ITS Gene Sequences Research Articles

Ultrastructural and Molecular Identification of the sarcocysts of Sarcocystis tenella and Sarcocystis arieticanis Infecting Domestic Sheep (Ovis aries) from Egypt.

In spite of the global economic significance of sheep production, little is known about the prevalence of various Sarcocystis spp. infecting the domestic sheep (Ovis aries) in Egypt. Muscle samples were collected from 175 sheep (>2years) slaughtered at El-Mahalla El-Kubra abattoir, Gharbia governorate, Egypt. Samples were initially examined by naked eye for the existence of macrosarcocysts. The microscopic sarcocysts were detected and identified using the light microscopy and the Transmission electron microscopy (TEM). Different microscopic species of ovine Sarcocystis were molecularly confirmed by PCR, sequence analyses and phylogeny. Preliminary light microscopic inspection of the muscle specimens revealed the existence of only the microscopic sarcocysts of Sarcocystis tenella and Sarcocystis arieticanis in 152 (86.8%) out of the175 examined animals. Sarcoysts of S.tenella had striated thick cyst wall that amounted from 3.5-5.5 μm in thickness whereas, S.arieticanis sarcocysts had a thin cyst wall that ranged from 1-3 μm in thickness. S.tenella sarcocysts were detected in 115 sheep (65.7%), and were more prevalent than those of S.arieticanis, observed only in 68 sheep (38.8%). No macroscopic sarcocysts were observed in any of the examined carcasses. Transmission electron microscopy (TEM) of the cyst wall of S.tenella revealed the existence of the short stubby villar protrusions (VP) with the characteristic disk-like structures at the tips of the (VP). While, TEM of S.arieticanis showed that the cyst wall had elongated tubular protrusions that measured approximately 5-7 μm in length. Each (VP) consisted of a dome-shaped base (0.3-0.9 μm in diameter), a relatively thick middle portion (0.1-0.3 μm) in width, and a thin hair-like distal portion that measured about (0.03 x 1-4.5 μm). Comparative analyses of the sequences of the four genetic markers (18S rRNA, 28S rRNA, mitochondrial cox1 and ITS-1) for S.tenella and S.arieticanis isolates detected herein, revealed genetic variations of 95% and 95- 96% among the different isolates on the level of the 18S rRNA and 28S rRNA, respectively. Whereas, the cox1 and ITS-1 shared sequence identities of 76-78% and 70-73%, respectively. S.tenella was strongly related to S.capracanis infecting goats (Capra hircus). Sequence identity of 98% on the level of 18S rRNA, 28S rRNA genes was observed between the currently identified isolates of S.tenella and the formerly GenBank deposited isolates of S.capracanis. While, cox1 sequences shared identities of 92-93%. Furthermore, S.arieticanis isolates identified here were closely related to the formerly published sequences of S.hircicanis. The 18S rRNA and 28S rRNA sequences of S.arieticanis shared 98% and 94-95% identities with those of S.hircicanis, respectively. However, 87-88% homologies were observed between the cox1 sequences of S.arieticanis and S.hircicanis. Consequently, cox1 and ITS-1 gene sequences act as better genetic markers than 18S rRNA and 28S rRNA sequences for the characterization of ovine Sarcocystis spp. Maximum parsimony analyses based on the sequences of three genetic markers, (18S rRNA, 28S rRNA and mitochondrial cox1), yielded the same placement of the currently identified isolates of the two taxa (S.tenella and S.arieticanis) within a clade of Sarcocystis species with carnivorous animals as known, or assumed, final hosts.

