminuir la contaminación microbiana en el material vegetal cosechado de Artemisia annua L y Tagetes lucida Cav. Objetivos: garantizar la calidad microbiológica de las drogas vegetales obtenidas de estas 2 especies medicinales. Métodos: se utilizó material vegetal procedente de las parcelas establecidas en la Estación Experimental de Plantas Medicinales Dr Juan Tomás Roig, en suelo ferralítico rojo hidratado (ferralsols). Se emplearon 2 testigos, uno sin tratar y otro tratado con agua potable; para las pruebas a nivel de laboratorio las muestras de 300 g en A. annua y de 250 g de T lucida fueron lavadas con agua potable y después sometidas a un proceso de desinfección en una solución de hipoclorito de sodio 0,5 por ciento y 1,0 por ciento, sumergidas durante 5 y 10 min en ambos casos; para las pruebas de escalado se seleccionó el tratamiento químico que a nivel de laboratorio resultó más efectivo, se emplearon 3 réplicas de la droga cruda de 10 kg en el caso de A annua y de 6 kg en T lucida, con posterioridad las muestras se escurren sobre bastidores y después pasan a un proceso de secado en estufa de 35 a 40 ºC durante 2 d Luego de secadas se realizan los correspondientes análisis microbiológico y físico-químico. En el primer caso se determinó el conteo total de bacterias, conteo total de hongos y otras enterobacterias, además de microorganismos aislados. En el control físico- químico, los porcentajes de materia orgánica extraña; materia inorgánica extraña y hojas ennegrecidas; los índices numéricos como humedad, cenizas totales, sustancias solubles en agua, sustancias solubles en etanol 30 por ciento, sustancias solubles en etanol 70 por ciento, así como mediante tamizaje fitoquímico, la presencia de metabolitos secundarios...

Introduction: the use of washing and chemical disinfection as a method allows reducing microbial pollution in the harvested vegetal material of Artemisia annua L. and Tagetes lucida Cav Objectives: to assure the microbiological quality of vegetal drugs from these two medicinal species. Methods: there was used some vegetal material from lots on red hydrated ferralitic soils (ferrasols) located in Dr Juan Tomás Roig Experimental Center of Medicinal Plants. Two controls, one treated with drinking water and the other untreated, were used. For lab tests, the 300g A. annua and 250g T lucida samples were washed with drinking water and then were subjected to disinfection process using 0,5 and 1,0 percent sodium hypochloride in which both samples were submerged for 5 and 10 minutes. The most effective chemical treatment at lab was selected for the scaling test of three replicas of the crude drug from 10 kg of A annua and from 6 kg T lucida respectively. The samples were placed upon frames to be dried later at 35 to 40 ºC for 2 hours. After drying, the corresponding microbiological and physical-chemical analyses were performed. The microbiological analysis determined the total count of bacteria, of fungi and other enterobacteria in addition to isolated microorganisms. The physical and chemical analysis estimated the percentages of foreign organic matter, foreign inorganic matter and blackened leaves; and the indexes of humidity, total ashes, soluble substances in water, soluble substances in 30 percent etanol and soluble substances in 70 percent etanol...

The fungal fast lane: common mycorrhizal networks extend bioactive zones of allelochemicals in soils.

Allelopathy, a phenomenon where compounds produced by one plant limit the growth of surrounding plants, is a controversially discussed factor in plant-plant interactions with great significance for plant community structure. Common mycorrhizal networks (CMNs) form belowground networks that interconnect multiple plant species; yet these networks are typically ignored in studies of allelopathy. We tested the hypothesis that CMNs facilitate transport of allelochemicals from supplier to target plants, thereby affecting allelopathic interactions. We analyzed accumulation of a model allelopathic substance, the herbicide imazamox, and two allelopathic thiophenes released from Tagetes tenuifolia roots, by diffusion through soil and CMNs. We also conducted bioassays to determine how the accumulated substances affected plant growth. All compounds accumulated to greater levels in target soils with CMNs as opposed to soils without CMNs. This increased accumulation was associated with reduced growth of target plants in soils with CMNs. Our results show that CMNs support transfer of allelochemicals from supplier to target plants and thus lead to allelochemical accumulation at levels that could not be reached by diffusion through soil alone. We conclude that CMNs expand the bioactive zones of allelochemicals in natural environments, with significant implications for interspecies chemical interactions in plant communities.

