Seasonal analysis of the tropane alkaloid ecgonine methyl ester and the occurrence of other highly-valued tropanes in the South African <Emphasis Type="Italic">Erythroxylum</Emphasis> trees

The coca family (Erythroxylaceae) consists of trees and shrubs sub-divided into four genera: Aneulophus, Nectaropetalum, Pinacopodium, and Erythroxylum, which include species with highly valuable medicinal compounds. E. delagoense, E. emarginatum, and E. pictum are endemic to southern Africa and have great pharmaceutical potential based on their traditional uses. Previous studies have shown certain inconsistencies in terms of the presence or absence of tropane alkaloids in these species, resulting in a need for further research and clarification. Therefore, the aim of this study was to determine the seasonal variation of the immediate biosynthetic precursor of cocaine, the tropane alkaloid, ecgonine methyl ester in the three South African Erythroxylum species by means of gas chromatography–mass spectrometry, as well as to conduct a phytochemical screening for observing the presence of other potential compounds and tropane alkaloids. We found significant differences in tropane concentrations from the seasonal variation study, explaining the discrepancies in previous reports on its presence/absence in these species. Furthermore, we report for the first time on the occurrence of selected highly valuable tropane alkaloids in E. emarginatum currently used in ‘blockbuster medicine’.     

Plant growth promoting potential of bacterial endophytes in novel association with <Emphasis Type="Italic">Olea ferruginea</Emphasis> and <Emphasis Type="Italic">Withania coagulans</Emphasis>

Microbially unexplored medicinal plants can have a genetically diverse microbial population with multi-functional plant growth promoting traits. In this aspect, 75 endophytic bacterial isolates with plant growth promoting traits were isolated from Withania coagulans Dunal and Olea ferruginea Royal. Many of these bacteria were able to solubilize phosphate, produce indole-3-acetic acid, ammonia as well as hydrogen cyanide, synthesize extracellular enzymes and show antagonistic activities against plant pathogenic fungi under in vitro conditions. These isolates were also characterized by morphological and biochemical analysis. Furthermore, four representative isolates with pronounced plant growth promoting activities were identified as Enterobacter cloacae, Enterobacter dissolvens, Enterobacter hormaechei and Cronobacter sakazakii by 16S rDNA sequencing analysis. This work for the first time, reported the isolation of endophytic bacteria, the novel association form selected plants, Withania coagulans and Olea ferruginea. The explored endophytes might have great potential in the field of biocontrol and plant growth promoting for sustainable agricultural practices.     

Four novel <Emphasis Type="Italic">Talaromyces</Emphasis> species isolated from leaf litter from Colombian Amazon rain forests

Various Talaromyces strains were isolated during a survey of fungi involved in leaf litter decomposition in tropical lowland forests in the Caquetá and Amacayacu areas of the Colombian Amazon. Four new Talaromyces species are described using a polyphasic approach, which includes phenotypic characters, extrolite profiles and phylogenetic analysis of the internal transcribed spacer region (ITS) barcode, and beta-tubulin (BenA) and calmodulin (CaM) gene regions. Talaromyces amazonensis sp. nov., T. francoae sp. nov. and T. purgamentorum sp. nov. belong to Talaromyces section Talaromyces, and T. columbiensis sp. nov. is located in section Bacillispori. The new species produce several bioactive compounds: T. amazonensis produces the potential anticancer agents duclauxin, berkelic acid and vermicillin, and T. columbiensis produces the effective anticancer agent wortmannin (together with duclauxin). In addition to the new species, T. aculeatus and T. macrosporus were isolated during this study on leaf litter decomposition.     

Micropropagation and genetic transformation of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merr.: a review

Key message

This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties.

Abstract

Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.

