Significant enhancement of lignan accumulation in hairy root cultures of Linum album using biotic elicitors

Linum album has been shown to accumulate some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, we examined the effects of fungal elicitors on the production of lignans in L. album hairy root cultures. The biosynthesis of lignans was differentially affected by fungal elicitors. Fusarium graminearum extract induced the highest increase of PTOX, 190 μg g^?1 dry weight (DW), and lariciresinol, 260 μg g^?1 DW, which was two-fold and three-fold greater than the untreated control, respectively, while Trichoderma viride extract enhanced the accumulation of MPTOX, instead of PTOX, up to 160 µg g^?1 DW, which was 2.4-fold greater than the control. The enhancing effects of fungal elicitors on lignans production was correlated with the increased expression of some key genes involved in the biosynthesis of these compounds, phenylalanine ammonia-lyase, cinnamoyl-CoA reductase, cinnamyl-alcohol dehydrogenase and pinoresinol-lariciresinol reductase.     

Synthesis,characterization and evaluation of antimicrobial and cytotoxic activities of biogenic silver nanoparticles synthesized from <Emphasis Type="Italic">Streptomyces xinghaiensis</Emphasis> OF1 strain

We report synthesis of silver nanoparticles (AgNPs) from Streptomyces xinghaiensis OF1 strain, which were characterised by UV–Vis and Fourier transform infrared spectroscopy, Zeta sizer, Nano tracking analyser, and Transmission electron microscopy. The antimicrobial activity of AgNPs alone, and in combination with antibiotics was evaluated against bacteria, namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis, and yeasts viz., Candida albicans and Malassezia furfur by using micro-dilution method. The minimum inhibitory concentration (MIC) and minimum biocidal concentration of AgNPs against bacterial and yeast strains were determined. Synergistic effect of AgNPs in combination with antibacterial and antifungal antibiotics was determined by FIC index. In addition, MTT assay was performed to study cytotoxicity of AgNPs alone and in combination with antibiotics against mouse fibroblasts and HeLa cell line. Biogenic AgNPs were stable, spherical, small, polydispersed and capped with organic compounds. The variable antimicrobial activity of AgNPs was observed against tested bacteria and yeasts. The lowest MIC (16 µg ml^?1) of AgNPs was found against P. aeruginosa, followed by C. albicans and M. furfur (both 32 µg ml^?1), B. subtilis and E. coli (both 64 µg ml^?1), and then S. aureus and Klebsiella pneumoniae (256 µg ml^?1). The high synergistic effect of antibiotics in combination with AgNPs against tested strains was found. The in vitro cytotoxicity of AgNPs against mouse fibroblasts and cancer HeLa cell lines revealed a dose dependent potential. The IC₅₀ value of AgNPs was found in concentrations of 4 and 3.8 µg ml^?1, respectively. Combination of AgNPs and antibiotics significantly decreased concentrations of both antimicrobials used and retained their high antibacterial and antifungal activity. The synthesis of AgNPs using S. xinghaiensis OF1 strain is an eco-friendly, cheap and nontoxic method. The antimicrobial activity of AgNPs could result from their small size. Remarkable synergistic effect of antibiotics and AgNPs offer their valuable potential in nanomedicine for clinical application as a combined therapy in the future.     

Reduced sensitivity of tomato early blight pathogen (Alternaria solani) isolates to protectant fungicides,and implication on disease control

The sensitivity of Alternaria solani isolates to the fungicides mancozeb and chlorothalonil was evaluated, to determine if inadequate disease management by these fungicides could be attributed to reduced sensitivity of A. solani isolates to these fungicides. The sensitivity of 60 isolates of A. solani was assessed using the inhibition of radial mycelial growth (RG) method, using fungicide concentrations of 0, 1.0, 10, 100, 500 and 1000 μg a.i ml^?1 medium. EC₅₀ was calculated for each isolate and fungicide combination. The EC₅₀ values of different A. solani isolates to mancozeb ranged from 9.05 to 712.65 μg ml^?1. EC₅₀ values of different isolates to chlorothalonil ranged from 4.25 to 849.4 μg ml^?1. The percentage of isolates with reduced sensitivity was 46.7 and 53.3% for mancozeb and chlorothalonil, respectively. Results of the in vivo tests demonstrated decline in disease control by the two fungicides with the reduced-sensitivity isolates compared to the sensitive ones.     