Biological decolorization of azo dyes from textile wastewater effluent by Aspergillus niger

In this study, fungal isolate D2-1 was isolated from contaminated soil collected from textile industry companies and showed high potentiality for decolorization of two different azo dyes. This isolate was identified as Aspergillus niger D2-1 using morphological and cultural characteristic as well ITS gene sequencing. Decolorization process under different optimized condition of azo dye concentration, pH, incubation periods, temperature, inoculum size and different carbon and nitrogen sources were investigated. The maximum decolorization efficiency against 100 ppm dye concentration of reactive yellow (4GL) and reactive red (4BL) dyes of 98.62 % and 92.42 %, respectively, were recorded for Aspergillus niger D2-1 at pH 9.0, in presence of 2 % glucose and 0.5% yeast extract as carbon and nitrogen sources respectively at room temperature after 7 days on shaking conditions. The decolorization percentage was confirmed by ultraviolet-visible (UV-Vis) spectrum analysis of untreated/treated reactive yellow (4GL) and reactive red (4BL) dyes, which showed complete disappearance of peaks at ~ 425 nm and at ~ 520 nm, indicates the degradation of dyes due to fungal activity. Treatment of textile wastewater effluent by Aspergillus niger D2-1 showed high decolorization percentage (59%) for effluent, also physico-chemical characteristics of textile effluent such as chemical oxygen demand (COD), total dissolved solids (TDS) and total suspended solids (TSS) were decreased from 756 mg/L, 1597 mg/L and 821 mg/L to 391 mg/L, 845 mg/L and 362 mg/L respectively. Moreover, Gas chromatography - mass spectroscopy (GC-MS) of textile effluent before and after treatment were recorded and confirmed the potentiality of Aspergillus niger D2-1 in dyes wastewater treatment.

Rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real-time PCR and Sanger sequencing

Analyses of short subunit gene sequences have been established for taxonomic classification and identification of bacteria and fungi. To produce partial bacterial ribosomal 16S rRNA and rpoB and fungal ribosomal ITS/LSU gene sequences for DNA sequencing, real-time PCR assays supplemented with the nucleic acid stain SYBR Green were created. Generation of PCR products was monitored based on amplification and melting curves. The PCR products were subsequently subjected to Sanger sequencing on demand for identification of bacteria and fungi in routine microbiological diagnostics within a period of two days.From a total of 78 bacterial isolates 40 (51%) or 67 (86%) could be identified at species level using only partial 16S rRNA or additionally rpoB gene sequences based on BLASTN (NCBI) database queries, respectively. Using partial 16S rRNA and rpoB gene sequencing unambiguous assignment was not possible for the closely related species of the Bacillus (B.) cereus group, Bordetella (B.) pertussis/ B. parapertussis/ B. bronchiseptica, Brucella spp., Enterobacter cloacae complex, Escherichia/ Shigella spp., Staphylococcus (S.) hyicus/ S. agnetis and Yersinia (Y.) pseudotuberculosis/ Y. pestis. However, partial rpoB gene sequencing succeeded in identifying 27 bacterial isolates at species level in addition to 16S rRNA gene sequencing.Regarding ITS/LSU gene sequencing, best results could be achieved by ITS gene sequencing followed by LSU gene sequencing, resulting in 32 (63%) and 21 (43%) of a total of 51 fungal isolates that could be identified at species level, respectively. Insufficient identification at species level was observed for the genera Apiotrichum, Aspergillus, Cladosporium, Cryptococcus, Microsporum, Nannizziopsis, Penicillium, Trichosporon, and Tolypocladium included in this study.The concept of this procedure is suitable for rapid and reasonable molecular identification of bacteria and fungi within two days and is therefore applicable in routine microbiological diagnostic laboratories.

An efficient strategy for screening polyunsaturated fatty acid-producing oleaginous filamentous fungi from soil

Screening oleaginous microorganisms capable of accumulating considerable lipids is essential for industrial lipid production. Here we demonstrated forty-seven filamentous fungal isolates were obtained from eight soil samples using a new screening strategy with both triphenyltetrazolium chloride (TTC), a redox indicator used for testing oil presence, and cerulenin, an inhibitor of fatty acid synthase (FAS), supplemented in screening medium. Among these fungal isolates, nineteen have high lipid content (>20% dry biomass weight) and were affiliated with the genus Mortierella by morphology identification and phylogenetic analysis based on ITS gene sequences. Notably, one strain designated as SL-4 reached 32% of its biomass weight as lipid, displaying the highest potential. Two candidates with high lipid content as well as biomass production were selected for exploring the effect of different carbon and nitrogen sources on morphology, biomass and lipid accumulation.

