Fungal Elicitor Research Articles

Fusarium oxysporum cell elicitor enhances betalain content in the cell suspension culture of Celosia cristata.

We started a cell suspension culture from magenta coloured calli of cockscomb to study the effect of biotic and abiotic elicitors on the biosynthesis of betalain pigments. The cultures were grown in a flask containing 30ml MS media fortified with 13.5μM 2,4-D and 0.44μM BAP. These cultures were elicited during its log-phase of growth using fungal elicitors (prepared from mycelia of Fusarium oxysporum), yeast extract, copper sulphate and cobalt chloride. The elicitation reduced the cell count, cell viability and percent pigmented cell in the suspension culture. Similarly, it also resulted in reduced betalain content by all the elicitors except 0.125×10-3% fungal elicitor. Rather, fungal elicitor at this concentration significantly enhanced the amaranthin, betanin, betalamic acid and betaxanthin content in the culture. Besides this, copper sulphate doubled the pigment contribution (ratio of particular pigment content to total pigment content) of betaxanthin at all the concentrations. Therefore, we conclude that fungal elicitor can further be investigated to enhance the content of betalain pigments in suspension culture at a larger scale.

Transcriptome profiling of lentil (Lens culinaris) through the first 24 hours of Ascochyta lentis infection reveals key defence response genes

BackgroundAscochyta blight, caused by the fungus Ascochyta lentis, is one of the most destructive lentil diseases worldwide, resulting in over $16 million AUD annual loss in Australia alone. The use of resistant cultivars is currently considered the most effective and environmentally sustainable strategy to control this disease. However, little is known about the genes and molecular mechanisms underlying lentil resistance against A. lentis.ResultsTo uncover the genetic basis of lentil resistance to A. lentis, differentially expressed genes were profiled in lentil plants during the early stages of A. lentis infection. The resistant ‘ILL7537’ and susceptible ‘ILL6002’ lentil genotypes were examined at 2, 6, and 24 h post inoculation utilising high throughput RNA-Sequencing. Genotype and time-dependent differential expression analysis identified genes which play key roles in several functions of the defence response: fungal elicitors recognition and early signalling; structural response; biochemical response; transcription regulators; hypersensitive reaction and cell death; and systemic acquired resistance. Overall, the resistant genotype displayed an earlier and faster detection and signalling response to the A. lentis infection and demonstrated higher expression levels of structural defence-related genes.ConclusionsThis study presents a first-time defence-related transcriptome of lentil to A. lentis, including a comprehensive characterisation of the molecular mechanism through which defence against A. lentis is induced in the resistant lentil genotype.

Cloning and Characterization of Promoters of the Fungal Immunomodulatory Protein Genes from Ganoderma spp. (Agaricomycetes) and Their Response to Methyl Jasmonate and Salicylic Acid.

Ganoderma mushrooms for medicinal use contain various bioactive compounds, but the genetic elements available for these medicinal mushrooms are still limited. In this study we cloned and analyzed the promoters of fungal immunomodulatory protein (FIP) genes from G. lucidum and G. atrum. FIP gene expression was induced by different concentrations of methyl jasmonate (MeJA) and salicylic acid (SA), and messenger RNA expression was detected by quantitative reverse-transcription polymerase chain reaction. The results provided 5' upstream sequences of FIP genes from G. lucidum and G. atrum. Sequence analysis showed that the FIP-glu promoter sequence contained 11 CAAT boxes, 3 TATA boxes, 3 MeJA-responsive elements, 3 MYB binding site (MBS) motifs, 1 abscisic acid responsive element, 1 TGA, 1 anaerobic inducible element, 2 circadian elements, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. The 5' flanking region of FIP-gat included 9 CAAT boxes, 4 TATA boxes, 3 MeJA-responsive elements, 1 AuxRR core, 1 GC motif, 1 MBS, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. On the transcriptional level, both FIP-glu and FIP-gat reached their highest expression after treatment with MeJA at 500 μmol/L. FIP-glu expression depended on the concentration of SA (0-1000 mg/L); the expression of the FIP-gat gene was highest at a concentration of 100 mg MeJA/L. This research lays the foundation to use Ganoderma mycelia as bioreactors for producing FIPs.

