Colonization with non-mycorrhizal culturable endophytic fungi enhances orchid growth and indole acetic acid production.

BACKGROUND: Symbiotic associations of endophytic fungi have been proved by possessing an ability to produce hormones and metabolites for their host plant. Members of the Orchidaceae are obligate mycorrhizal species but a non-mycorrhizal association needs more investigation for their ability to promote plant growth and produce plant growth hormones. In the present study, endophytic fungi were isolated from the roots of Dendrobium longicornu Lindl., to investigate the root colonizing activity and role in plant growth and development. RESULTS: Among 23 fungal isolates were identified both by morphological and molecular technique as Penicillium sp., Fusarium sp., Coniochaeta sp., Alternaria sp., and Cladosporium sp. The dominate species were Coniochaeta sp. and Cladosporium sp. The dominant species as per the isolation was Coniochaeta sp. These fungal strains were screened for growth-promoting activity of Cymbidium aloifolium (plantlet) consider as cross genus interaction and Dendrobium longicornu (protocorms) as a host plant in in-vitro condition. Importantly, Cladosporium sp., and Coniochaeta sp. showed successful colonization and peloton formation with roots of C. aloifolium. Moreover, it also enhanced acclimatization of plantlets. Fungal elicitors from nine fungal isolates enhanced the growth of the in vitro grown protocorms of D. longicornu. Key bioactive compounds detected in the fungal colonized plant extract were 2H-pyran-2-one, Cyclopropanecarboxylic acid, Oleic Acid and d-Mannitol, which may have a potential role in plant-microbe interaction. All fungal endophytes were able to synthesize the indole acetic acid (IAA) in presence of tryptophan. Moreover, fungal extract DLCCR7 treated with DL-tryptophan yielded a greater IAA concentration of 43 µg per ml than the other extracts. The iaaM gene involved in IAA synthesis pathway was amplified using iaaM gene primers successfully from Alternaria sp., Cladosporium sp., and Coniochaeta sp. CONCLUSIONS: Hence, this study confirms the production of IAA by endophytes and demonstrated their host as well as cross-genus plant growth-promoting potential by producing metabolites required for the growth of the plant.

A prospectus of plant growth promoting endophytic bacterium from orchid (Vanda cristata).

BACKGROUND: A plant growth-promoting endophytic bacterium PVL1 isolated from the leaf of Vanda cristata has the ability to colonize with roots of plants and protect the plant. PVL1 was isolated using laboratory synthetic media. 16S rRNA gene sequencing method has been employed for identification before and after root colonization ability. RESULTS: Original isolated and remunerated strain from colonized roots were identified as Bacillus spp. as per EzBiocloud database. The presence of bacteria in the root section of the plantlet was confirmed through Epifluorescence microscopy of colonized roots. The in-vitro plantlet colonized by PVL1 as well as DLMB attained higher growth than the control. PVL1 capable of producing plant beneficial phytohormone under in vitro cultivation. HPLC and GC-MS analysis suggest that colonized plants contain Indole Acetic Acid (IAA). The methanol extract of Bacillus spp., contains 0.015 µg in 1 µl concentration of IAA. PVL1 has the ability to produce antimicrobial compounds such as ethyl iso-allocholate, which exhibits immune restoring property. One-way ANOVA shows that results were statistically significant at P ≤ 0.05 level. CONCLUSIONS: Hence, it has been concluded that Bacillus spp. PVL1 can promote plant growth through secretion of IAA during root colonization and ethyl iso-allocholate to protect plants from foreign infections. Thus, this study supports to support Koch's postulates of bacteria establishment.

Antioxidant and cytotoxic activities of Dendrobium moniliforme extracts and the detection of related compounds by GC-MS.