     

Bioprospecting of <Emphasis Type="Italic">Diaporthe terebinthifolii</Emphasis> LGMF907 for antimicrobial compounds

Antibiotic-resistant bacteria have been observed with increasing frequency over the past decades, driving the search for new drugs and stimulating the interest in natural products sources. Endophytic fungi from medicinal plants represent a great source of novel bioactive compounds useful to pharmaceutical and agronomical purposes. Diaporthe terebinthifolii is an endophytic species isolated from Schinus terebinthifolius, a plant used in popular medicine for several health problems. The strain D. terebinthifolii LGMF907 was previously reported by our group to produce secondary metabolites with biological activity against phytopathogens. Based on these data, strain LGMF907 was chosen for bioprospecting against microorganisms of clinical importance and for characterization of major secondary metabolites. In this study, different culture conditions were evaluated and the biological activity of this strain was expanded. The crude extracts demonstrated high antibacterial activity against Escherichia coli, Micrococcus luteus, Saccharomyces cerevisiae, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant S. aureus. The compounds diaporthin and orthosporin were characterized and also showed activity against the clinical microorganisms evaluated. This study discloses the first isolation of diaporthin and orthosporin from D. terebinthifolii, and revealed the potential of this endophytic fungus to produce secondary metabolites with antimicrobial activity.     

Physiological and biochemical parameters: new tools to screen barley root exudate allelopathic potential (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L. subsp. <Emphasis Type="Italic">vulgare</Emphasis>)

Morphological markers/traits are often used in the detection of allelopathic stress, but optical signals including chlorophyll a fluorescence emission could be useful in developing new screening techniques. In this context, the allelopathic effect of barley (Hordeum vulgare subsp. vulgare) root exudates (three modern varieties and three landraces) were assessed on the morphological (root and shoot length, biomass accumulation), physiological (F_v/F_m and F₀), and biochemical (chlorophyll and protein contents) variables of great brome (Bromus diandrus Roth., syn. Bromus rigidus Roth. subsp. gussonii Parl.). All the measured traits were affected when great brome was grown in a soil substrate in which barley plants had previously developed for 30 days before being removed. The response of receiver plants was affected by treatment with activated charcoal, dependent on barley genotype and on the nature of the growing substrate. The inhibitory effect was lower with the addition of the activated charcoal suggesting the release of putative allelochemicals from barley roots into the soil. The barley landraces were more toxic than modern varieties and their effect was more pronounced in sandy substrate than in silty clay sand substrate. In our investigation, the chlorophyll content and F_v/F_m were the most correlated variables with barley allelopathic potential. These two parameters might be considered as effective tools to quantify susceptibility to allelochemical inhibitors in higher plants.     

Illumina-based analysis of the rhizosphere microbial communities associated with healthy and wilted Lanzhou lily (<Emphasis Type="Italic">Lilium davidii</Emphasis> var. <Emphasis Type="Italic">unicolor</Emphasis>) plants grown in the field

Lanzhou lily (Liliumdavidii var. unicolor) is the best edible lily as well as a traditional medicinal plant in China. The microbes associated with plant roots play crucial roles in plant growth and health. However, little is known about the differences of rhizosphere microbes between healthy and wilted Lanzhou lily (Lilium davidii var. unicolor) plants. The objective of this study was to compare the rhizosphere microbial community and functional diversity of healthy and wilted plants, and to identify potential biocontrol agents with significant effect. Paired end Illumina Mi-Seq sequencing of 16S rRNA and ITS gene amplicons was employed to study the bacterial and fungal communities in the rhizosphere soil of Lanzhou lily plants. BIOLOG technology was adopted to investigate the microbial functional diversity. Our results indicated that there were major differences in the rhizosphere microbial composition and functional diversity of wilted samples compared with healthy samples. Healthy Lanzhou lily plants exhibited lower rhizosphere-associated bacterial diversity than diseased plants, whereas fungi exhibited the opposite trend. The dominant phyla in both the healthy and wilted samples were Proteobacteria and Ascomycota, i.e., 34.45 and 64.01 %, respectively. The microbial functional diversity was suppressed in wilted soil samples. Besides Fusarium, the higher relative abundances of Rhizoctonia, Verticillium, Penicillium, and Ilyonectria (Neonectria) in the wilted samples suggest they may pathogenetic root rot fungi. The high relative abundances of Bacillus in Firmicutes in healthy samples may have significant roles as biological control agents against soilborne pathogens. This is the first study to find evidence of major differences between the microbial communities in the rhizospheric soil of healthy and wilted Lanzhou lily, which may be linked to the health status of plants.     