The chitinase activity of oil palm (Elaeis guineensis Jacq.) roots against fungal endophytes and pathogenic Ganoderma boninense

Application of fungal endophytes can be an alternative to control basal stem rot disease in oil palm, caused by 'Ganoderma boninense'. Chitinase is a type of defensive protein synthesized by plants in response to biotic factors. The purpose of this study was to analyze the chitinase activity of oil palm as a defensive mechanism to fungal endophytes and pathogenic 'G. boninense'. Four species of fungal endophytes, 'Trichoderma harzianum' MTP 10 (Th-MTP10), Trichoderma longibrachiatum KBA 31 (Tl-KBA 31), 'Lasiodiplodia venezuelensis' MJP 28 (Lv-MJP 28), 'Dothidiomycetes' sp. MTD 29 (Dt-MTD 29) and one species of fungal pathogen 'G. boninense' and with their each cell wall suspension were introduced to oil palm plantlets in axenic condition. Chitinase activity was observed from the root of oil palm plantlets inoculated with both living cell and cell wall suspension of endophytic fungi and pathogenic 'G. boninense'. Results showed that chitinase activities varied in each fungal treatment and were significantly differed from control. Fungal cell wall elicitors were able to significantly induce chitinase activity after 1 week post treatment (wpt). Statistically, only the chitinase activity from fungal endophyte Lv-MJP 28 was significantly higher from others for 8 and 12 days. Pre-treatment of oil palm plantlet with fungal cell wall suspension for 1 wpt could induce the chitinase activity higher than control when, oil palm infected with fungal pathogen of 'Ganoderma boninense'.

MiR858-Mediated Regulation of Flavonoid-Specific MYB Transcription Factor Genes Controls Resistance to Pathogen Infection in Arabidopsis.

MicroRNAs (miRNAs) are a class of short endogenous non-coding small RNAs that direct post-transcriptional gene silencing in eukaryotes. In plants, the expression of a large number of miRNAs has been shown to be regulated during pathogen infection. However, the functional role of the majority of these pathogen-regulated miRNAs has not been elucidated. In this work, we investigated the role of Arabidopsis miR858 in the defense response of Arabidopsis plants to infection by fungal pathogens with necrotrophic (Plectosphaerella cucumerina) or hemibiotrophic (Fusarium oxysporum and Colletotrichum higginsianum) lifestyles. Whereas overexpression of MIR858 enhances susceptibility to pathogen infection, interference with miR858 activity by target mimics (MIM858 plants) results in disease resistance. Upon pathogen challenge, stronger activation of the defense genes PDF1.2 and PR4 occurs in MIM858 plants than in wild-type plants, whereas pathogen infection induced weaker activation of these genes in MIR858 overexpressor plants. Reduced miR858 activity, and concomitant up-regulation of miR858 target genes, in MIM858 plants, also leads to accumulation of flavonoids in Arabidopsis leaves. The antifungal activity of phenylpropanoid compounds, including flavonoids, is presented. Furthermore, pathogen infection or treatment with fungal elicitors is accompanied by a gradual decrease in MIR858 expression in wild-type plants, suggesting that miR858 plays a role in PAMP (pathogen-associated molecular pattern)-triggered immunity. These data support that miR858 is a negative regulator of Arabidopsis immunity and provide new insights into the relevant role of miR858-mediated regulation of the phenylpropanoid biosynthetic pathway in controlling Arabidopsis immunity.

Selinene Volatiles Are Essential Precursors for Maize Defense Promoting Fungal Pathogen Resistance.