The effect of the symbiosis between Tagetes erecta L. (marigold) and Glomus intraradices in the uptake of Copper(II) and its implications for phytoremediation.

Phytoremediation is an environmental biotechnology that seeks to remediate pollution caused by bioaccumulative toxins like copper (Cu). Symbiotic mycorrhizal associations can increase the uptake and delivery of low mobility nutrients and micronutrients to the host plant because they solubilize these substances and increase their catchment area. To analyze the effect of mycorrhizae on the phytoaccumulation of Cu, we studied their ability to solubilize Cu(II) and enhance its absorption by the plant Tagetes erecta L. colonized with the arbuscular mycorrhizal fungus Glomus intraradices. Plants were grown for nine weeks in a growth chamber under controlled conditions of temperature, relative humidity and photoperiod. Cu was added in the insoluble form of CuO to simulate the insoluble Cu-O affixed species in soil. The biotic and abiotic parameters of colonization, foliar area, biomass and the pH of leachates were determined as functions of the Cu concentration that was measured in the roots, shoots and leachates by AAS. The results of Cu absorption showed that the colonized plants accumulated more Cu in the roots as well as the whole plant and that both the colonized and non-colonized plants displayed the typical behavior of Cu excluders. Mycorrhizal colonization of the roots resulted in a proliferation of vesicles and this was observed to scale with root tissue Cu concentrations. Also, the G. intraradices-T. erecta system displayed a higher resistance to the toxicity induced by Cu while nonetheless improving the indices of phytoaccumulative yields. These results suggest that G. intraradices possibly accumulates Cu in its vesicles thereby enhancing the Cu tolerance of T. erecta even while increasing root Cu accumulation. The parameters of bioconcentration factor and translocation factor measured in this work suggest that the system T. erecta-G. intraradices can potentially phytostabilize Cu in contaminated soils.

Arbuscular mycorrhizal fungi in terms of symbiosis-parasitism continuum.

Arbuscular mycorrhizal fungi are forming the most wide-spread mycorrhizal relationships on Earth. Mycorrhiza contributes to phosphorous acquisition, water absorption and resistance to diseases. The fungus promotes the absorption of nutrients and water from soil, meanwhile the host plant offers photosynthetic assimilates in exchange, like carbohydrates, as energy source. The plant benefits from the contribution of symbiotic partner only when nutrients are in low concentrations in soil and the root system would not be able to absorb sufficiently the minerals. When the help of mycorrhizal fungi is not necessarily needed, the host plant is making an economy of energy, suppressing the development of fungi in the internal radicular space. In this moment, the nature of relationship turns from symbiotic to parasitic, triggering a series of defensive reactions from the plant. Also, there were several cases reported when the presence of arbuscular mycorrhizal fungi negatively influenced the host plant. For example, in adverse environmental conditions, like very high temperatures, instead of determining a higher plant biomass and flowering, the mycorrhiza reduces the growth of the host plant. We conducted a pot experiment with hydroponic culture to examine the effect of arbuscular mycorrhiza on development of French marigold as a host plant. As experimental variants, the phosphorous content in nutrient medium and temperature varied. Plants were artificially infected with arbuscular mycorrhizal fungi using a commercial inoculum containing three fungal species, as following: Glomus intraradices, Glomus etunicatum and Glomus claroideum. Colonization intensity and arbuscular richness were checked using root staining with aniline blue and estimation with the Trouvelot method. To observe the differences between plants from the experimental variants, we examined the number of side shoots, flower buds and fully developed flowers, fresh biomass and total leaf area. Results show that adverse climatic conditions, like temperature shock at the beginning of growing period modified the nature of symbiosis. In this case, the physiological parameters were reduced at colonized plants, while usual, constant growing conditions permitted the normal, efficient and beneficial development of symbiosis.

