Isolation and characterization of endophytic fungi having plant growth promotion traits that biosynthesizes bacosides and withanolides under in vitro conditions.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 µg mL-1), Jujubogenin isomer of Bacopasaponin C (65,339 µg mL-1), and Bacopasaponin C (1325 µg mL-1) while Bacopaside II (13,030 µg mL-1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 µg mL-1), and Withanolide B (1024 µg mL-1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.

Trichoderma lixii (IIIM-B4), an endophyte of Bacopa monnieri L. producing peptaibols.

BACKGROUND: Exploration of microbes isolated from north western Himalayas for bioactive natural products. RESULTS: A strain of Trichoderma lixii (IIIM-B4) was isolated from Bacopa monnieri L. The ITS based rDNA gene sequence of strain IIIM-B4 displayed 99% sequence similarity with different Trichoderma harzianum species complex. The highest score was displayed for Hypocrea lixii strain FJ462763 followed by H. nigricans strain NBRC31285, Trichoderma lixii strain CBS 110080, T. afroharzianum strain CBS124620 and Trichoderma guizhouense BPI:GJS 08135 respectively. Position of T. lixii (IIIM-B4) in phylogenetic tree suggested separate identity of the strain. Microbial dynamics of T. lixii (IIIM-B4) was investigated for small peptides. Medium to long chain length peptaibols of 11 residue (Group A), 14 residue (Group B) and 17 residue (Group C) were identified using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometer. Optimization is undeniably a desideratum for maximized production of desirable metabolites from microbial strain. Here optimization studies were carried out on T. lixii (IIIM-B4) using different growth media through Intact Cell Mass Spectrometry (ICMS). A multifold increase was obtained in production of 11 residue peptaibols using rose bengal medium. Out of these, one of them named as Tribacopin AV was isolated and sequenced through mass studied. It was found novel as having unique sequence Ac-Gly-Leu-Leu-Leu-Ala-Leu-Pro-Leu-Aib-Val-Gln-OH. It was found to have antifungal activity against Candida albicans (25 µg/mL MIC). CONCLUSION: In this study, we isolated a strain of T. lixii (IIIM-B4) producing medium and long chain peptaibols. One of them named as Tribacopin AV was found novel as having unique sequence Ac-Gly-Leu-Leu-Leu-Ala-Leu-Pro-Leu-Aib-Val-Gln-OH, which had antifungal properties.

Plant-microbe interactions endorse growth by uplifting microbial community structure of Bacopa monnieri rhizosphere under nematode stress.

The modification of rhizosphere microbial diversity and ecological processes are of rising interest as shifting in microbial community structure impacts the mutual role of host-microbe interactions. Nevertheless, the connection between host-microbial community diversity, their function under biotic stress in addition to their impact on plant performances is poorly understood. The study was designed with the aim to analyze the tripartite interactions among Chitiniphilus sp., Streptomyces sp. and their combination with indigenous rhizospheric microbial population of Bacopa monnieri for enhancing the plant growth and bacoside A content under Meloidogyne incognita stress. Overall, plants treated with the microbial combination recorded enhanced growth as illustrated by significantly higher biomass (2.0 fold), nitrogen uptake (1.8 fold) and bacoside A content (1.3 fold) along with biocontrol efficacy (58.5%) under nematode infected field. The denaturing gradient gel electrophoresis (DGGE) fingerprints of 16S-rDNA revealed that microbial inoculations are major initiators of bacterial community structure in the plant rhizosphere. Additionally, the plants treated with microbial combination showed maximum diversity viz., Shannon's (3.29), Margalef's (4.21), and Simpson's (0.96) indices. Likewise the metabolic profiling data also showed a significant variation among the diversity and evenness indices upon microbial application on the native microflora. We surmise that the application of beneficial microbes in combinational mode not only helped in improving the microbial community structure but also successfully enhanced plant and soil health under biotic stress.

Microbial interference mitigates Meloidogyne incognita mediated oxidative stress and augments bacoside content in Bacopa monnieri L.

Microbial interference plays an imperative role in plant development and response to various stresses. However, its involvement in mitigation of oxidative stress generated by plant parasitic nematode in plants remains elusive. In the present investigation, the efficacy of microbe's viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 single and in combinations was examined to mitigate oxidative stress generated by M. incognita in medicinal plant, Bacopa monnieri. Microbial combination with and without pathogen also enhanced the growth parameters along with secondary metabolites (bacoside) of B. monnieri than the pathogen inoculated control. The study showed that initially the production of hydrogen peroxide (H2O2) was higher in dual microbes infected with pathogen which further declined over M. incognita inoculated control plants. Superoxide dismutase and free radical scavenging activity were also highest in the same treatment which was linearly related with least lipid peroxidation and root gall formation in B. monnieri under the biotic stress. Microscopic visualization of total reactive oxygen species (ROS), H2O2, superoxide radical and programmed cell death in host plant further extended our knowledge and corroborated well with the above findings. Furthermore, scanning electron microscopy confirmed good microbial colonization on the host root surface around nematode penetration sites in plants treated with dual microbes under pathogenic stress. The findings offer novel insight into the mechanism adopted by the synergistic microbial strains in mitigating oxidative stress and simultaneously stimulating bacoside production under pathogenic stress.

Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress.

Plant-associated beneficial microbes have been explored to fulfill the imperative function for plant health. However, their impact on the host secondary metabolite production and nematode disease management remains elusive. Our present work has shown that chitinolytic microbes viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 singly as well as in combination modulated the biosynthetic pathway of bacoside A and systemic defense mechanism against Meloidogyne incognita in Bacopa monnieri. Interestingly, expression of bacoside biosynthetic pathway genes (3-Hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate diphosphate decarboxylase, and squalene synthase) were upregulated in plants treated with the microbial combination in the presence as well as in absence of M. incognita stress. These microbes not only augmented bacoside A production (1.5 fold) but also strengthened host resistance via enhancement in chlorophyll a, defense enzymes and phenolic compounds like gallic acid, syringic acid, ferulic acid and cinnamic acid. Furthermore, elevated lignification and callose deposition in the microbial combination treated plants corroborate well with the above findings. Overall, the results provide novel insights into the underlying mechanisms of priming by beneficial microbes and underscore their capacity to trigger bacoside A production in B. monnieri under biotic stress.

Analysis of propagation of Bacopa monnieri (L.) from hairy roots, elicitation and Bacoside A contents of Ri transformed plants.

Agrobacterium rhizogenes mediated transformation has been experimented in leaf explants of the memory herb Bacopa monnieri in order to assess the regeneration potential of hairy roots (HR) followed by the elicitation of transformed plants for increased Bacoside A production. Out of the four strains tested, A4 and MTCC 532 derived HR exhibited regrowth in MS basal medium while MTCC 2364 derived HR showed regeneration in MS medium supplemented with suitable phyto hormones. R1000 derived HR possessed no regeneration potential. Comparable to A4, MTCC 532 derived HR displayed maximum regrowth frequency of about 85.71 ± 1.84 % with an increase in biomass to threefold. Therefore, five HR plant lines (MTCC 532 derived) were generated and maintained in MS basal liquid medium in which HR3 topped the others in producing a huge biomass of about 67.09 ± 0.66 g FW. PCR amplification and southern hybridization analysis of rol A gene (280 bp) has been performed in order to confirm the transformation process. Moreover, HR3 plant line has accumulated highest total phenolic content of about 165.68 ± 0.82 mg GAE/g DW and highest total flavonoid content of about 497.78 ± 0.57 mg QRE/g DW when compared to other lines and untransformed controls. In addition, HR3 plant extract showed 85.58 ± 0.14 % of DPPH (2, 2-diphenyl-1-picryl hydrazyl) inhibition displaying its reliable anti oxidant potential. Further on elicitation with 10 mg/L chitosan for 2 weeks, HR3 has produced 5.83 % of Bacoside A which is fivefold and threefold increased production when compared to untransformed and transformed unelicited controls respectively. This is the first report on eliciting HR plants for increased metabolite accumulation in B. monnieri.

Surfactin, Iturin, and Fengycin Biosynthesis by Endophytic Bacillus sp. from Bacopa monnieri.

Endophytic microorganisms which are ubiquitously present in plants may colonize intracellularly or intercellularly without causing any diseases. By living within the unique chemical environment of a host plant, they produce a vast array of compounds with a wide range of biological activities. Because of this, natural products of endophytic origin have been exploited for antimicrobial, antiviral, anticancer, and antioxidant properties. Also, they can be considered to function as an efficient microbial barrier to protect plants from various pathogens. In the present study, endophytic bacterium BmB 9 with antifungal and antibacterial activity isolated from the stem tissue of Bacopa monnieri was studied for the molecular and chemical basis of its activity. PCR-based genome mining for various biosynthetic gene clusters proved the presence of surfactin, iturin, and type I polyketide synthase (PKS) genes in the isolate. The LC-MS/MS based analysis of the extract further confirmed the production of surfactin derivatives (M + H(+)-1008.6602, 1022.6755), iturin (M + H(+)-1043.5697), and fengycin (M + H(+)-1491.8195, 1477.8055) by the selected bacterial isolate. The 16S rDNA sequence similarity based analysis identified the isolate BmB 9 as Bacillus sp. with 100 % identity to Bacillus sp. LCF1 (KP257289).

Metabolite and Mechanistic Basis of Antifungal Property Exhibited by Endophytic Bacillus amyloliquefaciens BmB 1.