To ensure food security, maize (Zea mays) is a model crop for understanding useful traits underlying stress resistance. In contrast to foliar biochemicals, root defenses limiting the spread of disease remain poorly described. To better understand belowground defenses in the field, we performed root metabolomic profiling and uncovered unexpectedly high levels of the sesquiterpene volatile β-selinene and the corresponding nonvolatile antibiotic derivative β-costic acid. The application of metabolite-based quantitative trait locus mapping using biparental populations, genome-wide association studies, and near-isogenic lines enabled the identification of terpene synthase21 (ZmTps21) on chromosome 9 as a β-costic acid pathway candidate gene. Numerous closely examined β-costic acid-deficient inbred lines were found to harbor Zmtps21 pseudogenes lacking conserved motifs required for farnesyl diphosphate cyclase activity. For biochemical validation, a full-length ZmTps21 was cloned, heterologously expressed in Escherichia coli, and demonstrated to cyclize farnesyl diphosphate, yielding β-selinene as the dominant product. Consistent with microbial defense pathways, ZmTps21 transcripts strongly accumulate following fungal elicitation. Challenged field roots containing functional ZmTps21 alleles displayed β-costic acid levels over 100 μg g-1 fresh weight, greatly exceeding in vitro concentrations required to inhibit the growth of five different fungal pathogens and rootworm larvae (Diabrotica balteata). In vivo disease resistance assays, using ZmTps21 and Zmtps21 near-isogenic lines, further support the endogenous antifungal role of selinene-derived metabolites. Involved in the biosynthesis of nonvolatile antibiotics, ZmTps21 exists as a useful gene for germplasm improvement programs targeting optimized biotic stress resistance.

The Role of Cell Wall Degrading Enzymes in Pathogenesis of Magnaporthe oryzae.

The plant cell wall is always the physical barrier in which phytopathogenic fungi must overcome by producing an array of cell wall degrading enzymes (CWDEs) that allow them to invade host tissues through the degradation of cell wall components of plants. Magnaporthe oryzae is a causal agent of blast disease, one of the most devastating disease in rice resulting significant crop losses worldwide. The penetration of plant cuticle and cell walls induced by infection structures of M. oryzae has been known to be acquired by the association of turgor pressure and CWDEs for successful infection of M. oryzae. In this review, we focus on recent discoveries of M. oryzae CWDEs, gene regulation and their biological roles as fungal virulence factors and elicitors of host defense response leading to plant resistance against fungal pathogens.

Identification and functionality prediction of pathogenesis-related protein 1 from legume family.

The production and accumulation of pathogenesis-related (PR) proteins in plants is one of the important responses to biotic and abiotic stress. Large number of identified PR proteins has been categorized into 17 functional families based on their structure, phylogenetics, and biological activities. However, they are not widely studied in legume crops. Using 29 PR1 proteins from Arabidopsis thaliana, as query, here we have predicted 92 candidate PR1 proteins through the PSI-BLAST and HMMER programs. These candidate proteins were comprehensively analyzed with, multiple sequence alignment, domain architecture studies, signal peptide, and motif extraction followed by phylogenetic analysis. Further, response of two candidate PR1 proteins from chickpea against Fusarium oxysporum f.sp.ciceri attack was validated using qRT-PCR followed by their 3D structure prediction. To decipher mode of action for PR1s, docking of pathogen extracellular matrix components along with fungal elicitors was performed with two chickpea PR1 proteins. Based on these findings, we propose carbohydrate to be the unique pathogen-recognition feature for PR1 proteins and β-glucanase activity via β-glucan binding or modification.

Physicochemical and microbial responses of Streptomyces natalensis HW-2 to fungal elicitor.

The effects of fungal elicitor on the physicochemical and microbial responses of Streptomyces natalensis HW-2 were investigated. The results showed that the elicitor could decrease dry cell weight (DCW) by 17.7% and increase the utilization of glucose, while the curve of pH was not obviously altered. The elicitor enhanced the yield of natamycin from 1.33 to 2.49g/L. The morphology of the colony and the mycelium treated with elicitor showed significant differences from that of control. The level of intracellular reactive oxygen species (ROS) increased to 333.8ng/L, which was a twofold increase comparing with the control. The concentration of Ca2+ reached 421.1nmol/L, which increased by 32.8% after the addition of the elicitor. The activities of pyruvic carboxylase and phosphoenol pyruvate carboxylase were enhanced by 27.8 and 11.9%, respectively, while citrate synthase activity decreased by 23.1% in comparison with the control.

CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis.

