An environmentally benign process to synthesize vanillin and other substituted phenyl aldehydes using natural phenylpropenes.

The limited vanillin (3a) production from plant sources requires identifying some renewable and sustainable approaches for its synthesis. This study aimed to develop an efficient, eco-friendly process for synthesizing vanillin (3a) from eugenol (1a) and eugenol-rich essential oils. The chemical methodology for vanillin (3a) synthesis involved base-mediated isomerization of eugenol (1a) to isoeugenol (2a), followed by OsO4/NaIO4 mediated oxidation of isoeugenol to vanillin (3a) using different additives such 1,4-diazabicyclo[2.2.2]octane (DABCO) and substituted pyridines in reusable environment-friendly solvents. Use of 2,6-dimethylpyridine and 2,6-dimethylpyridine N-oxide as additives in the oxidation step offered a significantly higher product yield (vanillin 3a, 70 %). The process synthesized vanillin (3a) irrespective of the cis/ trans stereochemistry of isoeugenol (2a). The peculiarity of the method relates to converting eugenol (1a) to vanillin (3a) without phenolic group protection, which offers step economy. Besides efficient vanillin (3a) synthesis, the process's general implications involve converting other naturally occurring phenylpropenes or phenylpropenes-enriched oils to the corresponding phenyl aldehydes (59-82 % yield).

A sustainable bioprocess technology for producing food-flavour (+)-γ-decalactone from castor oil-derived ricinoleic acid using enzymatic activity of Candida parapsilosis: Scale-up optimization and purification using novel composite.

Ricinoleic acid (RA) from castor oil was employed in biotransformation of peach-flavoured γ-decalactone (GDL), using a Candida parapsilosis strain (MTCC13027) which was isolated from waste of pineapple crown base. Using four variables-pH, cell density, amount of RA, and temperature-the biotransformation parameters were optimized using RSM and BBD. Under optimized conditions (pH 6, 10 % of microbial cells, 10 g/L RA at 28°C), the conversion was maximum and resulted to 80 % (+)-GDL (4.4 g/L/120 h) yield in shake flask (500 mL). Furthermore, optimization was achieved by adjusting the aeration and agitation parameters in a 3 L bioreactor, which were then replicated in a 10 L bioreactor to accurately determine the amount of (+)-GDL. In bioreactor condition, 4.7 g/L (>85 %) of (+)-GDL is produced with 20 % and 40 % dissolved oxygen (1.0 vvm) at 150 rpm in 72 h and 66 h, respectively. Further, a new Al-Mg-Ca-Si composite column-chromatography method is developed to purify enantiospecific (+)-GDL (99.9 %). This (+)-GDL is 100 % nature-identical as validated through 14C-radio-carbon dating. Thorough chemical investigation of enantiospecific (+)-GDL is authenticated for its use as flavour. This bioflavour has been developed through a cost-effective biotechnological process in response to the demand from the food industry on commercial scale.

Temperature-induced lipocalin-mediated membrane integrity: Possible implications for vindoline accumulation in Catharanthus roseus leaves.

Plant lipocalins perform diverse functions. Recently, allene oxide cyclase, a lipocalin family member, has been shown to co-express with vindoline pathway genes in Catharanthus roseus under various biotic/abiotic stresses. This brought focus to another family member, a temperature-induced lipocalin (CrTIL), which was selected for full-length cloning, tissue-specific expression profiling, in silico characterization, and upstream genomic region analysis for cis-regulatory elements. Stress-mediated variations in CrTIL expression were reflected as disturbances in cell membrane integrity, assayed through measurement of electrolyte leakage and lipid peroxidation product, MDA, which implicated the role of CrTIL in maintaining cell membrane integrity. For ascertaining the function of CrTIL in maintaining membrane stability and elucidating the relationship between CrTIL expression and vindoline content, if any, a direct approach was adopted, whereby CrTIL was transiently silenced and overexpressed in C. roseus. CrTIL silencing and overexpression confirmed its role in the maintenance of membrane integrity and indicated an inverse relationship of its expression with vindoline content. GFP fusion-based subcellular localization indicated membrane localization of CrTIL, which was in agreement with its role in maintaining membrane integrity. Altogether, the role of CrTIL in maintaining membrane structure has possible implications for the intracellular sequestration, storage, and viability of vindoline.

Role of ACC-deaminase synthesizing Trichoderma harzianum and plant growth-promoting bacteria in reducing salt-stress in Ocimum sanctum.

