Effects of methyl jasmonate on the growth and triterpenoid production of diploid and tetraploid Centella asiatica (L.) Urb. hairy root cultures.

In this study, the effects of methyl jasmonate (MeJA) on the phytomass and triterpenoid production of diploid and tetraploid Centella asiatica hairy roots were investigated. Hairy root cultures were obtained from diploid and induced tetraploid plants of C. asiatica infected by Agrobacterium rhizogenes strain ATCC 43057. MeJA triggered triterpenoid production in both ploidy hairy roots, whereas triterpenoids were not produced in the untreated hairy roots. Among the treatments, the 50 µM MeJA treatment yielded the maximum triterpenoid production in diploid hairy roots of 27.25 ± 0.27 µg/mg Dry weight (DW) total triterpenoid at day 21. For the tetraploid hairy root cultures, the 28th-day hairy root culture produced a maximum amount of triterpenoids of 16.29 ± 6.32 µg/mg DW in response to the 50 µM MeJA treatment, whereas the 100 µM MeJA treatment produced a similar triterpenoid amount (16.31 ± 9.24 µg/mg DW) at day 14. Moreover, in response to 50 µM MeJA, we obtained different ratios of aglycone to glycoside, i.e., 1:7 and 1:2, between the diploid and tetraploid hairy root cultures. Asiaticoside was the dominant phytochemical, followed by asiatic acid and madecassic acid. This study provides valuable information for producing triterpenoids for C. asiatica commercial products and preparations by using hairy root cultures.

Optimization of Direct Blue-14 dye degradation by Bacillus fermus (Kx898362) an alkaliphilic plant endophyte and assessment of degraded metabolite toxicity.

Alkaliphilic bacteria possesses the ability to survive in the extreme conditions with high salt concentrations. The adaptability of alkaliphilic bacteria to extreme conditions has made them predominant degrader in the field of biodegradation. A moderately alkaliphilic endophyte was isolated from Centella asiatica with a potential to degrade a di-azo dye Direct Blue-14(DB-14). The isolate was identified as Bacillus fermus with 97% similarity strain Xmb064. On optimization, maximum of 92.76% biodegradation was attained with dye concentration at 68.78 ppm supplemented with 1 g of sucrose and 2.5% (v/v) of inoculum for 72 h incubation. Characterization of the biodegraded product carried out using UV-vis spectrophotometry, FT-IR and LC-MS confirmed the destabilization of di-azo bond followed with the degradation of DB-14. Cytogenotoxicity studies revealed the biodegraded products to be less toxic. The current study is the first report on the optimization, biotransformation and cytogenotoxicity of DB-14 by B. fermus strain Centella.

Determination and quantification of asiaticoside in endophytic fungus from Centella asiatica (L.) Urban.

Centella asiatica (L.) Urban is a highly considered medicinal plant owing to its secondary metabolites asiaticoside, madecassoside, asiatic acid, and madecassic acid. The asiaticoside, one of the most important constituents of the plant, is a triterpenoid saponin having memory enhancement property. Given its medicinal properties, we isolated and characterized endophytic fungi from this plant with the aim to screen these microorganisms for asiaticoside production. In total, we isolated 13 endophytic fungi from the leaves of the plant, out of which one of the isolates produced asiaticoside. This asiaticoside producing isolate was identified as Colletotrichum gloeosporioides by internal transcribed spacer-based rDNA sequencing. The presence of asiaticoside in ethyl acetate extract of C. gloeosporioides was confirmed by LC-MS. The production of asiaticoside measured in relation to incubation time and subculture generation revealed presence of 62.29 ± 3.36 µg/100 mL of asiaticoside by C. gloeosporioides on the 15th day in first subculture generation followed by a decrease in subsequent generations. A similar trend was also shown by yield and growth curve of C. gloeosporioides. The asiaticoside production and yield were found to be positively correlated. This paper reported the production of asiaticoside by an endophytic fungus C. gloeosporioides for the first time. The present findings definitely provide an impetus to the production of asiaticoside by utilizing the endophytic source. Chemical compound studied in this article: Asiaticoside (PubChemCID: 108062).

Isolation and identification of an antifungal compound from endophytic Streptomyces sp. CEN26 active against Alternaria brassicicola.

UNLABELLED: 2,5-Bis(hydroxymethyl)furan monoacetate (BHMF-OAc) compound was isolated from the culture filtrate secreted by Streptomyces sp. CEN26, an endophytic actinomycete found in the root nodes of Centella asiatica (L.) Urban. The compound was purified using various chromatographic techniques and bioassay-guided fractionation. The compound was then identified through a structural characterization using (1) H, (13) C-NMR and MS analysis. After the purification and identification process, a series of experiments were conducted in order to test whether the compound affected conidial germination of Alternaria brassicicola. Findings show that the compound inhibited and deformed the conidial germination of A. brassicicola. Its minimal inhibitory concentrations of MIC90 and MIC50 were found to be 100 and 25 ppm respectively. Further experimentation show that the aforementioned concentrations of MIC90 and MIC50 completely suppressed the appressorium formation of the fungal pathogen. Therefore, the BHMF-OAc compound does have antifungal properties that help protect plants from infection by A. brassicicola. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic Streptomyces sp. CEN26 produces 2,5-bis(hydroxymethyl)furan monoacetate (BHMF-OAc), which is a compound that inhibits the infection process of Alternaria brassicicola in cabbage. Such an antifungal property would allow for the use of the BHMF-OAc as an alternative to chemical fungicides.

Study in vitro of the impact of endophytic bacteria isolated from Centella asiatica on the disease incidence caused by the hemibiotrophic fungus Colletotrichum higginsianum.