BACKGROUND: The medicinal orchid Dendrobium moniliforme contains water-soluble polysaccharides, phenanthrenes, bibenzyl derivatives, and polyphenol compounds. This study explored the antioxidant and cytotoxic activities of D. moniliforme extracts and detected their bioactive compounds. METHODS: Plant material was collected from the Daman of Makawanpur district in central Nepal. Plant extracts were prepared from stems using hexane, chloroform, acetone, ethanol and methanol. The total polyphenol content (TPC) in each extract was determined using Folin-Ciocalteu's reagent and the total flavonoid content (TFC) in each extract was determined using the aluminium chloride method. The in vitro antioxidant and cytotoxic activities of each extract were determined using DPPH (2,2-diphenyl-1-picrylhydrazyl) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays respectively. Gas chromatography and mass spectrometry (GC-MS) analysis was used to detect bioactive compounds. RESULTS: TPC content was highest (116.65 µg GAE/mg of extract) in D. moniliforme chloroform extract (DMC) and TFC content was highest (116.67 µg QE/mg of extract) in D. moniliforme acetone extract (DMA). D. moniliforme hexane extract (DMH) extract showed the highest percentage of DPPH radical scavenging activity (94.48%), followed closely by D. moniliforme ethanol extract (DME) (94.45%), DMA (93.71%) and DMC (94.35%) at 800 µg/ml concentration. The antioxidant capacities of DMC, DMA, DMH and DME, which were measured in IC50 values, were much lower 42.39 µg/ml, 49.56 µg/ml, 52.68 µg/ml, and 58.77 µg/ml respectively than the IC50 of D. moniliforme methanol extract (DMM) (223.15 µg/ml). DMM at the concentration of 800 µg/ml most inhibited the growth of HeLa cells (78.68%) and DME at the same concentration most inhibited the growth of U251 cells (51.95%). The cytotoxic capacity (IC50) of DMM against HeLa cells was 155.80 µg/ml of extract and that of DME against the U251 cells was 772.50 µg/ml of extract. A number of bioactive compounds were detected in both DME and DMM. CONCLUSION: The fact that plant extract of D. moniliforme has a number of bioactive compounds which showed antioxidant and cytotoxic activities suggests the potential pharmacological importance of this plant.

Application of plant cell and tissue culture for the production of phytochemicals in medicinal plants.

Approximately 80% of the world inhabitants depend on the medicinal plants in the form of traditional formulations for their primary health care system well as in the treatment of a number of diseases since the ancient time. Many commercially used drugs have come from the information of indigenous knowledge of plants and their folk uses. Linking of the indigenous knowledge of medicinal plants to modern research activities provides a new reliable approach, for the discovery of novel drugs much more effectively than with random collection. Increase in population and increasing demand of plant products along with illegal trade are causing depletion of medicinal plants and many are threatened in natural habitat. Plant tissue culture technique has proved potential alternative for the production of desirable bioactive components from plants, to produce the enough amounts of plant material that is needed and for the conservation of threatened species. Different plant tissue culture systems have been extensively studied to improve and enhance the production of plant chemicals in various medicinal plants.

Genetic diversity and population structure analysis of Paris polyphylla Sm. revealed by SSR marker.

Paris polyphylla Sm. is a vulnerable medicinal plant distributed in the Himalayan countries. This plant has numerous pharmacological benefits, including anticancer, anti-inflammatory, analgesic, and antipyretic properties. The distribution, conservation status, and traditional usage of this species are fairly known in Nepal. However, its diversity and population structure at the molecular level are unexplored. This study analyzes, the genetic diversity and population structure of 32 P. polyphylla germplasms collected from Central, Eastern and Western regions of Nepal using 15 simple sequence repeat (SSR) markers. All the SSR primers were polymorphic and amplified 60 alleles ranging from 50 bp to 900 bp. The polymorphic information content (PIC) value ranged from 0 to 0.75. The average value of the observed heterozygosity (Ho), expected heterozygosity (He), Shannon's information index (I), and total heterozygosity (Ht) were 0.63, 0.53, 0.92 and 0.32, respectively. The analysis of molecular variance (AMOVA), showed a maximum variation of 74% within the individual in a population and only 26% variation among the population. In the population STRUCTURE analysis two clusters were formed where Eastern germplasms (EN) were separated far from the Central and Western germplasms (CWN), this clustering was in complete correspondence to the unweighted pair group method based on arithmetic average (UPGMA) and principle coordinate analysis (PCoA). Furthermore, in the UPGMA and PCoA, germplasms collected from the same or relatively similar geographic origin were closer. These findings are critical for developing conservation policies, facilitating evolutionary research, sustainable utilization and commercial cultivation of this pharmacologically important and threatened species.

Bioactive secondary metabolites in Paris polyphylla Sm. and their biological activities: A review.

Paris polyphylla Sm. is an important medicinal plant used to treat a variety of diseases through traditional medicine systems such as Ayurveda, Tibetan traditional medicines, Chinese traditional medicines, and others around the world. The IUCN red list has designated it as "vulnerable" due to a decline in wild population by over-exploitation, habitat degradation, illegal collection for trade and traditional use. This review paper aims to summarize the bioactive secondary metabolites in Paris polyphylla. Paris saponins or steroidal saponins are the main bioactive chemical constituents from this plant that account for more than 80% of the total compounds. For instance, polyphyllin D, diosgenin, paris saponins I, II, VI, VII, and H are steroidal saponins having anticancer activity comparable to synthetic anticancer medicines. Antioxidant, anticancer, anti-leishmaniasis, antibacterial, antifungal, anthelmintic, antityrosinase, and antiviral effects of extracts and pure compounds were also demonstrated in vivo and in vitro. In conclusion, this review summarizes the bioactive components from the P. polyphylla which will be useful to researchers and scientists, and for the development of potential drugs.