Revelation and cloning of valinomycin synthetase genes in Streptomyces lavendulae ACR-DA1 and their expression analysis under different fermentation and elicitation conditions

Streptomyces species are amongst the most exploited microorganisms due to their ability to produce a plethora of secondary metabolites with bioactive potential, including several well known drugs. They are endowed with immense unexplored potential and substantial efforts are required for their isolation as well as characterization for their bioactive potential. Unexplored niches and extreme environments are host to diverse microbial species. In this study, we report Streptomyces lavendulae ACR-DA1, isolated from extreme cold deserts of the North Western Himalayas, which produces a macrolactone antibiotic, valinomycin. Valinomycin is a K+ ionophoric non-ribosomal cyclodepsipeptide with a broad range of bioactivities including antibacterial, antifungal, antiviral and cytotoxic/anticancer activities. Production of valinomycin by the strain S. lavendulae ACR-DA1 was studied under different fermentation conditions like fermentation medium, temperature and addition of biosynthetic precursors. Synthetic medium at 10°C in the presence of precursors i.e. valine and pyruvate showed enhanced valinomycin production. In order to assess the impact of various elicitors, expression of the two genes viz. vlm1 and vlm2 that encode components of heterodimeric valinomycin synthetase, was analyzed using RT-PCR and correlated with quantity of valinomycin using LC–MS/MS. Annelid, bacterial and yeast elicitors increased valinomycin production whereas addition of fungal and plant elicitors down regulated the biosynthetic genes and reduced valinomycin production. This study is also the first report of valinomycin biosynthesis by Streptomyces lavendulae.

Metabolomics reveals biotic and abiotic elicitor effects on the soft coral Sarcophyton ehrenbergi terpenoid content

The effects of six biotic and abiotic elicitors, i.e. MeJA (methyl jasmonate), SA (salicylic acid), ZnCl2, glutathione and β-glucan BG (fungal elicitor), and wounding, on the secondary metabolite accumulation in the soft coral Sarcophyton ehrenbergi were assessed. Upon elicitation, metabolites were extracted and analysed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Except for MeJA, no differences in photosynthetic efficiency were observed after treatments, suggesting the absence of a remarkable stress on primary production. Chemometric analyses of UPLC-MS data showed clear segregation of SA and ZnCl2 elicited samples at 24 and 48 h post elicitation. Levels of acetylated diterpene and sterol viz., sarcophytonolide I and cholesteryl acetate, was increased in ZnCl2 and SA groups, respectively, suggesting an activation of specific acetyl transferases. Post elicitation, sarcophytonolide I level increased 132 and 17-folds at 48 h in 0.1 mM SA and 1 mM ZnCl2 groups, respectively. Interestingly, decrease in sarcophine, a major diterpene was observed only in response to ZnCl2, whereas no change was observed in sesquiterpene content following treatments. To the best of our knowledge, this study provides the first documentation for elicitation effects on a soft corals secondary metabolome and suggests that SA could be applied to increase diterpenoid levels in corals.

The rice LysM receptor-like kinase OsCERK1 is required for the perception of short-chain chitin oligomers in arbuscular mycorrhizal signaling.

The rice lysin-motif (LysM) receptor-like kinase OsCERK1 is now known to have a dual role in both pathogenic and symbiotic interactions. Following the recent discovery that the Oscerk1 mutant is unable to host arbuscular mycorrhizal (AM) fungi, we have examined whether OsCERK1 is directly involved in the perception of the short-chain chitin oligomers (Myc-COs) identified in AM fungal exudates and shown to activate nuclear calcium (Ca2+ ) spiking in the rice root epidermis. An Oscerk1 knockout mutant expressing the cameleon NLS-YC2.60 was used to monitor nuclear Ca2+ signaling following root treatment with either crude fungal exudates or purified Myc-COs. Compared with wild-type rice, Ca2+ spiking responses to AM fungal elicitation were absent in root atrichoblasts of the Oscerk1 mutant. By contrast, rice lines mutated in OsCEBiP, encoding the LysM receptor-like protein which associates with OsCERK1 to perceive chitin elicitors of the host immune defense pathway, responded positively to Myc-COs. These findings provide direct evidence that the bi-functional OsCERK1 plays a central role in perceiving short-chain Myc-CO signals and activating the downstream conserved symbiotic signal transduction pathway.