Screening for potential <Emphasis Type="Italic">Striga hermonthica</Emphasis> fungal and bacterial biocontrol agents from suppressive soils in Western Kenya

Striga hermonthica is a hemiparasitic weed that causes huge grain yield losses to small-scale farmers in Africa. Effective biocontrol agents against S. hermonthica can sustainably mitigate these losses. This study characterized the biocontrol potential of culturable fungal and bacterial isolates from S. hermonthica suppressive soils of western Kenya. These isolates were screened for their ability to produce antibiotic compounds and extra cellular enzymes and also their ability to cause S. hermonthica seed decay. Genomic DNA of the selected bacterial and fungal isolates was extracted and partial characterization of 16S rRNA and 18S rRNA genes performed respectively. Analysis show that antibiosis and enzymatic properties of potential biocontrol isolates correlated positively. Isolate KY041696 recorded high antibiosis, enzymatic and seed decay values. This study also revealed that bioactive bacterial isolates belonged to Bacillus, Streptomyces and Rhizobium genera. In this study, no fungal isolate caused S. hermonthica seed decay. This study therefore provides baseline information on the potential biocontrol microbes against S. hermonthica in Western Kenya that could be exploited further in the management of the weed.     

Isolation and functional analysis of apple MdHMGR1 and MdHMGR4 gene promoters in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cereal grains offer great potential as a storage system for production of highly valuable proteins using biotechnological approaches, but such applications require tight temporal and spatial control of transgene expression. Towards this aim, we have undertaken a detailed analysis of α-kafirin (α-kaf) promoter and α-kaf signal peptide (sp) in transgenic sorghum plants, using green fluorescent protein gene (gfp) as a reporter. Constructs containing either the α-kaf promoter or the constitutive maize ubiquitin-1 (ubi) promoter driving either gfp or sp-gfp translational fusion were introduced into Sorghum bicolor inbred line Tx430 by particle bombardment. We show for the first time that the α-kaf promoter directs endosperm-specific transgene expression, with activity first detected at 10 days post-anthesis (dpa), peaking at 20 dpa, and remaining active through to physiological maturity. Furthermore, we demonstrate for the first time that the α-kafirin sp is sufficient to direct foreign protein to protein bodies in the endosperm. The evidence is also provided for possible mis-targeting by α-kaf sp in vegetative tissues of transgenic lines with ubi-sp-gfp, resulting in loss of reporter gene translational activity that no GFP signal was observed. These results demonstrate that α-kaf promoter and α-kaf sp are well suited for seed bioengineering to produce recombinant proteins in sorghum endosperm or deposit foreign proteins into sorghum protein bodies.     

Extracellular targeting of an active endoxylanase by a TolB negative mutant of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis>

Gluconobacter (G.) oxydans strains have great industrial potential due to their ability to incompletely oxidize a wide range of carbohydrates. But there is one major limitation preventing their full production potential. Hydrolysis of polysaccharides is not possible because extracellular hydrolases are not encoded in the genome of Gluconobacter species. Therefore, as a first step for the generation of exoenzyme producing G. oxydans, a leaky outer membrane mutant was created by deleting the TolB encoding gene gox1687. As a second step the xynA gene encoding an endo-1,4-β-xylanase from Bacillus subtilis was expressed in G. oxydans ΔtolB. More than 70 % of the total XynA activity (0.91 mmol h^?1 l culture^?1) was detected in the culture supernatant of the TolB mutant and only 10 % of endoxylanase activity was observed in the supernatant of G. oxydans xynA. These results showed that a G. oxydans strain with an increased substrate spectrum that is able to use the renewable polysaccharide xylan as a substrate to produce the prebiotic compounds xylobiose and xylooligosaccharides was generated. This is the first report about the combination of the process of incomplete oxidation with the degradation of renewable organic materials from plants for the production of value-added products.     