Salinity is a significant concern in crop production, causing severe losses in agricultural yields. Ocimum sanctum, also known as Holy Basil, is an important ancient medicinal plant used in the Indian traditional system of medicine. The present study explores the use of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing strains of plant-growth-promoting bacteria (PGPB) namely Str-8 (Halomonas desiderata), Sd-6 (Brevibacterium halotolerans), Fd-2 (Achromobacter xylosoxidans), Art-7 (Burkholderia cepacia), and Ldr-2 (Bacillus subtilis), and T. harzianum (Th), possessing multi-functional properties like growth promotion, stress alleviation, and for enhancing O. sanctum yield under salt stress. The results showed that co-inoculation of Th and PGPBs enhanced plant height and fresh herb weight by 3.78-17.65% and 7.86-58.76%, respectively; highest being in Th + Fd-2 and Th + Art-7 compared to positive control plants. The doubly inoculated plants showed increased pigments, phenol, flavonoids, protein, sugar, relative water content, and nutrient uptake (Nitrogen and Phosphorous) as compared to monocultures and untreated positive control plants. In addition, co-inoculation in plants resulted in lower Na+, MDA, H2O2, CAT, APX activities, and also lower ACC accumulation (49.75 to 72.38% compared to non-treated salt- stressed plant) in O. sanctum, which probably played a significant role in minimizing the deleterious effects of salinity. Finally, multifactorial analysis showed that co-inoculation of Th and PGPBs improved O. sanctum growth, its physiological activities, and alleviated salt stress compared to single inoculated and positive control plants. These microbial consortia were evaluated for the first time on O. sanctum under salt stress. Therefore, the microbial consortia application could be employed to boost crop productivity in poor, marginalized and stressed agricultural fields. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01328-2.

Corrigendum: Calliterpenone, a natural plant growth promoter from a medicinal plant Callicarpa macrophylla, sustainably enhances the yield and productivity of crops.

[This corrects the article DOI: 10.3389/fpls.2022.960717.].

Towards the development of phytoextract based healthy ageing cognitive booster formulation, explored through Caenorhabditis elegans model.

The positive effect of herbal supplements on aging and age-related disorders has led to the evolution of natural curatives for remedial neurodegenerative diseases in humans. The advancement in aging is exceedingly linked to oxidative stress. Enhanced oxidative stress interrupts health of humans in various ways, necessitating to find stress alleviating herbal resources. Currently, minimal scientifically validated health and cognitive booster resources are available. Therefore, we explored the impact of plant extracts in different combinations on oxidative stress, life span and cognition using the multicellular transgenic humanized C. elegans, and further validated the same in Mus musculus, besides testing their safety and toxicity. In our investigations, the final product-the HACBF (healthy ageing cognitive booster formulation) thus developed was found to reduce major aging biomarkers like lipofuscin, protein carbonyl, lipid levels and enhanced activity of antioxidant enzymes. Further confirmation was done using transgenic worms and RT-PCR. The cognitive boosting activities analyzed in C. elegans and M. musculus model system were found to be at par with donepezil and L-dopa, the two drugs which are commonly used to treat Parkinson's and Alzheimer's diseases. In the transgenic C. elegans model system, the HACBF exhibited reduced aggregation of misfolded disease proteins α-synuclein and increased the health of nicotinic acetylcholine receptor, levels of Acetylcholine and Dopamine contents respectively, the major neurotransmitters responsible for memory, language, learning behavior and movement. Molecular studies clearly indicate that HACBF upregulated major genes responsible for healthy aging and cognitive booster activities in C. elegans and as well as in M. musculus. As such, the present herbal product thus developed may be quite useful for healthy aging and cognitive boosting activities, and more so during this covid-19 pandemic. Supplementary Information: The online version contains supplementary material available at 10.1007/s13237-022-00407-1.

Perspectives and potential applications of endophytic microorganisms in cultivation of medicinal and aromatic plants.

