Isolation and molecular identification of β-carotene producing strains of Dunaliella salina and Dunaliella bardawil from salt soil samples by using species-specific primers and internal transcribed spacer (ITS) primers

Dunaliella salina and Dunaliella bardawil are unique species of the genus Dunaliella that produce large amounts of â-carotene when cultivated under appropriate conditions. These include high light intensity, high sodium chloride concentration, nitrate deficiency and extreme temperatures. Under these conditions, only D. salina and D. bardawil can accumulate â-carotene to as much as 10% of the cellular dry weight. Because the morphological characterization is based on the environmental factors, the Dunaliella will change the shape, so identification and differentiation of Dunaliella species by morphology is very difficult. In this research study, we isolated, identified and discriminated the different Dunaliella β-carotene producing strains from salt soil samples, by using 18S rDNA and internal transcribed spacer (ITS) gene sequences. The soil samples were collected from four different provinces of the North Eastern part of Thailand.namely: UdonThani, BuriRam, AmnartCharoen and Chaiyaphum. Among the four isolates, only BuriRam KU01 and UdonThani KU01 were D. salina and D. bardawil, respectively whereas AmnartCharoen KU01 and Chaiyaphum KU01 were not these Dunaliella species. At 4 M NaCl, with deficiency of nitrate (KNO3) and phosphate (KH2PO4) in the medium, the D. salina strain BuriRam KU01 produced β-carotene at the level of 56.25 ± 0.97 pg·cell-1 and D. bardawil strain UdonThani KU01 produced β-carotene at the level of 52.91 ± 0.29 pg·cell-1 at the 25th day after inoculation. The 18S rDNA and ITS sequences of D. salina strain BuriRam KU01 and D. bardawil strain UdonThani KU01 were submitted to the National Center for Biotechnology Information (NCBI) database with accession numbers of JN052202, JN052203, JN034031 and JN052204, respectively. By using the species-specific primers and ITS primers the â-carotene producing strains of Dunaliella was identified.Key words: 18S rDNA, β-carotene, carotenoid, Dunaliella bardawil, Dunaliella salina, internal transcribed spacer (ITS), salinity

Effects of light intensity and the remaining nitrate concentration on the beta-carotene accumulation of a wild Dunaliella salina strain isolated from the saline soil

An isolated Dunaliella salina strain from northern Thailand was cultured in modified Johnson’s medium in column photobioreactor. The beta-carotene accumulation mainly depended on the quantities of cells entering into carotenogenesis condition that was significantly enhanced by high started KNO3 concentration. Low remaining nitrate concentration in the culture of each cell (RNCC) was suitable for algae to accumulate beta-carotene. Following the cultivation time extended, RNCC of all cultures decreased and tended to the same level (10-20 pg/cell) although the biomass or betacarotene content in the culture was higher in high started KNO3 concentration. High light intensity restrained the growth especially in low KNO3 concentration but improved betacarotene accumulation and RNCC. The highest biomass and beta-carotene dry weight (DW) were 2.25 g L-1 and 79.2 mg g-1 DW respectively. Above results indicated that increasing the biomass and as early as possible to strengthen the stress on each cell was important to improve the final beta-carotene yield

Short-term physiological responses to drought stress in seedling of tropical and temperate maize (Zea mays L.) cultivars

Understanding of the response of tropical and temperate maize (Zea mays L.) to drought is the first step for tolerant temperate maize improvement. Eight maize hybrids were used to investigate physiology responses under drought stress, four of them were tropical maize and the others were temperate maize. Results showed that there were different drought tolerances but similar trends in both tropical maize and temperate maize. Gas exchange parameters revealed different strategies of maize under the stress. In our study, most of the temperate hybrids maintained open stomata to keep a higher photosynthesis rate at the beginning of stress, while the other hybrids decreased stomatal conductance. Compared to temperate maize, the tropical maize had higher antioxidase activity and greater physiological parameter variation among hybrids. KS5731 and ZD309 had stronger drought resistance among tropical and temperate maize hybrids separately. Tolerant hybrids maintained active photosynthesis, have higher osmotic adjustment ability and antioxidase activities but lower malonaldehyde content than the sensitive ones. Our results led to a better understanding of the physiological responses of tropical and temperate maize plants to drought stress and may provide an insight of breeding for drought resistance in maize

Effects of phosphorus on the growth and chlorophyll fluorescence of a Dunaliella salina strain isolated from saline soil under nitrate limitation