Trichoderma asperellum T1 mediated antifungal activity and induced defense response against leaf spot fungi in lettuce (Lactuca sativa L.)

Leaf spot, caused by Corynespora cassiicola and Curvularia aeria, is a major disease of lettuce that results in the reduction of the quality and the quantity of lettuce production. Primary in vitro examination revealed that Trichoderma sp. T1 inhibited the mycelial growth of C. cassiicola and C. aeria by 83.79% and 85.71%, respectively, in dual culture assays. Gel diffusion showed cell-free culture filtrate of Trichoderma sp. T1 inhibited C. cassiicola and C. aeria by 50.38% and 53.97%, respectively. Based on the ITS gene sequence, an effective T1 isolate was identified as Trichoderma asperellum. Treatments of lettuce plants with spore suspension of T. asperellum T1 induced defense-related enzymes such as peroxidase and polyphenol oxidase, and the cell wall degrading enzymes β-1,3-glucanase and chitinase in the plants. The activities of β-1,3-glucanase, chitinase, peroxidase and polyphenol oxidase were higher at 24 and 48 h after treatment with spore suspension of T. asperellum T1 than in the control group. Crude extract from inoculated lettuce caused morphology changes in fungal mycelia. In addition, reduction of disease severity was observed in lettuce pretreated with T. asperellum T1. The obtained results suggest that T. asperellum T1 efficiently controlled leaf spot and induced defense responses in lettuce.

Finding out potent probiotic cultures from ayurvedic formulation Takrarishta through in-vitro probiotic characterization and principal component analysis

This study was intended to investigate the microbes involved in preparation Takrarishta. ‘Takrarishta’ is an ayurvedic fermented medicinal product used against inflammatory bowel disease (IBD). Total 13 cultures including 11 bacteria and 2 yeasts were isolated from actively fermenting Takrarishta. The isolates were characterized morphologically, biochemically and were further identified by 16S rRNA/18S rRNA ITS gene sequencing. Among the isolates, 8 isolates obeyed the probiotic characteristics while the principal component analysis (PCA) revealed that Lactobacillus plantarum TP5 and Kluyveromyces marxianus TP6 and TM1 were the most potent probiotics. The Takrarishta preparation is based upon strong scientific temperaments like modern concept of fermenting prebiotic, using probiotic microbes to get postbiotics for better efficacy. This outcome provides a strong evidence in support of Takrarishta being a fermented product for the treatment of IBD.

Herbivore-Diet Analysis Based on Illumina MiSeq Sequencing: The Potential Use of an ITS2-Barcoding Approach to Establish Qualitative and Quantitative Predictions of Diet Composition of Mongolian Sheep.

DNA-barcoding approaches to estimate the diet compositions of grazing animals have received significant attention, and particularly when combined with next-generation sequencing, these techniques have substantially improved in recent years. In this study, the identity and species composition of plant material ingested by Mongolian sheep were estimated through the use of 350 bp ITS2 gene sequences of the vegetation found in fecal samples. Four diets were formulated using varying amounts of eight plant species that are common in the grasslands of northern China. Sixteen Mongolian sheep were taken from pastures and randomly assigned to four groups, and each group received one of four diets. Each sheep was randomly assigned to one of 16 confinement pens and fed its respective diet for 12 consecutive days. Fecal samples were removed from each pen from days 7-12, preserved, and composited for each pen. All herbage species included in the daily diets were detected in each fecal sample, with the exception of Phragmites australis. Moreover, 12 additional different plant species were retrieved from feces of the experimental sheep. The obtained data provided preliminary support for the use of the ITS2 barcode to determine which plants were consumed. Moreover, the proportions of the herbage DNA sequences recovered from sheep feces and those of the herbage masses in the daily diets did not completely match. These results indicate that the non-Gramineae DNA sequences amplified with ITS2 primers (including those of Chenopodium album, Artemisia scoparia, Artemisia tanacetifolia, and Medicago sativa) far exceeded those of the Gramineae species (including Leymus chinensis and Puccinellia distans), which constitute the largest share of the experimental diets. A significant positive correlation (Spearman's ρ = 0.376, P = 0.003) between the actual herbage mass proportions in the experimental diets and the herbage-DNA-sequence proportions provided sufficiently favorable support for the further investigation of DNA barcoding for the quantification of plants in feces. A significant regression coefficient was found between the relative DNA-sequence proportions of L. chinensis ( R2 = 0.82, P < 0.0001), P. distans ( R2 = 0.64, P = 0.0017), and C. album ( R2 = 0.98, P < 0.0001) and their respective herbage mass proportions. The quantitative relationship can be expressed by the linear-regression equations y = 0.90 x - 0.22, y = 0.98 x - 0.03, and y = 5.00 x - 0.25, respectively. Thus, these results demonstrate that dietary-DNA-barcoding methods exhibited potential in providing valuable quantitative information regarding food-item components. However, it should be noted that this explorative data needs to be further improved by using additional genes and by creating a sophisticated reference database, thus enhancing both quality and accuracy of the obtained results.