Ensuring food and nutritional security, it is crucial to use chemicals in agriculture to boost yields and protect the crops against biotic and abiotic perturbations. Conversely, excessive use of chemicals has led to many deleterious effects on the environment like pollution of soil, water, and air; loss of soil fertility; and development of pest resistance, and is now posing serious threats to biodiversity. Therefore, farming systems need to be upgraded towards the use of biological agents to retain agricultural and environmental sustainability. Plants exhibit a huge and varied niche for endophytic microorganisms inside the planta, resulting in a closer association between them. Endophytic microorganisms play pivotal roles in plant physiological and morphological characteristics, including growth promotion, survival, and fitness. Their mechanism of action includes both direct and indirect, such as mineral phosphate solubilization, fixating nitrogen, synthesis of auxins, production of siderophore, and various phytohormones. Medicinal and aromatic plants (MAPs) hold a crucial position worldwide for their valued essential oils and several phytopharmaceutically important bioactive compounds since ancient times; conversely, owing to the high demand for natural products, commercial cultivation of MAPs is on the upswing. Furthermore, the vulnerability to various pests and diseases enforces noteworthy production restraints that affect both crop yield and quality. Efforts have been made towards enhancing yields of plant crude drugs by improving crop varieties, cell cultures, transgenic plants, etc., but these are highly cost-demanding and time-consuming measures. Thus, it is essential to evolve efficient, eco-friendly, cost-effective simpler approaches for improvement in the yield and health of the plants. Harnessing endophytic microorganisms as biostimulants can be an effective and alternative step. This review summarizes the concept of endophytes, their multidimensional interaction inside the host plant, and the salient benefits associated with endophytic microorganisms in MAPs.

Calliterpenone, a natural plant growth promoter from a medicinal plant Callicarpa macrophylla, sustainably enhances the yield and productivity of crops.

The global population is rising at an alarming rate, which is threatening food and nutritional security. Although chemical fertilizers and pesticides are important for achieving food security, their excessive usage critically affects soil health and adds up residues in the food chain. There is an increasing interest in identifying eco-friendly farm inputs that can improve crop productivity through sustainable agricultural practices. One of the most common approaches to reducing chemical inputs in agriculture is the use of plant growth regulators (PGRs). Here, we demonstrate the benefits of a natural and novel plant growth enhancer "calliterpenone," isolated from Callicarpa macrophylla, a medicinal plant, for increasing crop productivity in six crops, viz., rice, wheat, potato, tomato, chickpea, and onion. Results revealed that the application of calliterpenone (foliar spraying or seed soaking) enhanced the yield of rice (28.89%), onion (20.63%), potato (37.17%), tomato (28.36%), and chickpea (26.08%) at 0.001 mM and of wheat (27.23%) at 0.01 mM concentrations in comparison to control. This enhancement in yield was reflected through improvements in its growth attributes, viz., spike length, tillers plant-1, seeds spike-1, plant height, and biomass. Furthermore, the exogenous application of calliterpenone could increase the endogenous level of indole-3-acetic acid (IAA) in all tested crops and decrease the content of abscisic acid (ABA) in a few. Trials conducted at farmers' fields showed an overall ~12% increase in rice yield (mean of 11 farmers' fields ranging from 3.48 to 19.63%) and ~10% increase in wheat yield (ranging from 3.91 to 17.51%). The 0.001 mM of calliterpenone was the best effective dose for most crops except wheat, where a concentration of 0.01 mM was found to be the most optimal. This study indicates that calliterpenone is a natural plant growth promoter that can be used in boosting the yields of multiple crops and would be an important input component of organic farming.

Stress responsiveness of vindoline accumulation in Catharanthus roseus leaves is mediated through co-expression of allene oxide cyclase with pathway genes.

Vindoline is an important alkaloid produced in Catharanthus roseus leaves. It is the more important monomer of the scarce and costly anticancer bisindole alkaloids, vincristine, and vinblastine, as unlike catharanthine (the other monomer), its biosynthesis is restricted to the leaves. Here, biotic (bacterial endophyte, phytoplasma, virus) and abiotic (temperature, salinity, SA, MeJa) factors were studied for their effect on vindoline accumulation in C. roseus. Variations in vindoline pathway-related gene expression were reflected in changes in vindoline content. Since allene oxide cyclase (CrAOC) is involved in jasmonate biosynthesis and MeJa modulates many vindoline pathway genes, the correlation between CrAOC expression and vindoline content was studied. It was taken up for full-length cloning, tissue-specific expression profiling, in silico analyses, and upstream genomic region analysis for cis-regulatory elements. Co-expression analysis of CrAOC with vindoline metabolism-related genes under the influence of aforementioned abiotic/biotic factors indicated its stronger direct correlation with the tabersonine-to-vindoline genes (t16h, omt, t3o, t3r, nmt, d4h, dat) as compared to the pre-tabersonine genes (tdc, str, sgd). Its expression was inversely related to that of downstream-acting peroxidase (prx) (except under temperature stress). Direct/positive relationship of CrAOC expression with vindoline content established it as a key gene modulating vindoline accumulation in C. roseus.