Isolation and characterization of trichalasin-producing endophytic fungus from <Emphasis Type="BoldItalic">Taxus baccata</Emphasis>

An endophytic fungus (strain T1) isolated from Taxus baccata was studied for the production of metabolites with anticancer and antioxidant activities. This fungus was identified as Diaporthe sp. based on rDNA-internal transcribed spacer (ITS) sequence analysis. The crude extract showed cytotoxic activity against MCF-7 and HeLa cancer cell lines, with IC₅₀ (concentration inhibiting 50% of growth rate) values of 1058?±?44 and 1257?±?80 μg ml^?1, respectively. The scavenging activity of fungal extract increased significantly with increasing concentration [IC₅₀ (concentration required to scavenge 50% of free radicals) 482?±?9 μg ml^?1]. Ultra-high-performance liquid chromatography-quadrupole-time of flight analysis revealed the presence of three trichalasins (trichalasin E, F and H) in the crude extract of T1 which are known to have antitumour and antioxidant activities. These results suggest that Diaporthe sp. has the potential to be used for therapeutic purposes because of its antiproliferative and antioxidant potential and also for the production of cytochalasins.     

Secondary metabolites released into the rhizosphere by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> spp. as underestimated component of nonspecific replant disease

Background and aims

A study was performed to investigate the role of fungal metabolites released into the rhizosphere of replanted orchards as a potential biotic component of tree growth decline.

Methods

The phytotoxicity of the gamma ray-sterilized crude culture filtrates of sixteen fungal species originating from replanted apple orchards was tested in a bioassay. Low molecular weight compounds released by Fusarium spp. were analyzed.

Results

The fungal culture filtrates affected seedling growth and health with an activity that varied from growth inhibition to promotion. Three out of the six species of Fusarium tested produced species-specific mycotoxins such as equisetin and enniatin B and D (<1 μg ml^?1 and <6 μg ml^?1, respectively) associated with root-tip necrosis, whereas fusaric acid (80–230 μg ml^?1) was associated with asymptomatic plant growth inhibition. These findings were consistent with those obtained using pure compounds. Moreover, methoxyconidiol, paecilaminol, integrastatin B and other biologically active compounds, whose fungal origin and phytotoxicity have not yet been reported, were found. in all fungal filtrates.

Conclusions

Findings suggest that i) phytopathogenicity of soil borne fungi can be expressed regardless of root infection; ii) a synergistic interaction between co-occurring mycotoxins and other biologically active compounds may explain plant growth inhibition. Iii) fungal metabolites released into soil may represent an underestimated component of nonspecific replant disease.

     

Exogenous application of rutin and gallic acid regulate antioxidants and alleviate reactive oxygen generation in <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

The effect of rutin and gallic acid on growth, phytochemical and defense gene activation of rice (Oryza sativa L.) was investigated. The seeds of rice were primed with different concentrations of rutin and gallic acid (10–60 µg mL^?1) to explicate the effect on germination on water agar plates. Further, to study the effect of most effective concentrations of gallic acid (60 µg mL^?1) and rutin (50 µg mL^?1), greenhouse pot experiment was set up to determine the changes in growth, antioxidant and defense parameters. The results revealed more pronounced effect of gallic acid on total chlorophyll and carotenoids as well as on total flavonoid content and free radical scavenging activities. Gene expression analysis of OsWRKY71, PAL, CHS and LOX genes involved in strengthening the plant defense further validated the results obtained from the biochemical analysis. Microscopic analysis also confirmed reduction in total reactive oxygen species, free radicals like H₂O₂ and O₂ ^? by exogenous application of gallic acid and rutin. The data obtained thus suggest that both gallic acid and rutin can affect the growth and physiology of rice plants and therefore can be used to develop effective plant growth promoters and as substitute of biofertilizers for maximizing their use in field conditions.     