Bio-production of novel water-soluble yellow pigment from Aspergillus sp. and exploring its sustainable textile applications.

In the present study, 40 pigment-producing microbes were isolated from various soil sources. Among these, a novel water-soluble yellow pigment-producing fungal isolate (MBYP1) was identified as Aspergillus sp. through ITS gene sequencing. The maximum pigment yield (UA430nm, 12.45 ± 0.5g/l) was obtained when strain MBYP1 was cultured under optimum conditions (28°C and pH 5.5 under static condition). Subsequently, the pigment was purified through gel chromatography and high-performance liquid chromatography (HPLC). Characterization of purified pigment through UV-Vis and liquid chromatography-mass spectrometry (LC-MS) reveal maximum absorbance at 430nm and molecular mass of 301m/z, respectively. Further, the pigment exhibited a maximum dyeing capacity of up to 80% irrespective of mordant. Toxicity evaluation of purified pigment with zebra fish model system reported an IC50 value of 710µg/mL. Pigment antioxidant ability was established by DPPH (35.7µg/mL) and phosphomolybdenum assay (226.61mg/g) thus ascertaining improvised light fastness of dyed fabric. Moreover, lack of antimicrobial activity (up to 40µg/mL) improves pigment bio-degradability. In collective, the novel yellow pigment from Aspergillus sp. MBYP1 strain was found to be an eco-friendly alternative to synthetic dye for potential applications in textile industries.

Plant diversity and density predict belowground diversity and function in an early successional alpine ecosystem.

Despite decades of interest, few studies have provided evidence supporting theoretical expectations for coupled relationships between aboveground and belowground diversity and ecosystem functioning in non-manipulated natural ecosystems. We characterized plant species richness and density, soil bacterial, fungal and eukaryotic species richness and phylogenetic diversity (using 16S, ITS, and 18S gene sequencing), and ecosystem function (levels of soil C and N, and rates of microbial enzyme activities) along a natural gradient in plant richness and density in high-elevation, C-deficient soils to examine the coupling between above- and belowground systems. Overall, we observed a strong positive relationship between aboveground (plant richness and density) and belowground (bacteria, fungi, and non-fungal eukaryotes) richness. In addition to the correlations between plants and soil communities, C and N pools, and rates of enzyme activities increased as plant and soil communities became richer and more diverse. Our results suggest that the theoretically expected positive correlation between above- and belowground communities does exist in natural systems, but may be undetectable in late successional ecosystems due to the buildup of legacy organic matter that results in extremely complex belowground communities. In contrast, microbial communities in early successional systems, such as the system described here, are more directly dependent on contemporary inputs from plants and therefore are strongly correlated with plant diversity and density.