Comparative Cytotoxic Activity of Wild Harvested Stems and In Vitro-Raised Protocorms of Dendrobium chryseum Rolfe in Human Cervical Carcinoma and Glioblastoma Cell Lines.

From the medicinal orchid Dendrobium chryseum Rolfe, which is used in traditional and folk Chinese medicine, the protocorms were raised in Murashige and Skoog (MS) media in three strengths, full strength (FMS), half strength (1/2 MS), and quarter strength (1/4 MS), with or without the phytohormones 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) and coconut water (CW). The comparative cytotoxic activities of the wild and in vitro-raised protocorms were evaluated in human cervical carcinoma (HeLa) and human glioblastoma (U251) cell lines by MTT assay. In in vivo and in vitro, the methanol extracts of D. chryseum showed significant cytotoxic activities. Significant growth inhibition (%) and potent IC50 values were demonstrated in HeLa cell lines (49.79% (210.5 µg/mL) for in vitro-raised Dendrobium chryseum (DCT) versus 46.97% (226.5 µg/mL) for wild Dendrobium chryseum (DCW)). Similarly, activities against U251 cell lines exhibited also significant inhibition (28.76% (612.54 µg/mL) for DCW and 17.15% (1059.92 µg/mL) for DCT). The cytotoxic activities of both, wild and tissue-cultured samples, were superior in HeLa cells. In U251 cells, the wild sample was more active than the tissue-cultured one with a moderate cytotoxic effect. Hence, protocorm culture may therefore be a promising future tool for producing pharmacologically bioactive compounds in medicinal orchids. Such sustainable technology approach will minimize the pressure on the natural population of threatened but commercially important medicinal orchids.

Isolation, characterization, and plant growth-promoting activities of endophytic fungi from a wild orchid Vanda cristata.

Endophytism is one of the widely explored phenomena related to orchids and fungi. Endophytic fungi assist plants by supplementing nutrient acquisition, and synthesis of plant growth regulators. Vanda cristata is an epiphytic orchid that has a great diversity of endophytic fungi. Endophytic fungi were isolated from roots, stems, and leaves of V.cristata and identified by both morphological and molecular study. Furthermore, the isolated endophytic fungi were subjected to auxin synthesis, phosphate solubilization, ammonia synthesis, and elicitor growth test for understanding their growth-promoting effect in a qualitative and quantitative manner. Altogether, 12 different endophytic fungi were isolated from roots, stems, and leaves of V. cristata of which most species belonged to Ascomycota. Unidentified II fungi were found to be most effective for auxin synthesis and phosphate solubilization while Agaricus bisporous and Mycolepto discus were most effective for ammonia synthesis. We have tested the plant growth-promoting activity of the twelve isolated endophytic fungi on Cymbidium aloifolium protocorms (12 weeks old). All the endophytic fungi showed growth-promoting activity. Plant growth of Cymbidium aloifolium was found higher on the MS medium supplemented with all fungal elicitors. Fungal elicitor CVS4, however, showed the highest plant growth-promoting activity toward C. aloifolium.

Antioxidant, anticancer and antimicrobial effects of In vitro developed protocorms of Dendrobium longicornu.

In vitro seed germination and protocorms formation were successfully established in traditionally used Dendrobium longicornu. Fresh protocorms (178.34 g - 183.90 g) were produced on the elicitor of Alternaria sp, Bacillus subtilis and Fusarium solani supplemented MS-medium. Methanol extract of D. longicornu protocorms has scavenged 94.31 % of DPPH radicals at 1000 µg/mL. Its 117.56 µg/mL concentration has scavenged 50 % DPPH radical (IC50). Similarly, it inhibits 25.39 % and 27.80 % HeLa and U251 cells at 500 µg/mL. The IC50 was found as 350.06 µg/mL and 507.22 µg/mL for HeLa and U251 cells respectively. Further, it inhibited the growth of E. coli, K. pneumoniae and E. cloacae with the zone of inhibition 4, 2 and 2 mm respectively. In conclusion, protocorms developed through in vitro seeds culture have accumulated and synthesized bioactive secondary metabolites. Therefore, protocorms could be utilized to the isolation of compounds for formulation of herbal drugs without damaging natural populations.

Cytotoxic effect of selected wild orchids on two different human cancer cell lines.

Majority of the orchid species are used in the traditional medicines for the treatment of several diseases. They are the sources of polysaccharides, phenanthrenes, bibenzyl derivatives, revesteral, stilbenoids and polyphenol compounds. This study explored the cytotoxic activity of seven wild orchid species and identification of medicinally active compounds. The extracts of orchid species were screened for cytotoxic effect on the human cervical cancer cells (HeLa) and human glioblastoma cells (U251) using an MTT assay. The medicinally active compounds of high cytotoxic extracts were identified by GC-MS resulting in many stilbenoids and phenolic derivatives. The extract of Dendrobium transparens (DTs) and Vanda cristata (VCw) showed high cytotoxic effect towards the HeLa and U251 cell lines (IC50 of DTs: 382.14 µg/ml and 75.84 µg/ml respectively and IC50 of VCw: 317.23 µg/ml and 163.66 µg/ml respectively). This study concludes that they could be used as cancer therapeutics.