Molecular insights into enhanced resistance of Papaver somniferum against downy mildew by application of endophyte bacteria Microbacterium sp. SMR1.

Downy mildew is one of the most serious diseases of Papaver somniferum. Endophytes isolated from different parts of P. somniferum were screened for their ability to enhance resistance against downy mildew caused by the obligate biotrophic oomycete Peronospora meconopsidis. Two endophytes (SMR1 and SMR2) reduced the downy mildew on three P. somniferum genotypes (Sampada, J-16, and I-14). SMR1 (Microbacterium sp.) also enhanced the resistance of P. somniferum against downy mildew under field conditions. The biochemical markers of plant susceptibility under biotic stresses (proline and malondialdehyde) were found to be reduced in P. somniferum upon SMR1 treatment. To understand the mechanisms underlying the enhanced resistance to downy mildew in SMR1 endophyte-treated P. somniferum genotype J-16, we compared the expression profiles using the next-generation RNA sequencing approach between P. somniferum pretreated with SMR1 and untreated endophyte-free control plants following exposure to downy mildew pathogen. Comparative transcriptome analysis revealed differential expression of transcripts belonging to broad classes of signal transduction, protein modification, disease/defense proteins, transcription factors, and phytohormones in SMR1-primed P. somniferum after infection with downy mildew pathogen. Furthermore, enhanced salicylic acid content was observed in SMR1-primed P. somniferum after exposure to downy mildew pathogen. This study sheds light on molecular mechanisms underlying enhanced resistance to downy mildew in SMR1-primed P. somniferum. Finally, we propose that the SA-dependent defense pathway, the hallmark of systemic acquired resistance, is activated in SMR1-primed P. somniferum, triggering the endophyte-induced resistance.

Endophytic bacterium CIMAP-A7 mediated amelioration of atrazine induced phyto-toxicity in Andrographis paniculata.

The presence of atrazine, a triazine herbicide, and its residues in agriculture soil poses a serious threat to human health and environment through accumulation in edible plant parts. Hence, the present study focused on atrazine induced stress amelioration of Andrographis paniculata, an important medicinal plant, by a plant growth promoting and atrazine degrading endophytic bacterium CIMAP-A7 inoculation. Atrazine has a non-significant effect at a lower dose while at a higher dose (lower: 25 and higher: 50 mg kg-1) 22 and 36% decrease in secondary metabolite content and plant dry weight of A. paniculata was recorded, respectively. Endophyte CIMAP-A7 inoculation significantly reduced atrazine soil content, by 78 and 51% at lower and a higher doses respectively, than their respective control treatments. Inoculation of CIMAP-A7 exhibited better plant growth in terms of increased total chlorophyll, carotenoid, protein, and metabolite content with reduced atrazine content under both atrazine contaminated and un-contaminated treatments. Atrazine induced oxidative stress in A. paniculata was also ameliorated by CIMAP-A7 by reducing stress enzymes, proline, and malondialdehyde accumulation under contaminated soil conditions than un-inoculated treatments. Furthermore, the presence of atrazine metabolites deisopropylatrazine (DIA) and desethylatrazine (DEA) strongly suggests a role of CIMAP-A7 in mineralization however, the absence of these metabolites in uninoculated soil and all plant samples were recorded. These findings advocate that the amelioration of atrazine induced stress with no/least pesticide content in plant tissues by plant-endophyte co-interactions would be efficient in the remediation of atrazine contaminated soils and ensure safe crop produce.

The Bioactive Potential of Culturable Fungal Endophytes Isolated From the Leaf of Catharanthus roseus (L.) G. Don.