Incidence of leaf spot disease on cotton caused by Curvularia verruculosa and role of its hydrolytic enzymes in pathogenesis

The finding described in this study is the first report of leaf spot disease of cotton caused by Curvularia verruculosa surveyed in the state of Maharashtra (India). The isolated phytopathogenic fungal strain was identified using morphological characteristics and molecular identification of ITS gene sequence (MF784436) and D1D2 region of LSU gene (KY978073). The ability of fungal strain to secrete hydrolytic enzymes viz., pectinase, xylanase, protease, cellulase and lipase was detected. The secretion profile of hydrolytic enzymes by C. verruculosa was also examined in planta and in vitro. The secretion of cellulase, xylanase and protease was found to be inducible on cotton-stalk powder containing media; while secretion of pectinase and lipase was constitutive in glucose containing medium. The hydrolytic enzymes secretion during etiological progression of disease was detected on cotton leaves at regular interval of 24h up to 10days. A significant correlation (P<0.05) was observed between hydrolytic enzymes secretion and disease severity index. The increased level of hydrolytic enzymes in infected plant sample indicates their role in disease progression. The newly documented fungal phytopathogen Curvularia verruculosa was deposited at National Fungal Culture Collection of India, Pune with accession number of NFCCI-4119.

Mycobacterial infections in carcasses of ruminants slaughtered at the two slaughterhouses of Kassala, Sudan

Tuberculosis (TB) is a chronic bacterial disease of humans and animals. It is characterized by the progressive development of specific granulomatous lesions in affected organs. Human TB is endemic in Eastern Sudan. However, knowledge on the epidemiology of TB in ruminants is scarce. In a six-month study from June to November 2014, a total of 2304 carcasses of cattle, sheep, goats and camels slaughtered at the East and West Gaash slaughterhouses of Kassala were inspected to investigate TB prevalence. Only 0.1% (n = 2) of the carcasses had suspicious TB lesions. These lesions were solely found in carcasses of sheep, in the liver, lungs, and peritoneal cavity. The samples collected from the lesions were investigated for the presence of mycobacteria, which were found in one of the two carcasses. The grown bacteria were subjected to a line probe assay (GenoType Mycobacterium CM), and to 16S rDNA and ITS gene sequencing, and whole genome sequencing (WGS). However, none of these methods identified this isolate as a valid Mycobacterium species. Nevertheless, 16S sequence allocated this isolate to slow growing mycobacteria. Neither Mycobacterium bovis nor M. caprae nor M. tuberculosis were found in the collected granulomatous lesions. In conclusion, the overall prevalence of TB-suggestive lesions in ruminants in Kassala was very low. Extended studies combining the use of a tuberculin skin test and slaughterhouse-based investigations should be adopted for TB surveillance in ruminants in Sudan.

Notes on powdery mildews (Erysiphales) in Thailand VI. Phyllactinia and Leveillula

Nine species of the genus Phyllactinia and one species of Leveillula on various hosts are recorded for Thailand. Morphological descriptions of powdery mildew specimens on hosts belonging to nine plant families and molecular phylogenetic analyses based on ITS and 28S rRNA gene sequences are provided. Phyllactinia mori-macrourae on Morus macroura (Moraceae) is proposed as new species. Phyllactinia mori-macrourae differs from Ph. moricola by having smaller chasmothecia, less chasmothecial appendages, less asci per chasmothecium, and longer ascospores. Ovulariopsis broussonetiae-papyriferae is combined to Phyllactinia broussonetiae-papyriferae. Phyllactinia alangii, Ph. dalbergiae, Phyllactinia cf. ehretiae, Ph. gmelinae, Ph. moricola, Ph. pyri-serotinae, Ph. Terminaliae, and Leveillula taurica are new records for Thailand. Ehretia laevis and S. aculeatissimum are new host records for powdery mildews species worldwide.

Porostereum sp., Associated with Saffron (Crocus sativus L.), is a Latent Pathogen Capable of Producing Phytotoxic Chlorinated Aromatic Compounds.

Saffron (Crocus sativus L.) is one of the most expensive spices in the world due to its medicinal and aromatic value. However, saffron production is severely affected by the corm rot disease throughout the saffron producing countries. In this study, we report a basidiomycetous latent pathogen of saffron, designated as CSE26, capable of producing phytotoxic compounds. CSE26 is a highly odorous basidiomycete with monomitic hyphal system. Molecular phylogeny of ITS and 28S ribosomal gene sequence of CSE26 assigned it as Porostereum spadiceum. It was found to produce corm rot in C. sativus under in vivo and field conditions, with a disease severity index of 0.7 and 0.5, respectively. CSE26 was found to produce chlorinated aromatic compounds (CAMs) having phytotoxic activity against Arabidopsis plants. Therefore, these compounds may be acting as pathogenic determinants of CSE26. However, there is a need to study the level of production of these CAMs by this fungus in the natural environment and their effects on plant health.