Plants are ubiquitously colonized by endophytic microorganisms which contribute significantly to plant health through production of plant growth regulators or disease suppression. In the present study, an endophytic bacterial isolate designated as BmB 1 with significant antifungal and plant growth promoting properties was isolated from the stem tissue of Bacopa monnieri (L.) Pennell. The isolate was studied in detail for the molecular and chemical basis of its bioactivity which proved it to have the presence of surfactin, iturin, and type I polyketide synthase (PKS) genes. For the analysis of the chemical basis of antifungal property, extract of the isolate was initially checked for its activity on test pathogens and LC-MS/MS based analysis further confirmed the presence of bacillomycin (m/z (M+H(+)) 1031.8) and surfactin (m/z (M+H(+)) 1008.6 and 1022.6) in the extract prepared. The light microscopic and SEM analysis of the treated and untreated mycelia of the pathogens clearly revealed the hypal destruction caused by the compounds produced by the selected isolate. This confirms the ability of the organism to directly inhibit the growth of the tested pathogens. The GC-MS analysis also confirmed the isolate to have the presence of volatile compounds with the expected role to induce induced systemic resistance (ISR) of the plant. Because of the multitargeted antifungal property, the isolate which was identified as Bacillus amyloliquefaciens can have potential biocontrol applications.

Cytotoxic and antimicrobial activities of endophytic fungi isolated from Bacopa monnieri (L.) Pennell (Scrophulariaceae).

BACKGROUND: Endophytes, which reside in plant tissues, have the potential to produce novel metabolites with immense benefits for health industry. Cytotoxic and antimicrobial activities of endophytic fungi isolated from Bacopa monnieri (L.) Pennell were investigated. METHODS: Endophytic fungi were isolated from the Bacopa monnieri. Extracts from liquid cultures were tested for cytotoxicity against a number of cancer cell lines using the MTT assay. Antimicrobial activity was determined using the micro dilution method. RESULTS: 22% of the examined extracts showed potent (IC50 of <20 µg/ml) cytotoxic activity against HCT-116 cell line. 5.5%, 11%, 11% of the extracts were found to be cytotoxic for MCF-7, PC-3, and A-549 cell lines respectively. 33% extracts displayed antimicrobial activity against at least one test organism with MIC value 10-100 µg/ml. The isolate B9_Pink showed the most potent cytotoxic activity for all the cell lines examined and maximum antimicrobial activity against the four pathogens examined which was followed by B19. CONCLUSIONS: Results indicated the potential for production of bioactive agents from endophytes of Bacopa monnieri.

Root endophyte Piriformospora indica DSM 11827 alters plant morphology, enhances biomass and antioxidant activity of medicinal plant Bacopa monniera.

Unorganized collections and over exploitation of naturally occurring medicinal plant Bacopa monniera is leading to rapid depletion of germplasm and is posing a great threat to its survival in natural habitats. The species has already been listed in the list of highly threatened plants of India. This calls for micropropagation based multiplication of potential accessions and understanding of their mycorrhizal associations for obtaining plants with enhanced secondary metabolite contents. The co-cultivation of B. monniera with axenically cultivated root endophyte Piriformospora indica resulted in growth promotion, increase in bacoside content, antioxidant activity and nuclear hypertrophy of this medicinal plant.

Exiguobacterium oxidotolerans, a halotolerant plant growth promoting rhizobacteria, improves yield and content of secondary metabolites in Bacopa monnieri (L.) Pennell under primary and secondary salt stress.

Brahmi (Bacopa monnieri), an integral component of Indian Ayurvedic medicine system, is facing a threat of extinction owing to the depletion of its natural populations. The present study investigates the prospective of exploitation of halotolerant plant growth promoting rhizobacteria (PGPR) in utilising the salt stressed soils for cultivation of B. monnieri. The effects of two salt tolerant PGPR, Bacillus pumilus (STR2) and Exiguobacterium oxidotolerans (STR36) on the growth and content of bacoside-A, an important pharmaceutical compound in B. monnieri, were investigated under primary and secondary salinity conditions. The herb yields of un-inoculated plants decreased by 48 % under secondary salinization and 60 % under primary salinization than the non salinised plants. Among the rhizobacteria treated plants, E. oxidotolerans recorded 109 and 138 %, higher herb yield than non-inoculated plants subjected to primary and secondary salinity respectively. E. oxidotolerans inoculated plants recorded 36 and 76 % higher bacoside-A content under primary and secondary salinity respectively. Higher levels of proline content and considerably lower levels of lipid peroxidation were noticed when the plants were inoculated with PGPR under all salinity regimes. From the results of this investigation, it can be concluded that, the treatments with salt tolerant PGPR can be a useful strategy in the enhancement of biomass yield and saponin contents in B. monnieri, as besides being an eco-friendly approach; it can also be instrumental in cultivation of B. monnieri in salt stressed environments.