A rationally assembled microbial community for growing Tagetes patula L. in a lunar greenhouse.

Well-defined plant-associated bacteria were used for growing French marigolds (Tagetes patula L.) in anorthosite, a substrate of low bioavailability, analogous to a lunar rock. The consortium was composed mainly of plant growth-promoting rhizobacteria and biocontrol agents that were used for seed inoculation. Simultaneously, the sterile substrate was inoculated with the siliceous bacterium Paenibacillus sp. IMBG156. The plant benefited from bacterial activity which resulted in stimulation of seed germination, better plant development, and finally in flowering of inoculated tagetes. In contrast, control plants grew poorly in sterile anorthosite and never flowered. Analysis of bacterial community composition showed that both species colonized plant roots, and there were no shifts in the consortium structure in the rhizosphere of French marigolds within 6 weeks. Paenibacillus sp. IMBG156 was able to release some elements (Ca, Fe, Si) from substrate anorthosite. It was assumed that a rationally assembled consortium of bacterial strains supported growth and development of the model plant under growth-limiting conditions, at least by means of bioleaching and delivering of essential nutritional elements to the plants, and by promoting plant growth.

An optimization study of solid-state fermentation: xanthophylls extraction from marigold flowers.

Marigold flowers are the main natural source of xanthophylls, and marigold saponified extract is used as an additive in several food and pharmaceutical industries. In this work, the use of a solid-state fermentation (ensilage) process for increasing the yield of xanthophylls extracted from fermented marigold flowers was examined. The process consisted of a mixed culture of three microorganisms (Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans), part of the normal microbiota associated with the marigold flower. These microorganisms had been previously isolated, and were identified as relevant for the ensilage process due to their capacity to produce cellulolytic enzymes. Based on experimental design strategies, optimum operation values were determined for aeration, moisture, agitation, and marigold-to-inoculum ratio in the proposed solid-state fermentation equipment, leading to a xanthophylls yield of 17.8-g/kg dry weight. The optimum achieved represents a 65% increase with respect to the control. HPLC analysis indicated conservation of extracted oleoresin. Based on the experimental results, interactions were identified that could be associated with the heat and mass-transfer reactions taking place within the bioreactor. The insight gained allows conditions that limit growth and metabolic activity to be avoided.

Mixed culture optimization for marigold flower ensilage via experimental design and response surface methodology.

Endogenous microorganisms isolated from the marigold flower (Tagetes erecta) were studied to understand the events taking place during its ensilage. Studies of the cellulase enzymatic activity and the ensilage process were undertaken. In both studies, the use of approximate second-order models and multiple lineal regression, within the context of an experimental mixture design using the response surface methodology as optimization strategy, determined that the microorganisms Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans are the most significant in marigold flower ensilage and exhibit high cellulase activity. A mixed culture comprised of 9.8% Flavobacterium IIb, 41% A. anitratus, and 49.2% R. nigricans used during ensilage resulted in an increased yield of total xanthophylls extracted of 24.94 g/kg of dry weight compared with 12.92 for the uninoculated control ensilage.

[Effects of different arbuscular mycorrhizal fungi on Tagetes erecta growth and diesel degradation].

The effects of G. mosseae, G. geospora, G. constrictum and bacteria on diesel tolerance of Tagetes erecta were investigated under greenhouse conditions. The results showed that AM fungi could still develop mycorrhizal assosiations with mum when the diesel concentration was 5,000 mg.kg-1. White mum was better than yellow mum in diesel tolerance, with 63.1% total biomass increased. The colonization rate of inoculating AM fungi treatment was 3.5%-29.9% higher than the control. G. mosseae and G. geospora were better strains, their biomass increasing 9.0% and 42.7% than the control, respectively, while the effect of inoculating mixed AM fungi was not obvious. Bacteria inhibited the colonization of arbuscular mycorrhizal fungi on white mum, but promoted the vegetative and reproductive growth of mycorrhizal mum. Among 5 inoculation treatments, treatments of inoculating G. geospora and inoculating mixed AM fungi and bacteria were better, with 16.51% and 14.05% more diesel degradation rate than that of the control, respectively.