Thirty-one endophytic bacteria isolated from healthy leaves of Centella asiatica were screened in vitro for their ability to reduce the growth rate and disease incidence of Colletotrichum higginsianum, a causal agent of anthracnose. Isolates of Cohnella sp., Paenibacillus sp. and Pantoea sp. significantly stimulated the growth rate of C. higginsianum MUCL 44942, while isolates of Achromobacter sp., Acinetobacter sp., Microbacterium sp., Klebsiella sp. and Pseudomonas putida had no influence on this plant pathogen. By contrast, Bacillus subtilis BCA31 and Pseudomonas fluorescens BCA08 caused a marked inhibition of C. higginsianum MUCL 44942 growth by 46 and 82 %, respectively. Cell-free culture filtrates of B. subtilis BCA31 and P. fluorescens BCA08 were found to contain antifungal compounds against C. higginsianum MUCL 44942. Inoculation assays on in vitro-cultured plants of C. asiatica showed that foliar application of B. subtilis BCA31, three days before inoculation with C. higginsianum MUCL 44942, significantly reduced incidence and severity of the disease. The role of endophytic bacteria in maintaining the apparent inactivity of C. higginsianum MUCL 44942 in C. asiatica grown in the wild is discussed.

Induction of root colonization by Piriformospora indica leads to enhanced asiaticoside production in Centella asiatica.

Centella asiatica (Indian pennywort) has wide application in Indian and Chinese traditional medicines with documented evidence for wound healing and neuroprotective and anti-aging potential. Asiaticoside, a trisaccharide triterpene, is the most medicinally active compound in the plant. ß-Amyrin synthase and squalene synthase have been identified as the two key genes in the triterpenoid pathway which regulate the production of asiaticoside in C. asiatica. The paper reports salient findings of our study utilizing the growth-promoting endophytic fungus Piriformospora indica to successfully colonize roots of C. asiatica in vitro cultures for investigating the effect of the mutualistic association on asiaticoside production. Co-cultivation of P. indica resulted in the rapid enhancement of root and shoot biomass of host plant, which was visible after 7 days of culture and continued up to 45 days. P. indica co-cultivation also favored the synthesis of asiaticosides, as evidenced by HPLC analysis which indicated about twofold increase (0.53% (w/w) in leaves and 0.23% (w/w) in whole plant) over control (0.33% (w/w) in leaves and 0.14% (w/w) in whole plant). Real-time PCR results confirmed the strong upregulation of squalene synthase and ß-amyrin synthase transcripts in P. indica-challenged plants compared with the control. Our data demonstrate the potential use of P. indica as a means to enhance plant secondary metabolite production in planta with scope for further field evaluation.

Optimization of an efficient semi-solid culture protocol for sterilization and plant regeneration of Centella asiatica (L.) as a medicinal herb.

The present study investigates the effects of different concentrations, as well as type of plant growth regulators (PGRs) and medium (MS, Duchefa) on the growth and development of Centella asiatica in semi-solid culture. In addition, a protocol for successful sterilization of C.asiatica explants prepared from field-grown plants highly exposed to fungal and bacterial contamination was determined. Results for sterilization treatments revealed that applying HgCl2 and Plant Preservative Mixture (PPM) with cetrimide, bavistin and trimethoprim which were included after washing with tap water, followed by the addition of PPM in the medium, produced a very satisfactory result (clean culture 90 ± 1.33%) and TS5 (decon + cetrimide 1% + bavistin 150 mg/L + trimethoprim 50 mg/L + HgCl20.1% + PPM 2% soak and 2 mL/L in medium) was hence chosen as the best method of sterilization for C.asiatica. The synergistic combination of 6 benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) in concentrations of 2 mg/L and 0.1 mg/L, respectively, in Duchefa medium compared with MS induced the most optimal percentage of sprouted shoots (93 ± 0.667), number of shoots (5.2 ± 0.079) and nodes (4 ± 0.067) per explant, leaf per explant (14 ± 0.107) and shoot length (4.1 ± 0.67 cm). Furthermore, optimum rooting frequency (95.2 ± 0.81%), the number of roots/shoot (7.5 ± 0.107) and the mean root length (4.5 ± 0.133 cm) occurred for shoots that were cultured on full-strength MS medium containing 0.5 mg/L indole-3-butyric acid (IBA). In this study, the acclimatized plantlets were successfully established with almost 85% survival. The findings of this study have proven an efficient medium and PGR concentration for the mass propagation of C.asiatica. These findings would be useful in micropropagation and ex situ conservation of this plant.

Endophytic fungi from leaves of Centella asiatica: occurrence and potential interactions within leaves.

Fungal endophytes were isolated from leaves of Centella asiatica (Apiaceae) collected at Mangoro (middle eastern region of Madagascar, 200 km from Antananarivo). Forty- five different taxa were recovered. The overall foliar colonization rate was 78%. The most common endophytes were the non-sporulating species 1 (isolation frequency IF 19.2%) followed by Colletotrichum sp.1 (IF 13.2%), Guignardia sp. (IF 8.5%), Glomerella sp. (IF 7.7%), an unidentified ascomycete (IF 7.2%), the non-sporulating species 2 (IF 3.7%) and Phialophora sp. (IF 3.5%). Using sequences of the ribosomal DNA internal transcribed spacer (ITS) regions, major endophytes (IF > 7%) were identified as xylariaceous taxa or as Colletotrichum higginsianum, Guignardia mangiferae and Glomerella cingulata. Results from in vitro fungal disk experiments showed a strong inhibitory activity of the xylariaceous non-sporulating species 1 against G. mangiferae and C. higginsianum and of C. higginsianum against G. mangiferae. This can be explained by antagonism between dominant taxa.