A fungal elicitor induces Sclerotium rolfsii sacc resistance in Atractylodis maceocephalae koidz

The enzymatic activities (POD, PPO, CAT, PAL), as protective enzymes in the leaves of Atractylodis maceocephalae koidz, were tested by treating different concentrations of polysaccharides isolated from Sclerotium rolfsii sacc (P. S. rolfsii) at 0 mg/l, 20 mg/l, 200 mg/l and 400 mg/l against S. rolfsii, and atractylenolides as a phytoalexin in the rhizome of A. maceocephalae were evaluated in compared with control. It was evident that the plant under stress by pathogen has instigated the significant synthesis and accumulation of atractylenolides and the higher enzymes activities were described on the eight day after fungal elicitor inoculation than the control group. Furthermore, the treatments of A. maceocephalae seedlings with P. S.rolfsii increased disease index development caused by S. rolfsii. The disease index is lowest when inoculated at a concentration as low as 20 mg/ml. In general, these results indicated that P. S. rolfsii may be useful as a fungal elicitor, which can enhance resistance and triggered innate immunity in A. maceocephalae, and had the potential to suppress the disease on A. maceocephalae when P. S. rolfsii at 20 mg/l, were used to inoculate the root of A. maceocephalae.

Genome-Wide Dissection of the Heat Shock Transcription Factor Family Genes in Arachis.

Heat shock transcription factors (Hsfs) are important transcription factors (TFs) in protecting plants from damages caused by various stresses. The released whole genome sequences of wild peanuts make it possible for genome-wide analysis of Hsfs in peanut. In this study, a total of 16 and 17 Hsf genes were identified from Arachis duranensis and A. ipaensis, respectively. We identified 16 orthologous Hsf gene pairs in both peanut species; however HsfXs was only identified from A. ipaensis. Orthologous pairs between two wild peanut species were highly syntenic. Based on phylogenetic relationship, peanut Hsfs were divided into groups A, B, and C. Selection pressure analysis showed that group B Hsf genes mainly underwent positive selection and group A Hsfs were affected by purifying selection. Small scale segmental and tandem duplication may play important roles in the evolution of these genes. Cis-elements, such as ABRE, DRE, and HSE, were found in the promoters of most Arachis Hsf genes. Five AdHsfs and two AiHsfs contained fungal elicitor responsive elements suggesting their involvement in response to fungi infection. These genes were differentially expressed in cultivated peanut under abiotic stress and Aspergillus flavus infection. AhHsf2 and AhHsf14 were significantly up-regulated after inoculation with A. flavus suggesting their possible role in fungal resistance.

Differential abilities of Korean soybean varieties to biosynthesize glyceollins by biotic and abiotic elicitors.

Glyceollins synthesized in soybeans that are exposed to biotic or abiotic stress have been reported to have health benefits. Considering that glyceollins are de novo synthesized from daidzein via several enzymatic steps and that isoflavone concentration widely varies among soybean varieties, the abilities of 60 soybean cultivars to synthesize glyceollins were compared under different elicitation conditions. Soybeans accumulated glyceollins differentially depending upon the cultivar when elicited with Aspergillus sojae. Contrary to our hypothesis that high isoflavone varieties may accumulate glyceollins more efficiently upon elicitation, glyceollin accumulation in response to fungal elicitation was not related with the concentration of either total isoflavones or daidzein in soybeans. Rather the glyceollin levels were significantly affected by soybean cultivar and most effectively increased by fungal infection. The data suggest that the selection of a strong fungal elicitor and a soybean cultivar with genotype that highly expresses the genes involved in glyceollin biosynthesis is essential for efficient glyceollin production.

The position of prenylation of isoflavonoids and stilbenoids from legumes (Fabaceae) modulates the antimicrobial activity against Gram positive pathogens.

The legume plant family (Fabaceae) is a potential source of antimicrobial phytochemicals. Molecular diversity in phytochemicals of legume extracts was enhanced by germination and fungal elicitation of seven legume species, as established by RP-UHPLC-UV-MS. The relationship between phytochemical composition, including different types of skeletons and substitutions, and antibacterial properties of extracts was investigated. Extracts rich in prenylated isoflavonoids and stilbenoids showed potent antibacterial activity against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus at concentrations between 0.05 and 0.1% (w/v). Prenylated phenolic compounds were significantly (p<0.01) correlated with the antibacterial properties of the extracts. Furthermore, the position of the prenyl group within the phenolic skeleton also influenced the antibacterial activity. Overall, prenylated phenolics from legume seedlings can serve multiple purposes, e.g. as phytoestrogens they can provide health benefits and as natural antimicrobials they offer preservation of foods.