INTRODUCTION: Endophyte is considered a source of natural bioactive secondary metabolites that provides an array of bioactive lead compounds. The present study was aimed to determine the antimicrobial and anti-inflammatory potential of fungal endophytes isolated from Catharanthus roseus. METHODS: A total of seven fungal endophytes crude extract were screened against bacterial pathogens. Of these, Curvularia geniculata CATDLF7 crude extract exhibited the most potent inhibitory activity against bacterial pathogens. Hence, CATDLF7 crude extract was subjected to chromatographic separation. This purification leads to the isolation of six pure compounds (1PS - 6PS). Of these, 3PS was found to be a major constituent and most effective against clinical isolates of methicillin- resistant Staphylococcus aureus (MRSA) with minimum inhibitory concentration (MIC) values ranging from 100 to 200 µg/ml. Based on the spectroscopic data, 3PS was characterized as α,ß- dehydrocurvularin. This compound also showed synergistic interaction with norfloxacin and reduced its MIC up to 32-folds with a fractional inhibitory concentration index (FICI) of 0.09. RESULTS: To understand the possible antibacterial mechanism of action, α,ß-dehydrocurvularin alone (100 µg/ml) exhibited efflux pump inhibitory potential by 0.84 fold decreasing in ethidium bromide (EtBr) fluorescence. In addition, α,ß-dehydrocurvularin inhibited inflammatory cytokines TNF-α and IL-6 production, which is further validated by molecular docking scores -4.921 and -5.641, respectively, for understanding orientation and binding affinity. CONCLUSION: Overall, the results highlighted identifying bioactive compound α,ß-dehydrocurvularin, which could be used as an antimicrobial and anti-inflammatory agent.

Bioremediation of arsenic by soil methylating fungi: Role of Humicola sp. strain 2WS1 in amelioration of arsenic phytotoxicity in Bacopa monnieri L.

Fungi mediated arsenic (As) stress modulation has emerged as an important strategy for the mitigation of As mediated stress management in plants for reducing As contamination to the food chain. In the present study, total of 45 fungal strains were isolated from the three As contaminated sites of West Bengal, India. These strains were morphologically different and inhibited variable As tolerance (10 to 5000 mg l-1As). Total 21 fungal isolates, tolerant up to 5000 mg l-1 AsV, were investigated for As removal (10 mg l-1 As) after 21 d of cultivation under laboratory conditions. The As bioaccumulation in fungal biomass ranged between 0.146 to 11.36 g kg-1 biomass. Range of volatilized As was between 0.05 to 53.39 mg kg-1 biomass. Most promising bioaccumulation and biovolatilization potential were observed in strains viz., 2WS1, 3WS1 and 2WS9. Strain 2WS1 showed highest As biovolatilization (53.39 mg kg-1 biomass) and was identified as Humicola sp. using ITS/5.8S rDNA gene sequencing. This is the first report of Humicola sp. having As biomethylation property. Best first 8 As biomethylating fungal strains were further tested for their As remediation and PGP potential in Bacopa monnieri plant grown in As contaminated soil (20 mg kg-1) in a pot experiment under greenhouse conditions. The highest leaf stem ratio and lowest As content in leaf tissues were observed in 2WS1 inoculated Bacopa monnieri plants. The presence of arsM gene in 2WS1 strain suggests As biovolatilization as possible bioremediation and As stress mitigation strategy of 2WS1. Therefore, application of this strain of Humicola sp. strain 2WS1 in As contaminated soils could be a potential and realistic mitigation strategy for reducing As contamination to cropping system coupled with enhanced productivity.

Vulnerability of Soil Microbiome to Monocropping of Medicinal and Aromatic Plants and Its Restoration Through Intercropping and Organic Amendments.

Cultivation of medicinal and aromatic plants (MAPs) is persistently increasing due to excessive demands of naturals. Agricultural land and its microbial diversity are primarily adapted to conventional crops, and introduction of MAP and their continuous monocropping may disturb the ecological stability of soil microbiome. Here, the effect of cultivation of MAPs on soil microbial diversity was studied. The aim of the study is to examine the effects of cultivation of MAPs on the possible shift in soil microbial diversity and to restore such impacts by using organic amendments or intercropping. Terminal restriction fragments polymorphism (TRFLP) and next-generation sequencing (NGS) studies showed that of the various selected MAPs, maximal modulation in the soil microbial diversity patterns was noticed in fields of Mentha arvensis and Artemisia annua, and the traces of essential oil/phytochemicals were detected in bulk and rhizospheric soil. In both Artemisia- and Mentha-cultivated soil, the total operating taxonomic unit (OTU) declined in both bulk and rhizospheric soil in comparison to control (Zea mays), but the bacterial richness of Mentha soil was slightly higher than that of control. However, cultivation of Mentha improved the evenness of the microbial community. The inclusion of crops like Sesbania and Chlorophytum and the application of vermicompost (VC) enhanced the microbial richness and evenness, thereby restoring the soil microbial state shift and resulting in higher productivity in the continuously Mentha cropped field. Our study concludes that long-term cultivation of some MAPs may affect the richness but promote the evenness of microbial diversity. The state shift could be restored to some extent, and crop productivity could be enhanced by the inclusion of selected crops and organic manures in cropping systems.