The Usefulness of Non-Toxic Plant Metabolites in the Control of Bacterial Proliferation

The effect of generally recognised as safe (GRAS) plant metabolites in regulating the growth of human pathogenic and probiotic bacteria and in the formation of biofilm was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against both pathogenic and probiotic microorganisms, at a subinhibitory concentration (SIC) of ≤50 μg ml^?1. Genistein, hydroquinone, p-hydroxybenzoic acid and resveratrol also showed antibacterial effects but at a wide concentration range (SIC = 50–1000 μg ml^?1). Catechin, gallic acid, protocatechuic acid and cranberry extracts were the most biologically compatible molecules (SIC ≥ 1000 μg ml^?1). Regarding the effect on biofilm, it was observed that thymol, carvacrol and eugenol showed antibiofilm activity against all potential pathogenic bacteria tested whilst specifically enhancing probiotic aggregation. Catechin, genistein and cranberry extracts did not inhibit the pathogenic aggregation but they stimulated probiotic biofilm formation, whilst gallic acid, protocateuchic acid, hydroquinone, p-hydroxybenzoic acid and resveratrol did not show opposite effect on biofilm formation between pathogenic and probiotic microorganisms. These results indicate that an appropriate combination of GRAS plant metabolites, which have traditionally been used as dietary constituents due to their health-promoting characteristics, can also be extremely useful in the regulation of bacterial proliferation in the intestinal microbiota. Hence, it is suggested to apply these natural GRAS molecules as dietary supplements in the food industry in order to promote probiotic viability and to prevent or reduce colonisation or proliferation of intestinal pathogens.     

Polyhydroxyalkanoate biosynthesis and simultaneous remotion of organic inhibitors from sugarcane bagasse hydrolysate by Burkholderia sp.

Burkholderia sp. F24, originally isolated from soil, was capable of growth on xylose and removed organic inhibitors present in a hemicellulosic hydrolysate and simultaneously produced poly-3-hydroxybutyrate (P3HB). Using non-detoxified hydrolysate, Burkholderia sp. F24 reached a cell dry weight (CDW) of 6.8 g L^?1, containing 48 % of P3HB and exhibited a volumetric productivity (P_P3HB) of 0.10 g L^?1 h^?1. Poly-3-hydroxybutyrate-co-3-hydroxyvalerate copolymers (P3HB-co-3HV) were produced using xylose and levulinic acid (LA) as carbon sources. In shake flask cultures, the 3HV content in the copolymer increased from 9 to 43 mol% by adding LA from 1.0 to 5.0 g L^?1. In high cell density cultivation using concentrated hemicellulosic hydrolysate F24 reached 25.04 g L^?1 of CDW containing 49 % of P3HB and P_P3HB of 0.28 g L^?1 h^?1. Based on these findings, second-generation ethanol and bioplastics from sugarcane bagasse is proposed.     

Non-toxic plant metabolites regulate Staphylococcus viability and biofilm formation: a natural therapeutic strategy useful in the treatment and prevention of skin infections

In the present study, the efficacy of generally recognised as safe (GRAS) antimicrobial plant metabolites in regulating the growth of Staphylococcus aureus and S. epidermidis was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against these microorganisms, at a subinhibitory concentration (SIC) of ≤ 50 μg ml^?1. Genistein, hydroquinone and resveratrol showed antimicrobial effects but with a wide concentration range (SIC = 50–1,000 μg ml^?1), while catechin, gallic acid, protocatechuic acid, p-hydroxybenzoic acid and cranberry extract were the most biologically compatible molecules (SIC ≥ 1000 μg ml^?1). Genistein, protocatechuic acid, cranberry extract, p-hydroxybenzoic acid and resveratrol also showed anti-biofilm activity against S. aureus, but not against S. epidermidis in which, surprisingly, these metabolites stimulated biofilm formation (between 35% and 1,200%). Binary combinations of cranberry extract and resveratrol with genistein, protocatechuic or p-hydroxibenzoic acid enhanced the stimulatory effect on S. epidermidis biofilm formation and maintained or even increased S. aureus anti-biofilm activity.     

Characterization of a novel asparaginase from soil metagenomic libraries generated from forest soil

Objectives

To screen soil metagenomic libraries for novel enzymes with enhanced activities.