Chemical solution is an efficient method to induce the formation of 2-(2-phenylethyl) chromone derivatives in Aquilaria sinensis

Agarwood is a highly valuable resinous and fragrant heartwood used worldwide as advanced tea, incense for religious ceremonies, traditional Chinese medicine and perfume. Healthy trees do not produce agarwood; wounding and infectious disease initiate its formation. In this study, mechanical wounds, fungal elicitors and salt solution were applied to induce the formation of 2-(2-phenylethyl) chromones, which are the principal compounds in agarwood. In this research, chemical solution was the best inducer that could induce Aquilaria sinensis calli to produce the highest amount of chromones E and M at 79.20 and 41.62μgg−1, respectively. Chemical solution was then applied on three-year-old saplings by transfusion to verify its induction effect. Chromones E and M were detected in treated saplings after 15 d and then increased with increasing pruning time; their contents reached 22.05 and 3.42μgg−1, respectively. Our results suggested that chemical solution was the most promising chemical inducer for agarwood production on the industrial scale.

Effects of Polysaccharide Elicitors from Endophytic Fusarium oxysporum Fat9 on the Growth, Flavonoid Accumulation and Antioxidant Property of Fagopyrum tataricum Sprout Cultures.

The purpose of this study was to evaluate the effects of four different fungal polysaccharides, named water-extracted mycelia polysaccharide (WPS), sodium hydroxide-extracted mycelia polysaccharide (SPS), hydrochloric-extracted mycelia polysaccharide (APS), and exo-polysaccharide (EPS) obtained from the endophytic Fusarium oxysporum Fat9 on the sprout growth, flavonoid accumulation, and antioxidant capacity of tartary buckwheat. Without visible changes in the appearance of the sprouts, the exogenous polysaccharide elicitors strongly stimulated sprout growth and flavonoid production, and the stimulation effect was closely related with the polysaccharide (PS) species and its treatment dosage. With application of 200 mg/L of EPS, 200 mg/L of APS, 150 mg/L of WPS, or 100 mg/L of SPS, the total rutin and quercetin yields of buckwheat sprouts were significantly increased to 41.70 mg/(100 sprouts), 41.52 mg/(100 sprouts), 35.88 mg/(100 sprouts), and 32.95 mg/(100 sprouts), respectively. This was about 1.11 to 1.40-fold compared to the control culture of 31.40 mg/(100 sprouts). Moreover, the antioxidant capacity of tartary buckwheat sprouts was also enhanced after treatment with the four PS elicitors. Furthermore, the present study revealed the polysaccharide elicitation that caused the accumulation of functional flavonoid by stimulating the phenylpropanoid pathway. The application of beneficial fungal polysaccharide elicitors may be an effective approach to improve the nutritional and functional characteristics of tartary buckwheat sprouts.

Response surface modeling of natural alizarin production in hairy root cultures of Rubia tinctorum L. upon elicitation with fungal mycelia

The roots of Rubia tinctorum L., the common madder contain natural red dye known as alizarin. In the current study, central composite design of response surface methodology was employed for modeling of fungal elicitor treatment on natural alizarin production in uniform hairy root cultures of common madder in liquid 1/2 B5 medium. Upon fungal elicitation assay, using two fungal mycelia elicitors ( Aspergillus niger and Bipolaris maydis ) at three different times (0, 12 and 24h), the production of alizarin was determined. According to the results, after 24h; modeling and optimization conditions, including combination of 2 % of both elicitors for alizarin production equal to 10.0 mg.g -1 DW was evaluated. Optimal process parameters have been determined by using a high desirability value of 1.00 in Design-Expert ® software. Our results, altogether, offer a promising method regarding to the improvement of the alizarin production, as a pivotal natural dye in industrial applications.