Drastic anthocyanin increase in response to <Emphasis Type="Italic">PAP1</Emphasis> overexpression in <Emphasis Type="Italic">fls1</Emphasis> knockout mutant confers enhanced osmotic stress tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

pap1 - D/fls1ko double mutant plants that produce substantial amounts of anthocyanin show tolerance to abiotic stress.

Abstract

Anthocyanins are flavonoids that are abundant in various plants and have beneficial effects on both plants and humans. Many genes in flavonoid biosynthetic pathways have been identified, including those in the MYB-bHLH-WD40 (MBW) complex. The MYB gene Production of Anthocyanin Pigment 1 (PAP1) plays a particularly important role in anthocyanin accumulation. PAP1 expression in many plant systems strongly increases anthocyanin levels, resulting in a dark purple color in many plant organs. In this study, we generated double mutant plants that harbor fls1ko in the pap1-D background (i.e., pap1-D/fls1ko plants), to examine whether anthocyanins can be further enhanced by blocking flavonol biosynthesis under PAP1 overexpression. We also wanted to examine whether the increased anthocyanin levels contribute to defense against osmotic stresses. The pap1-D/fls1ko mutants accumulated higher anthocyanin levels than pap1-D plants in both control and sucrose-treated conditions. However, flavonoid biosynthesis genes were slightly down-regulated in the pap1-D/fls1ko seedlings as compared to their expression in pap1-D seedlings. We also report the performance of pap1-D/fls1ko seedlings in response to plant osmotic stresses.

     

An improved method of DNA purification from secondary metabolites rich medicinal plants using certain chaotropic agents

The presented work describes good quality DNA isolation method from mature leaves of some medicinally important plant species, viz. Asparagus racemosus, Withania somnifera, Abrus precatorius, Commiphora wightii and Carissa carandas. These plants hold immense medicinal values due to presence of certain secondary metabolites like polyphenols, terpenes, flavonoids, alkaloids, gums, resins, etc. Although these metabolites are accountable for important medicinal properties and authorize these plants to precedence over others, the same compounds disappoint the researcher while isolating high quality DNA. To overcome this problem, we propose a simple method in which DNA is adroitly bounded to diatomaceous earth in a solution of different chaotropic agent and alienated from intrusive compounds. Presented method affirms that secondary products, along with polysaccharides and proteins, can be perceptibly reduced by using silica matrix along with chaotropic agents. The described method is fast, simple and highly reliable for the isolation of DNA from obstinate plant species.     

Phytochemicals and biological activities of <Emphasis Type="Italic">Artemisia sieversiana</Emphasis>

This review discusses a whole plant’s chemical nature and biological effects of Artemisia sieversiana Ehrhart ex willd (ASS). Several types of chemical compositions have been isolated from A. sieversiana, including 26 terpenoids (21 guaiane-type sesquiterpenes, 3 germacrane-type sesquiterpenes, 1 muurolane-type sesquiterpene, and 1 diterpenoid), 16 sesamin-type lignans, 9 flavonoids, 3 steroids, and 3 alkaloids. Some of them have shown promising bioactivities, such as anti-tumour, anti-inflammatory, antioxidant functions and so on. Herein, we have summarized the phytochemical and pharmacological progress of ASS.     

Antibacterial,Anticancer and Neuroprotective Activities of Rare Actinobacteria from Mangrove Forest Soils