Results

To screen soil metagenomic libraries for novel enzymes with enhanced activities. A novel l-asparaginase was identified from forest soil metagenome and its characteristics were studied. The purified protein had a specific activity of 696 IU mg^?1 and optimum activity at pH 7 and 35 °C. Enhanced enzyme activities were observed in the presence of Mg²⁺, Ca²⁺ and K⁺. The K_m value, 2 mM, and enzyme specificity constant 7.7 mM^?1s^?1 indicated that the recombinant enzyme has good substrate affinity to l-asparagine compared with commercially-available Escherichia coli asparaginase. The IC₅₀ value of 0.78 µg ml^?1 (0.47 IU ml^?1) was observed with HL60 cell line and 0.39 µg ml^?1(0.23 IU ml^?1) with MOLT-3 and MOLT-4 cell lines, which is better than that of commercially-available drugs.

Conclusion

The soil metagenome derived l-asparaginase with enhanced activities could be a potential candidate to develop as a drug in Acute Lymphoblastic Leukemia (ALL) therapy.

     

Optimization of culture medium for microsclerotia production by Nomuraea rileyi and analysis of their viability for use as a mycoinsecticide

Microsclerotia (MS) formation was successfully induced in Nomuraea rileyi (Ascomycetes: Hypocreales) in liquid amended medium (AM) culture. To improve MS production, the culture medium was varied. Three quantitative variables, carbon, nitrogen, and basal salt sources, were investigated using the AM. An orthogonal experiment [L₉ (3)³] design was applied to optimize the culture medium. Maximum MS production (21.9 × 10⁴ MS ml^?1) was obtained using the optimized culture medium (32.0 g l^?1 of glucose, 2.0 g l^?1 of ammonium citrate, and 0.15 g l^?1 of ferrous sulfate). Subsequently, viability, including thermotolerance, shelf-life, and infection, were investigated to evaluate the potential of MS for use as a mycoinsecticide. The MS, collected from the optimized culture medium, exhibited virulence and greater thermotolerance than conidia, and maintained 86.4 % germinability after one-year room temperature storage. These results suggested that the MS obtained through optimization production can be used as a mycoinsecticide.     

Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants

Aflatoxin B1 (AFB1) is a highly toxic fungal metabolite having carcinogenic, mutagenic and teratogenic effects on human and animal health. Accidental feeding of aflatoxin-contaminated rice straw may be detrimental for ruminant livestock and can lead to transmission of this toxin or its metabolites into the milk of dairy cattle. White-rot basidiomycetous fungus Pleurotus ostreatus produces ligninolytic enzymes like laccase and manganese peroxidase (MnP). These extracellular enzymes have been reported to degrade many environmentally hazardous compounds. The present study examines the ability of P. ostreatus strains to degrade AFB1 in rice straw in the presence of metal salts and surfactants. Laccase and MnP activities were determined spectrophotometrically. The efficiency of AFB1 degradation was evaluated by high performance liquid chromatography. Highest degradation was recorded for both P. ostreatus MTCC 142 (89.14 %) and P. ostreatus GHBBF10 (91.76 %) at 0.5 µg mL^?1 initial concentration of AFB1. Enhanced degradation was noted for P. ostreatus MTCC 142 in the presence of Cu²⁺ and Triton X-100, at toxin concentration of 5 µg mL^?1. P. ostreatus GHBBF10 showed highest degradation in the presence of Zn²⁺ and Tween 80. Liquid chromatography-mass spectrometric analysis revealed the formation of hydrated, decarbonylated and O-dealkylated products. The present findings suggested that supplementation of AFB1-contaminated rice straw by certain metal salts and surfactants can improve the enzymatic degradation of this mycotoxin by P. ostreatus strains.     

Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 μg ml^?1 iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 μg ml^?1 iodide (I^?). When 10⁶ CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I₂), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 μg ml^?1, respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I^?), the molecular iodine concentration was estimated to be 0.76 μg ml^?1 after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 μg ml^?1.     

Culture Media Statistical Optimization for Biomass Production of a Ligninolytic Fungus for Future Rice Straw Degradation