Endophytic Consortium With Diverse Gene-Regulating Capabilities of Benzylisoquinoline Alkaloids Biosynthetic Pathway Can Enhance Endogenous Morphine Biosynthesis in Papaver somniferum.

Secondary metabolite biosynthesis in medicinal plants is multi-step cascade known to be modulated by associated endophytes. While a single endophyte is not able to upregulate all biosynthetic steps, limiting maximum yield achievement. Therefore to compliment the deficient characteristics in an endophyte we tried consortium of endophytes to achieve maximum yield. Here, efforts were made to maximize the in planta morphine yield, using consortium of two endophytes; SM1B (Acinetobacter sp.) upregulating most of the genes of morphine biosynthesis except T6ODM and CODM, and SM3B (Marmoricola sp.) upregulating T6ODM and CODM in alkaloid-less Papaver somniferum cv. Sujata. Consortium-inoculation significantly increased morphine and thebaine content, and also increased the photosynthetic efficiency of poppy plants resulted in increased biomass, capsule weight, and seed yields compared to single-inoculation. The increment in morphine content was due to the modulation of metabolic-flow of key intermediates including reticuline and thebaine, via upregulating pertinent biosynthetic genes and enhanced expression of COR, key gene for morphine biosynthesis. This is the first report demonstrating the endophytic-consortium complimenting the functional deficiency of one endophyte by another for upregulating multiple genes of a metabolic pathway similar to transgenics (overexpressing multiple genes) for obtaining enhanced yield of pharmaceutically important metabolites.

Application of essential oils as a natural and alternate method for inhibiting and inducing the sprouting of potato tubers.

Use of harmful chemicals and expensive maintenance of cold-storage conditions for controlling sprouting are among the major problems in potato storage. Here, 20 essential oils (EOs) were tested for their sprouting-inhibiting and sprouting-inducing activities. Overall, treatments of lemon grass (LG) and clove (CL) oils could induce sprouting whereas palmarosa (PR) and ajwain (AZ) oils could inhibit sprouting of potato tubers at normal-room-temperature (25 ±â€¯2 °C) storage. Selected-EOs treatments affected sprouting by modulation of accumulation of reducing sugars, ethylene, and expression of genes involved in tuber-sprouting such as ARF, ARP, AIP and ERF. Surprisingly, 7-days AZ-treatments could inhibit sprouting for 30-days which was mediated via damaging apical meristem. However, LG- and CL-treated tubers could produce enhanced potato yield as well. Present work clearly demonstrates that selected-EOs can be used as a promising eco-friendly approach for inducing/inhibiting sprouting of potato tubers during potato storage and those enhancing sprouting can be used for enhancing productivity.

Innate endophytic fungus, Aspergillus terreus as biotic elicitor of withanolide A in root cell suspension cultures of Withania somnifera.

In the present study, root cell suspension cultures of W. somnifera were elicited with mycelial extract (1% w/v) and culture filtrate (5% v/v) of their native endophytic fungus Aspergillus terreus 2aWF in shake flask. Culture filtrate of A. terreus 2aWF significantly elicits withanolide A at 6H (12.20 ± 0.52 µg/g FCB). However, with A. terreus 2aWF mycelial extract, withanolide A content was higher at 24H (10.29 µg/g FCB). Withanolide A content was maximum with salicylic acid (0.1 mM) treatment at 24H (8.3 ± 0.20 µg/g FCB). Further, expression analysis of withanolide pathway genes, hydrogen peroxide production, and lipid peroxidation was carried out after 48H of elicitation with 2aWF mycelial extract and culture filtrate. The expression levels of withanolides biosynthetic pathway genes, viz. HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were quantified by real time PCR at 48H of elicitation. In all the treatments, the expression levels of key genes were significantly upregulated as compared to untreated suspension cells. Hydrogen peroxide was noticeably enhanced in SA, mycelia extract and culture filtrate, at 20% (115 ± 4.40 nM/g FCB), 42% (137.5 ± 3.62 nM/g FCB), and 27% (122.8 ± 1.25 nM/g FCB) respectively; however, lipid peroxidation was 0.288 ± 0.014, 0.305 ± 0.041 and 0.253 ± 0.007 (µM/gm FCB) respectively, higher than the control (0.201 ± 0.007 µM/gm FCB).