Mangrove is a complex ecosystem that contains diverse microbial communities, including rare actinobacteria with great potential to produce bioactive compounds. To date, bioactive compounds extracted from mangrove rare actinobacteria have demonstrated diverse biological activities. The discovery of three novel rare actinobacteria by polyphasic approach, namely Microbacterium mangrovi MUSC 115^T, Sinomonas humi MUSC 117^T and Monashia flava MUSC 78^T from mangrove soils at Tanjung Lumpur, Peninsular Malaysia have led to the screening on antibacterial, anticancer and neuroprotective activities. A total of ten different panels of bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, ATCC 70069, Pseudomonas aeruginosa NRBC 112582 and others were selected for antibacterial screening. Three different neuroprotective models (hypoxia, oxidative stress, dementia) were done using SHSY5Y neuronal cells while two human cancer cells lines, namely human colon cancer cell lines (HT-29) and human cervical carcinoma cell lines (Ca Ski) were utilized for anticancer activity. The result revealed that all extracts exhibited bacteriostatic effects on the bacteria tested. On the other hand, the neuroprotective studies demonstrated M. mangrovi MUSC 115^T extract exhibited significant neuroprotective properties in oxidative stress and dementia model while the extract of strain M. flava MUSC 78^T was able to protect the SHSY5Y neuronal cells in hypoxia model. Furthermore, the extracts of M. mangrovi MUSC 115^T and M. flava MUSC 78^T exhibited anticancer effect against Ca Ski cell line. The chemical analysis of the extracts through GC–MS revealed that the majority of the compounds present in all extracts are heterocyclic organic compound that could explain for the observed bioactivities. Therefore, the results obtained in this study suggested that rare actinobacteria discovered from mangrove environment could be potential sources of antibacterial, anticancer and neuroprotective agents.     

Overexpression of <Emphasis Type="Italic">Zm-HINT1</Emphasis> Confers Salt and Drought Tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Histidine triad nucleotide-binding protein 1 (HINT1) is highly conserved in many species and plays important roles in various biological processes. However, little is known about the responses of HINT1 to abiotic stress in plants. Salt and drought stress are major limiting factors for plant growth and development, and their negative effects on crop productivity may threaten the world’s food supply. Previously, we identified a maize gene, Zm-HINT1, which encodes a 138-amino-acid protein containing conserved domains including the HIT motif, helical regions, and β-strands. Here, we demonstrate that overexpression of Zm-HINT1 in Arabidopsis confers salt and drought tolerance to plants. Zm-HINT1 significantly regulated Na⁺ and K⁺ accumulation in plants under salt stress. The improve tolerance characteristics of Arabidopsis plants that were overexpressing Zm-HINT1 led to increased survival rates after salt and drought treatments. Compared with control plants, those plants that overexpressed Zm-HINT1 showed increased proline content and superoxide dismutase activity, as well as lower malondialdehyde and hydrogen peroxide accumulation under salt and drought treatments. The expression patterns of stress-responsive genes in Arabidopsis plants that overexpressed Zm-HINT1 significantly differed from those in control lines. Taken together, these results suggest that Zm-HINT1 has potential applications in breeding and genetic engineering strategies that are designed to produce new crop varieties with improved salt and drought tolerance.     

The obtainment and characteristics of <Emphasis Type="Italic">Kalanchoe pinnata</Emphasis> L. plants expressing the artificial gene of the cecropin P1 antimicrobial peptide

Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays.     

<Emphasis Type="Italic">Lecanicillium muscarium</Emphasis> and <Emphasis Type="Italic">Adalia bipunctata</Emphasis> combination for the control of black bean aphid<Emphasis Type="Italic">, Aphis fabae</Emphasis>

The predator Adalia bipunctata (Coleoptera: Coccinellidae) and the entomopathogenic fungus Lecanicillium muscarium, have been considered as potential biological control against aphids. While it can be difficult to achieve a high control level of Aphis fabae Scopoli (Hemiptera: Aphididae) using only a single beneficial agent, the research presented here aimed to determine the interaction between L. muscarium and A. bipunctata potential for control against A. fabae. Lecanicillium muscarium was found to cause about 30% mortality in A. bipunctata and with a reduction in feeding by about 15%. However, co-application of A. bipunctata and L. muscarium can cause an addititive effect in reducing aphid populations, resulting in about 90% reduction in aphid populations compared with control treatment. Thus, these two biocontrol agents have the potential to be complementary. This research study demonstrates that it is possible to combine A. bipunctata with L. muscarium to provide a sustainable method for management of A. fabae on broad bean cropping system and that field studies are required.