An isolated Dunaliella salina (D. salina) KU XI from saline soils in northeastern Thailand was cultured in f/2 medium in column photobioreactor. The variations of the growth, chlorophyll and beta-carotene content and the maximum quantum yield of PS II photochemistry (Fv/Fm) under different NaH2PO4 concentrations were studied. Based on the results, the growth kinetics of D. salina KU XI was established, which could simulate the algae growth rate under different phosphate concentrations and temperatures. The phosphorus could significantly affect the growth and pigments accumulations of this isolated strain. Increasing NaH2PO4 concentration improved the biomass, the total chlorophyll and beta-carotene content, retarded the decrease of Fv/Fm value. The optimal phosphate concentration for the growth of D. salina KU XI was above 72.6 μM. The maximum biomass and beta-carotene were 0.24 g L-1 and 17.4 mg L-1 respectively when NaH2PO4 was 290.4 μM. The algae growth was restrained by phosphate or nitrate when NaH2PO4 below 12.1 μM or above 72.6 μM. It indicated that properly supplementing nitrate in the late growth stage with high phosphate concentration was favored for enhancing the growth and biomass production

β-carotene Production of UV-C Induced Dunaliella salina Under Salt Stress

Dunaliella salina is an important microalga for β-carotene production. In Thailand, the D. salina has not been commercially cultivated. Aim of this study was to screen high β-carotene producing mutants. D. salina KU11 cells were radiated with 0.4 mmol photons m-2·s-1 UV-C for 22 min. After 3 weeks, colonies which survived after radiation were allowed to grow on agar plates; fifty colonies out of 19,720 UV-mutagenized strains turned to orange. These colonies were picked up and transfer to modified Johnson medium. Four mutants out of fifty were KU5, KU18, KU20 and KU37 significantly produced high β-carotene at the stationary phase approximately up to 1.62, 2.03, 1.32 and 1.21 folds, respectively, compared to the wild type strain. In all three consecutive cycles of cultivation, the mutant KU18 accumulated 2.4-8.5 folds β-carotene (µg/ml) and 2.1-4.8 folds β-carotene (pg/cell) higher than the wild type, whereas cell density of the mutant KU18 was not different from the wild type. This is the new record of b-carotene producing mutant isolated from saline soil in Thailand

Changing in TSS, TA and Sugar Contents and Sucrose Synthase Activity in Ethephon-Treated 'Pattavia' Pineapple Fruit

ABSTRACT Exogenous ethylene increases endogenous ethylene which plays a crucial role on ripening in climacteric fruits. Although pineapple is a non-climacteric fruit, ethylene released from ethephon is effectively used to hasten the harvesting period. Effects from the use of a high concentration of ethephon on eating quality, fruit size and the reduction in harvesting period have been reported. In this paper, the effect of a low concentration of ethephon on pineapple fruit quality and sucrose synthase (SuSy) activity was investigated. Field experiment was arranged in split plot design. In the main plot, two levels of ethephon concentrations, i.e. 0 and 500 mg/l, were used by spraying at 110 days after forcing (DAF) fruits. The sub plot was harvesting time, i.e. 5 times of one-week intervals from 124 to 152 DAF. We found that the total soluble solid (TSS) was significantly increased in most of harvesting-treated fruits while the titratable acid (TA) was significantly increased at 131 DAF of harvesting-treated fruits. Only at 131 DAF harvesting time, the glucose content and SuSy activity of ethephon-treated fruits were significantly reduced and return to the control level afterward. However, ethephon had no effect on the fructose and sucrose contents at all harvesting times. In conclusion, fruit quality with shortening of harvesting time could be improved by applying 500 mg/l ethephon at 110 DAF since TSS content which is one of the parameter predicting eating quality of pineapple was increased without decreasing fruit quality

Stimulation of Hydrogen Photoproduction in Chlorella sorokiniana Subjected to Simultaneous Nitrogen Limitation and Sulfur- and/or Phosphorus-Deprivation

Photosynthetic hydrogen (H2 ) production by green algae has fascinated biologists and energy experts, due to the potential application of this process for renewable energy. In this study, H2 photoproduction and PSII photochemical activities were investigated in Chlorella sorokiniana exposed to simultaneous nitrogen limitation and sulfur (S-) and/ or phosphorus (P-) deprivation. Under S-deprivation, C. sorokiniana produced about 48.2 mL L-1 of H2 . Moreover, simultaneous nitrogen limitation (0.7 mM NH4 Cl) and sulfur- and/or phosphorus-deprivation significantly increased H2 production of C. sorokiniana over that of S-deprivation alone. Maximum H2 outputs of 77.3, 98.1 and 125.1 mL L-1 were obtained in the N-limited cultures exposed to P-deprivation (TAP-P), S-deprivation (TAP-S) and simultaneous S- and P-deprivation (TAP-S-P), respectively. The average rate of H2 production for the N-limited culture exposed to TAP-P, TAP-S and TAP-S-P was 1.07, 1.36 and 1.50 mL L-1 h-1, respectively. Interestingly, the H2 inducement time in the culture subjected to simultaneous N-limitation and S- and/or P-deprivation was much shorter than that of traditional S-deprivation. The photosynthetic inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropylp-benzoquinone (DBMIB) repressed H2 production in TAP-S-P (0.7 mM NH4 Cl) medium by 68.04% and 98.65%, respectively. The conditions of simultaneous N-limitation, S- and P-deprivation provided another efficient method for inducing H2 production in C. sorokiniana. In addition, we also found that two-thirds of the required electrons were generated from the splitting of H2 O in PSII and that the remaining onethird possibly came from some other substrate catabolism