The main objective of this study was to optimize a culture media for low scale biomass production of Pleurotus spp. Future applications of this optimization will be implemented for “in situ” rice straw degradation, increase soil nutrients availability, and lower residue and rice culture management costs. Soil samples were taken from different points in six important rice production cities in Colombia. For carbon and nitrogen source selection a factorial 4² design was carried out. The Plackett-Burman design permitted to detect carbon, nitrogen and inducer effects on fungus growth (response variable for all designs). This optimization was carried out by a Box-Behnken design. Finally a re-optimization assay for glucose concentration was performed by means of a One Factor design. Only 4/33 (12 %) isolates showed and important laccase or manganese peroxidase activity compared to Pleurotus ostreatus (HPB/P3). We obtained an increased biomass production in Pleurotus spp. (T1.1.) with glucose, followed by rice husk. Rice straw was considered an inducing agent for lignin degradation. Glucose was a significant component with positive effects, whereas Tween 80 and pH had negative effects. On the contrary, rice husk, yeast extract and CaCl₂ were not significant components for increase the biomass production. Final media composition consisted of glucose 25 g L^?1, yeast extract 5 g L^?1, Tween 80 0.38 % (v/v), Rice husk 10 g L^?1, CaCl₂ 1 g L^?1, and pH 4.88 ± 0.2. The Box-Behnken polynomial prediction resulted to be lower than the experimental validation of the model (6.59 vs. 6.91 Log₁₀ CFU ml^?1 respectively).     

Accumulation of arsenic in soil and rice under wetland condition in Bangladesh

Shallow tube well (STW) water, often contaminated with arsenic (As), is used extensively in Bangladesh for irrigating rice fields in the dry season, leading to potential As accumulation in soils. In the current study the consequences of arsenic from irrigation water and direct surface (0–15 cm) soil application were studied under field conditions with wetland rice culture over 2 years. Twenty PVC cylinders (30-cm length and 30-cm diameter) were installed in field plots to evaluate the mobility and vertical distribution of soil As, As mass balance, and the resulting influences on rice yield and plant-As concentration in Boro (dry season) and transplanted (T.) Aman (wet season) rice over the 2-year growth cycle. Treatments included irrigation-water As concentrations of 0, 1 and 2 mg L^?1 (Boro season only) and soil-As concentrations of 10 and 20 mg kg^?1. Following the 2-year cropping sequence the major portion (39.3–47.6%) of the applied arsenic was retained within the rooting zone at 0–15 cm depth, with 14.7–19.5% of the total applied As at the 5–10 cm and 10–15 cm soil depths compared to 1.3–3.6% at the 35–40 cm soil depth. These results indicate the relatively low mobility of applied As and the likely continued detrimental accumulation of As within the rooting zone. Arsenic addition in either irrigation water or as soil-applied As resulted in yield reductions from 21 to 74 % in Boro rice and 8 to 80 % in T. Aman rice, the latter indicating the strong residual effect of As on subsequent crops. The As concentrations in rice grain (0.22 to 0.81 µg g^?1), straw (2.64 to 12.52 µg g^?1) and husk (1.20 to 2.48 µg g^?1) increased with increasing addition of As. These results indicate the detrimental impacts of continued long-term irrigation with As-contaminated water on agricultural sustainability, food security and food quality in Bangladesh. A critical need exists for the development of crop and water management strategies to minimize potential As hazard in wetland rice production.     

Efficacy of bacterial seed treatments for the control of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> in maize

Maize colonization by the phytopathogenic fungi Fusarium verticillioides leads to economical and food quality losses and also implicates potential health risks. In order to control this fungal species different strategies are being considered. In the present work we investigated the in situ effects of the use of two in vitro proven bacterial biological control agents against Fusarium verticillioides, using maize seedlings grown in greenhouse conditions. The treatment of maize seeds with Fusarium verticillioides 10⁷ spores ml^?1 negatively affected the length of the stem and the weight of the root systems of resultant seedlings, and also reduced the numbers of non-rhizospheric organisms with ammonification and nitrification activities. The addition of Bacillus amyloliquefaciens or Microbacterium oleovorans, at a concentration of 10⁹ colony forming units ml^?1, to the seeds significantly reduced Fusarium verticillioides count at the root inner tissues of resultant seedlings. When testing the impact of bacterial treatments on soil populations, no alterations with respect to control numbers of organisms with nitrification, ammonification or cellulolytic potentials were observed. Culturable bacterial richness and diversity calculated at the rhizoplane and root inner tissues of maize seedlings neither changed in bacterized treatments when compared to control treatment. Our study showed that the Fusarium verticillioides in vitro proven antagonists, Bacillus amyloliquefaciens and Microbacterium oleovorans (at 10⁹ colony forming units ml^?1), were also effective at greenhouse conditions without causing major changes in culturable rhizospheric and endophytic microbial richness and diversity.     