Distribution and Genetic Variability of Fusarium oxysporum Associated with Tomato Diseases in Algeria and a Biocontrol Strategy with Indigenous Trichoderma spp.

Fifty fungal isolates were sampled from diseased tomato plants as result of a survey conducted in seven tomato crop areas in Algeria from 2012 to 2015. Morphological criteria and PCR-based identification, using the primers PF02 and PF03, assigned 29 out of 50 isolates to Fusarium oxysporum (Fo). The banding patterns amplified for genes SIX1, SIX3 and SIX4 served to identify races 2 and 3 of Fo f. sp. lycopersici (FOL), and Fo f. sp. radicis lycopersici (FORL) among the Algerian isolates. All FOL isolates showed pathogenicity on the susceptible tomato cv. “Super Marmande,” while nine of out 10 Algerian FORL isolates were pathogenic on tomato cv. “Rio Grande.” Inter simple sequence repeat (ISSR) fingerprints showed high genetic diversity among Algerian Fo isolates. Seventeen Algerian Trichoderma isolates were also obtained and assigned to the species T. asperellum (12 isolates), T. harzianum (four isolates) and T. ghanense (one isolate) based on ITS and tef1α gene sequences. Different in vitro tests identified the antagonistic potential of native Trichoderma isolates against FORL and FOL. Greenhouse biocontrol assays performed on “SM” tomato plants with T. ghanense T8 and T. asperellum T9 and T17, and three Fo isolates showed that isolate T8 performed well against FORL and FOL. This finding was based on an incidence reduction of crown and root rot and Fusarium wilt diseases by 53.1 and 48.3%, respectively.

First report of leaf spot caused by Phytophthora capsici on chili pepper (Capsicum frutescens L.) in Pakistan

A survey was conducted in chili and a leaf spot disease with approximately 46% incidence was observed in the field. Morphological characteristics of the isolates correspond to those reported for Phytophthora capsici. The resulting sequences of ITS and TEF1 gene had high similarity after BLAST analysis with those of P. capsici isolates. The pathogenicity of one isolate was tested by spraying the zoospore suspension and symptoms of leaf spots similar to those observed in the field developed on all the inoculated leaves.

Families and genera of diaporthalean fungi associated with canker and dieback of tree hosts

In this study we accept 25 families in Diaporthales based on phylogenetic analyses using partial ITS, LSU, rpb2 and tef1-α gene sequences. Four different families associated with canker and dieback of tree hosts are morphologically treated and phylogenetically compared. These include three new families (Diaporthostomataceae, Pseudomelanconidaceae, Synnemasporellaceae), and one new genus, Dendrostoma (Erythrogloeaceae). Dendrostoma is newly described from Malus spectabilis, Osmanthus fragrans and Quercus acutissima having fusoid to cylindrical, bicellular ascospores, with three new species namely D. mali, D. osmanthi and D. quercinum. Diaporthostomataceae is characterised by conical and discrete perithecia with bicellular, fusoid ascospores on branches of Machilus leptophylla. Pseudomelanconidaceae is defined by conidiogenous cells with apical collarets and discreet annellations, and the inconspicuous hyaline conidial sheath when mature on Carya cathayensis, compared to morphologically similar families Melanconidaceae and Juglanconidaceae. Synnemasporellaceae is proposed to accommodate fungi with synnematous conidiomata, with descriptions of S. toxicodendri on Toxicodendron sylvestre and S. aculeans on Rhus copallina.

Neurospora tetraspora D. Garcia, Stchigel & Guarro (= Gelasinospora tetrasperma Dowding) as a first record to Egypt

An interesting isolate of homothallic, ascosporic filamentous fungus having 4-spored asci, was recovered once from a non-rhizosphere soil sample collected from a grapevine plantation in the village of El-Khawaled, Sahel-Saleem city, Assiut. It was isolated on DYM agar plate at 25°C in June 2008. The isolate was identified phenotypically and genotypically as Neurospora tetraspora (= Gelasinospora tetrasperma ) and was deposited in the culture collection of Assiut University Mycological Centre as AUMC no. 6784 and ITS gene sequence of the strain was deposited at the National Center for Biotechnology Information (NCBI) and assigned a GenBank accession number JQ425383. N. tetraspora is being recorded in the current work for the first time in Egypt. By this addition, the genus is now known in Egypt by four species. A key is provided for the four species.

Ecological dyeing of silk fabric with Monascus

Monascus were cultured and applied in silk dyeing in this work. The bacterial morphology and cultural characteristics of Monascus were investigated. The rDNA D1/D2 and ITS gene sequences of this strain were identified. The effects of dyeing parameters on dye uptake were discussed and the dyeing method was optimized. Under the dyeing conditions (pH 3.6, 30 °C, 3 h), the dye uptake could reach 89.45%. Moreover, the soap washing and rubbing fastness of Monascus dyed fabric were both rated over Level 4. An ecological low temperature dyeing method was developed, which provides a basis for practical microbial dyeing of silk fabric.

Culturable Endophytes Diversity Isolated from Paeonia ostii and the Genetic Basis for Their Bioactivity.

Paeonia ostii is known for its excellent medicinal values as Chinese traditional plant. To date, the diversity of culturable endophytes associated with P. ostii is in its initial phase of exploration. In this study, 56 endophytic bacteria and 51 endophytic fungi were isolated from P. ostii roots in China. Subsequent characterization of 56 bacterial strains by 16S rDNA gene sequence analysis revealed that nine families and 13 different genera were represented. All the fungal strains were classed into six families and 12 genera based on ITS gene sequence. The biosynthetic potential of all the endophytes was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. The PCR screens were successful in targeting thirteen bacterial PKS, five bacterial NRPS, ten fungal PKS and nine fungal NRPS gene fragments. Bioinformatic analysis of these detected endophyte gene fragments facilitated inference of the potential bioactivity of endophyte bioactive products, suggesting that the isolated endophytes are capable of producing a plethora of secondary metabolites. These results suggest that endophytes isolated from P. ostii had abundant population diversity and biosynthetic potential, which further proved that endophytes are valuable reservoirs of novel bioactive compounds.

Isolation and Identification of Heavy Metals and Antibiotics Resistant Strains from Antananarivo Dumpsite, Madagascar

Heavy metals contamination is now widespread in the nature. At higher concentration, heavy metals become toxic and disturb the ecosystem including soil microorganisms. To adapt to such constraints, some microorganisms have developed tolerance mechanisms. Indeed, in the environment, the resistance of microorganisms to heavy metal often promotes to antibiotic resistance. This work aims to isolate strains from soil samples collected in Andralanitra landfill, to test their tolerance to heavy metals, to identify tolerant strains and to verify their resistance to antibiotics. According to the dilution method, a total of 48 strains were obtained, 14 were isolated on PDA medium, 10 on Sabouraud agar medium, 10 strains on Mossel agar medium, 7 on AS1 medium, 5 strains on TSA medium and 2 strains with King B medium. Resistance test to heavy metals performed by the wells method showed that out of the 48 isolated strains, 26 were capable to grow in the presence of heavy metals (solution composed of copper, zinc, cadmium, chromium, nickel, lead) at different concentrations. The highest number of tolerant strains was recorded at the concentration of 100mg/L ≤ C ≤ 1000mg/L. Four (4) strains were tolerant to the heavy metals solution at a concentration between 100mg/L and 1500mg/L. The molecular identification of these four most resistant strains by 16S rDNA gene sequencing and ITS gene sequencing allowed to classify them as belonging to the genera Ochrobactrum pseudogrignonense, Arthrobacter nicotianae, Penicillium crustosum and Penicillium commune. The antibiotic sensitivity test using disc diffusion method on Mueller-Hinton agar revealed that Ochrobactrum pseudogrignonense and Penicillium commune were resistant to Trimethoprim, Arthrobacter nicotianae showed resistance to Trimethoprim and Ciprofloxacin, Penicillium crustosum was resistant to all tested antibiotics.