<span style="font-size:15.0pt;mso-bidi-font-family:"Times New Roman";mso-bidi-font-weight: bold" lang="EN-GB">Screening, phenotypic and genotypic identification of β-carotene producing strains of <i style="mso-bidi-font-style:normal">Dunaliella</i> <i style="mso-bidi-font-style:normal">salina</i> from Thailand </span>

2198-2216<i style="mso-bidi-font-style: normal">Dunaliella salina is a salt-loving microalga that accumulates high amounts of β-carotene when cultivated under unfavorable conditions. In this study we aimed to screen <span style="mso-bidi-font-weight:bold; mso-bidi-font-style:italic">β-carotene producing strains of D.salina <span style="mso-bidi-font-style: italic">from salt soil samples collected from 19 provinces in the northeastern part of Thailand. For preliminary screening 70 pure isolated strains were screened by using 18S rDNA conserved primers (MA1&MA2), only 15 isolates produced a band (~2100 bp) as that of <i style="mso-bidi-font-style: normal">D.salina.  For the o<span style="mso-bidi-font-style: italic">bservation of β-carotene production, these 15 isolates were grown on agar plate containing modified Ramaraj medium with 1.0 M NaCl under continuous illumination.  After a month only 3 isolates completely turned red. However, by using the banding pattern produced by 18S rDNA primers, the 15 isolates could not be differentiated between the β-carotene and non β-carotene producing strains. Comparing the above results we found that the <span style="mso-bidi-font-style: italic">observation of β-carotene production on agar plate is more easy and suitable method than molecular technique to screen β-carotene producing strains. Morphological characteristics of the 3 screened Thai isolates clearly delineated that it belongs to the genus Dunaliella. ITS-RFLP banding pattern, 18S rDNA, ITS and RuBisCo large subunit (rbcL) sequences were used to confirm at the species level. These results indicated that these 3 isolates are D.salina and were named <i style="mso-bidi-font-style: normal">D.salina strain KU07, D.salina strain KU11 and D.salina strain KU13. Under non-stress conditions these 3 strains had an ability to accumulate β-carotene up to 5.61±0.25, 7.58±0.19 and 6.73±0.32 pg/cell, respectively. </span

Transcriptome analysis reveals defense-related genes and pathways against Xanthomonas campestris pv. vesicatoria in pepper (Capsicum annuum L.).

Bacterial spot (BS), incited by Xanthomonas campestris pv. vesicatoria (Xcv), is one of the most serious diseases of pepper. For a comparative analysis of defense responses to Xcv infection, we performed a transcriptomic analysis of a susceptible cultivar, ECW, and a resistant cultivar, VI037601, using the HiSeqTM 2500 sequencing platform. Approximately 120.23 G clean bases were generated from 18 libraries. From the libraries generated, a total of 38,269 expressed genes containing 11,714 novel genes and 11,232 differentially expressed genes (DEGs) were identified. Functional enrichment analysis revealed that the most noticeable pathways were plant-pathogen interaction, MAPK signaling pathway-plant, plant hormone signal transduction and secondary metabolisms. 1,599 potentially defense-related genes linked to pattern recognition receptors (PRRs), mitogen-activated protein kinase (MAPK), calcium signaling, and transcription factors may regulate pepper resistance to Xcv. Moreover, after Xcv inoculation, 364 DEGs differentially expressed only in VI037601 and 852 genes in both ECW and VI037601. Many of those genes were classified as NBS-LRR genes, oxidoreductase gene, WRKY and NAC transcription factors, and they were mainly involved in metabolic process, response to stimulus and biological regulation pathways. Quantitative RT-PCR of sixteen selected DEGs further validated the RNA-seq differential gene expression analysis. Our results will provide a valuable resource for understanding the molecular mechanisms of pepper resistance to Xcv infection and improving pepper resistance cultivars against Xcv