Fungal endophytes attune withanolide biosynthesis in Withania somnifera, prime to enhanced withanolide A content in leaves and roots.

Endophytes have been reported from all plant species from different parts of tissue including root, stem and leaves. Here we report, three fungal endophytes, Aspergillus terreus strain 2aWF (2aWF), Penicillium oxalicum strain 5aWF (5aWF), and Sarocladium kiliense strain 10aWF (10aWF) from Withania somnifera, which could enhance withanolides content in leaf and root. Upon treatment with the above endophytes to 4 weeks old plants in field conditions, W. somnifera elicited withanolide A content (97 to 100%) in leaves without considerable changes in withaferin A content. Furthermore, withanolide A content in roots of 5aWF and 10aWF endophyte treated W. somnifera plants increased up to 52% and 65% respectively. Incidentally, expression profile of withanolide and sterol biosynthetic pathway genes HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were significantly upregulated in 2aWF, 5aWF and 10aWF fungal endophyte treated plants. Besides, modulation of withanolide biosynthetic pathway genes, fungal endophytes also induce a host resistant related gene, NPR1 resulting in 2, 4 and 16 fold expression levels in 2aWF, 10aWF and 5aWF endophyte treatments respectively, compared to control plants. Overall, our results illustrate that application of native-fungal endophytes 2aWF (96.60%), 5aWF (95%) and 10aWF (147%) enhances plant biomass in addition to withanolide content.

Halotolerant Exiguobacterium profundum PHM11 Tolerate Salinity by Accumulating L-Proline and Fine-Tuning Gene Expression Profiles of Related Metabolic Pathways.

Salinity stress is one of the serious factors, limiting production of major agricultural crops; especially, in sodic soils. A number of approaches are being applied to mitigate the salt-induced adverse effects in agricultural crops through implying different halotolerant microbes. In this aspect, a halotolerant, Exiguobacterium profundum PHM11 was evaluated under eight different salinity regimes; 100, 250, 500, 1000, 1500, 2000, 2500, and 3000 mM to know its inherent salt tolerance limits and salt-induced consequences affecting its natural metabolism. Based on the stoichiometric growth kinetics; 100 and 1500 mM concentrations were selected as optimal and minimal performance limits for PHM11. To know, how salt stress affects the expression profiles of regulatory genes of its key metabolic pathways, and total production of important metabolites; biomass, carotenoids, beta-carotene production, IAA and proline contents, and expression profiles of key genes affecting the protein folding, structural adaptations, transportation across the cell membrane, stress tolerance, carotenoids, IAA and mannitol production in PHM11 were studied under 100 and 1500 mM salinity. E. profundum PHM11 showed maximum and minimum growth, biomass and metabolite production at 100 and 1500 mM salinity respectively. Salt-induced fine-tuning of expression profiles of key genes of stress pathways was determined in halotolerant bacterium PHM11.

Endophytes of Withania somnifera modulate in planta content and the site of withanolide biosynthesis.

Tissue specific biosynthesis of secondary metabolites is a distinguished feature of medicinal plants. Withania somnifera, source of pharmaceutically important withanolides biosynthesizes withaferin-A in leaves and withanolide-A in roots. To increase the in planta withanolides production, a sustainable approach needs to be explored. Here, we isolated endophytes from different parts of W. somnifera plants and their promising role in in planta withanolide biosynthesis was established in both in-vivo grown as well in in-vitro raised composite W. somnifera plants. Overall, the fungal endophytes improved photosynthesis, plant growth and biomass, and the root-associated bacterial endophytes enhanced the withanolide content in both in-vivo and in-vitro grown plants by modulating the expression of withanolide biosynthesis genes in leaves and roots. Surprisingly, a few indole-3-acetic acid (IAA)-producing and nitrogen-fixing root-associated endophytes could induce the biosynthesis of withaferin-A in roots by inducing in planta IAA-production and upregulating the expression of withanolide biosynthesis genes especially MEP-pathway genes (DXS and DXR) in roots as well. Results indicate the role of endophytes in modulating the synthesis and site of withanolides production and the selected endophytes can be used for enhancing the in planta withanolide production and enriching roots with pharmaceutically important withaferin-A which is generally absent in roots.