Aeromonas molluscorum Av27 is a potential tributyltin (TBT) bioremediator: phenotypic and genotypic characterization indicates its safe application

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 μg ml^?1), amoxicillin/clavulanic acid (30 μg ml^?1) and cephalothin (30 μg ml^?1) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.     

Phosphate solubilization potential and modeling of stress tolerance of rhizobacteria from rice paddy soil in northern Iran

The purposes of this study were to evaluate the phosphate solubilization activity of bacteria isolated from the rhizosphere of rice paddy soil in northern Iran, and to study the effect of temperature, NaCl and pH on the growth of these isolates by modeling. Three of the most effective strains from a total of 300 isolates were identified and a phylogenetic analysis was carried out by 16S rDNA sequencing. The isolates were identified as Pantoea ananatis (M36), Rahnella aquatilis (M100) and Enterobacter sp. (M183). These isolates showed multiple plant growth-promoting attributes such as phosphate solubilization activity and indole-3-acetic acid (IAA) production. The M36, M100 and M183 isolates were able to solubilize 172, 263 and 254 µg ml^?1 of Ca₃(PO₄)₂ after 5 days of growth at 28 °C and pH 7.5, and to produce 8.0, 2.0 and 3.0 μg ml^?1 of IAA when supplemented with l-tryptophan (1 mg ml^?1) for 72 h, at 28 °C and pH 7.0, respectively. The solubilization of insoluble phosphate was associated with a drop in the pH of the culture medium and there was an inverse relationship between pH and solubilized P (r = ?0.98, P < 0.0952). There were no significant differences among isolates in terms of acidity tolerance based on their confidence limits as assessed by segmented model analysis and all isolates were able to grow at pH 4.3–11 (with optimum at 7.0–7.5). Based on a sigmoidal trend of a three-parameter logistic model, the salt concentration required for 50 % inhibition was 8.15, 6.30 and 8.23 % NaCl for M36, M100 and M183 isolates, respectively. Moreover, the minimum and maximum growth temperatures estimated by the segmented model were 5.0 and 42.75 °C for M36, 12.76 and 40.32 °C for M100, and 10.63 and 43.66 °C for M183. The three selected isolates could be deployed as inoculants to promote plant growth in an agricultural environment.     

Feeding elicitors and precursors enhance colchicine accumulation in morphogenic cultures of <Emphasis Type="Italic">Gloriosa superba</Emphasis> L.

Morphogenic cultures of Gloriosa superba were initiated on Murashige and Skoog’s medium fortified with 2 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L^?1 naphthaleneacetic acid (NAA), 4% sucrose and 0.1% activated charcoal. To enhance the content of the alkaloid colchicine, morphogenic cultures were treated with different concentrations of abiotic elicitors like signalling compounds, metals, biotic elicitors, precursors and a combination of elicitors. Signalling molecules like acetyl salicylic acid (ASA) and sodium nitroprusside improved the production of colchicine. Abiotic elicitors have markedly (p?≤?0.05 or ≤?0.01) enhanced the colchicine content either at lower or higher concentrations. Among the metals, the highest amount of 11.67 mg of colchicine g^?1 dry wt was noticed at 60 mM rubidium chloride, followed by 60 mM NaCl (11.18 mg g^?1). Contrarily, in the presence of biotic elicitors such as Fusarium oxysporum, Alternaria solani, and Saccharomyces cerevisiae, colchicine content ranged only between 2 and 5.32 mg g^?1, but Bacillus subtilis repressed it. Among the aromatic amino acids, phenylalanine at 500 mg L^?1 influenced the highest accumulation of 19.48 mg g^?1 dry tissue, followed by tryptophan (12.47 mg g^?1), and tyrosine (9.87 mg g^?1), a direct precursor of colchicine biosynthesis, while intact tubers and leaves contained 4.65 and 4.16 mg of colchicine g^?1 dry tissue respectively. A combination of 10 µM AlCl₃ and 50 µM salicylic acid (SA) registered 17.34 mg g^?1 followed by 16.24 mg g^?1 tissue in presence of 1 µM HgCl₂ and 50 µM SA. The results suggest that the elicitor-stimulated colchicine accumulation was a stress response and can be exploited further for commercial production.