Assessment of Antioxidant and Cytotoxic Activities of Extracts of Dendrobium crepidatum.

Dendrobium crepidatum is an epiphytic orchid found in south Asia including Nepal and China. This orchid species is widely used in traditional Chinese medicine (TCM) for the treatment of cancer, diabetes, cataracts, and fever. The objectives of the present research were to assess the antioxidant and cytotoxic properties of its stem's extracts with the identification of bioactive secondary metabolites. The antioxidant and cytotoxic activities were evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, respectively, and compounds were identified using GC-MS (gas chromatography and mass spectrometry). Ethanol and acetone extracts scavenged 94.69 ± 0.10% and 93.41 ± 0.86% of DPPH free radicals, respectively. They showed 50% inhibition of DPPH free radicals (IC50) at concentrations of 73.90 µg/mL and 99.44 µg/mL, which were found to be statistically similar to that of ascorbic acid (control). Chloroform extract inhibited the growth of 81.49 ± 0.43% of HeLa (human cervical carcinoma) cells and hexane extract inhibited the growth of 76.45 ± 4.26% of U251 (human glioblastoma) cells at 800 µg/mL concentration. These extracts showed 50% inhibition of cell growth (IC50) toward both the HeLa and U251 cell lines at their high concentrations, which were found statistically significantly different from that of cisplatin drug (control). The above extracts showed antioxidant and cytotoxic properties, potentially due to the presence of tetracosane, triacontane, stigmasterol, and some phenol derivatives (2-methoxy-4-vinylphenol, 2-methoxy-5-(1-propenyl)-phenol, p-mesyloxyphenol, and 2,6-dimethoxy-4-(2-propenyl)-phenol). This study explores the potential of this orchid in alternative medicine toward the development of drugs from its medicinally active compounds.

Piriformospora indica promotes the growth of the in-vitro-raised Cymbidium aloifolium plantlet and their acclimatization.

Cymbidium aloifolium is known for its ornamental and medicinal values. It has been listed as threatened orchid species. In this study, in vitro propagated C. aloifolium plantlets were interacted with the Piriformospora indica. The growth assay was performed for 45 days; the plant growth pattern such as number and length of roots and shoots were measured. Microscopic study of the root section stained by trypan blue was done to detect the peloton formation. The methanol extracts of the fungal colonized plant as well as uncolonized (control) plant were prepared and various metabolites were identified by gas chromatography mass spectroscopy. Acclimatization was done in a substrate composition of coco peat: gravel: charcoal in ratio 2:2:1. P. indica-colonized plantlet showed the highest growth with the formation of clamdospore in the root section. The growth regulator such as auxin, ascorbic acid, andrographolide, hexadecanoic acid, and DL-proline were identified. After three months of field transfer, plantlet colonized by P. indica survived and remained healthy as compared to uncolonized control plantlet.

Isolation and Characterization of Plant Growth-Promoting Endophytic Fungi from the Roots of Dendrobium moniliforme.

The present study aims to identify the diverse endophytic fungi residing in the roots of Dendrobium moniliforme and their role in plant growth and development. Nine endophytic fungi were isolated from the root sections and characterized by molecular technique. Quantification of the indole acetic acid (IAA) compound by these endophytes was done. Further, Chemical profiling of R11 and R13 fungi was done by Gas Chromatography-Mass Spectroscopy (GC-MS). Asymbiotic seed derived protocorms of Rhynchostylis retusa was used for the plant growth assay to investigate the growth promoting activities of the fungal elicitor prepared from the isolated fungi from D. moniliforme. Among the isolated fungi, the relative dominant fungus was Fusarium sp. The R13 and R6 fungi were identified only at the genus level which concludes the fungi are of new species or strain. The indole acetic acid production was relatively higher in R10. Bioactive compound diversity was observed in the organic extract of R11 and R6. The presence of phenolic compound and essential oil suggest their contribution for the antimicrobial and antioxidant properties to their host plant, D. moniliforme. The plant growth assay result concluded, the fungal elicitor prepared from R10, Colletotrichum alatae was the best among all other for the plant growth activities.

Production of virus-free orchid Cymbidium aloifolium (L.) Sw. by various tissue culture techniques.

Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV) is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ») with or without 0.5 mg/l BAP (6-benzylaminopurine) and 0.5 mg/l NAA (Naphthalene acetic acid). To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo-grown source (mother) plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%). The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation.