Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.     

Biodegradation of kraft-lignin by <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from sludge of pulp and paper mill

Eight bacterial strains were isolated on kraft lignin (KL) containing mineral salt medium (L-MSM) agar with glucose and peptone from the sludge of pulp and paper mill. Out of these, ITRC-S8 was selected for KL degradation, because of its fast growth at highest tested KL concentration and use of various lignin-related low molecular weight aromatic compounds (LMWACs) as sole source of carbon and energy. The bacterium was identified by biochemical tests as Gram-positive, rod-shaped and non-motile. Subsequent 16S rRNA gene sequencing showed 95% base sequence homology and it was identified as Bacillus sp. In batch experiments, a decrease in pH was observed initially followed by an increase till it reached an alkaline pH, which did not alter the culture growth significantly. The bacterium reduced the colour and KL content of 500 mg l⁻¹ KL in MSM, in the presence of glucose and peptone, at pH 7.6, temperature 30°C, agitation of 120 rpm and 6 days of incubation by 65 and 37% respectively. Significant reduction in colour and KL content in subsequent incubations is indicative of a co-metabolism mechanism, possibly due to initial utilization of added co-substrates for energy followed by utilization of KL as a co-metabolic. The degradation of KL by bacterium was confirmed by GC-MS analysis indicating formation of several LMWACs such as t-cinnamic acid, 3, 4, 5-trimethoxy benzaldehyde and ferulic acid as degradation products, which were not present in the control (uninoculated) sample. This favours the idea of biochemical modification of the KL polymer to a single monomer unit.     

Free radical scavenging and antioxidant activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella sp. YS 4108

The reactive oxygen species (ROS) and free radical-initiated reactions are ascertained to play multiple roles in degenerative or pathological events such as aging, cancer, heart dysfunction and Alzheimer's disease. EPS2 with a mean molecular weight of 1.3 x 10(5) was characterized as an antioxidant exopolysaccharide from the broth of a marine filamentous fungus Keissleriella sp. YS 4108. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The radical eliminating and antioxidant actions of the glycan was assessed in different in vitro systems showing that EPS2 exhibited profound scavenging activities in superoxide radical. As a reinforcement of the action, similar radical scavenging effects of EPS2 were also discerned with both site-specific and non site-specific hydroxyl radical using the deoxyribose assay method. Moreover, EPS2 effectively blocked as well the non site-specific strand-breaking of DNA induced by the Fenton reaction at concentrations of 0.1 and 1 mg/mL. Further investigation of the effect of EPS2 on human low density lipoprotein (LDL) system demonstrated that it significantly inhibited copper-mediated oxidation of LDL in a dose-dependent manner. These results suggest that EPS2, possessing pronounced free radical scavenging and antioxidant activities, could be of considerable preventive and therapeutic significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer's disease which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules.     

Halorubellus salinus gen. nov., sp. nov. and Halorubellus litoreus sp. nov., novel halophilic archaea isolated from a marine solar saltern

Two extremely halophilic archaeal strains GX3(T) and GX26(T) were isolated from the Gangxi marine solar saltern near the Weihai city of Shandong Province, China. Cells from the two strains were pleomorphic and stained Gram-negative, colonies were red-pigmented. Strains GX3(T) and GX26(T) were able to grow at 25-50 °C (optimum 37 °C), at 1.4-5.1M NaCl (optimum 3.1M), at pH 5.5-9.5 (optimum pH 7.0) and neither strain required Mg(2+) for growth. Cells lyse in distilled water and the minimal NaCl concentration to prevent cell-lysis was 8% (w/v). The major polar lipids of the two strains were PA (phosphatidic acid), PG (phosphatidylglycerol), PGP-Me (phosphatidylglycerol phosphate methyl ester) and three major glycolipids (GL1, GL2 & GL3) chromatographically identical to S-TGD-1 (sulfated galactosyl mannosy glucosyl diether), S-DGD-1 (sulfated mannosyl glucosyl diether), and DGD-1 (mannosyl glucosyl diether) respectively, an unidentified lipid (GL4) was also detected in strain GX26(T). Phylogenetic analysis based on 16S rRNA gene revealed that strain GX3(T) and strain GX26(T) formed a distinct clade with the closest relative, Haladaptatus paucihalophilus (89.9-92.4% and 90.4-92.7, respectively). The rpoB' gene similarities between strains GX3(T) and GX26(T), and between the two strains and the closest relative, Halorussus rarus TBN4(T) are 96.5%, 84.3% and 83.9%, respectively. The DNA G+C contents of strain GX3(T) and strain GX26(T) are 67.3 mol% and 67.2 mol%, respectively. The DNA-DNA hybridization value between strain GX3(T) and strain GX26(T) was 44%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX3(T) and strain GX26(T) represent two novel species in a new genus within the family Halobacteriaceae, Halorubellus salinus gen. nov., sp. nov. (type strain GX3(T)=CGMCC 1.10384(T)=JCM 17115(T)) and Halorubellus litoreus sp. nov. (type strain GX26(T)=CGMCC 1.10386(T)=JCM 17117(T)).     

Microbacterium marinum sp. nov., isolated from deep-sea water

Two Gram-positive, rod-shaped bacterial strains, H101(T) and H207, were isolated from deep sea water collected from South-West Indian Ocean. Phylogenetic analysis of 16S rRNA gene sequences showed that the two strains were closely related to one another (100% similarity), and had the closest relationship with Microbacterium hominis NBRC 15708(T) and Microbacterium insulae KCTC 19247(T) (98.2-98.3% similarities). DNA-DNA hybridization value between strains H101(T) and H207 was 87.2 ± 3.7%, and the values between the two strains and the closely related type strains were well below 70%. The two strains also shared a number of physiological and biochemical characteristics that were distinct from the closely related species, and grew at 2-37 ° C, pH 5-11 and 0-8% (w/v) NaCl. Both strains contained MK-12, MK-13 and MK-11 as the detected menaquinones. The peptidoglycan was of type B1γ with an interpeptide bridge D-Glu(Hyg)→ Gly(2)→ l-Lys. The major cellular fatty acids were anteiso-C(15:0), anteiso-C(17:0), and iso-C(16:0). Based on the genetic and phenotypic properties, it is proposed that strains H101(T) and H207 be classified as representatives of a novel species of the genus Microbacterium, with the name Microbacterium marinum sp. nov. The type strain is H101(T) (= CGMCC 4.6941(T) = DSM 24947(T)).     

Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain

A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams (Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase α-chain (rpoA), RecA protein (recA), the α-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA–DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9^T(=CECT 7244^T=LMG 24045^T).     

Natrinema salaciae sp. nov., a halophilic archaeon isolated from the deep, hypersaline anoxic Lake Medee in the Eastern Mediterranean Sea

Two halophilic archaea, strains MDB25(T) and MDB20, were isolated from a sample of the brine from Lake Medee, at a depth of 3050m, in the Mediterranean Sea. Cells of the organisms were Gram-negative, non-motile and pleomorphic, and colonies were red pigmented. Strains MDB25(T) and MDB20 showed optimum growth at 45°C, in 2.6-3.4M NaCl and at pH 7.0-8.0. The major polar lipids of the two strains were phosphatidylglycerol (PG1 and PG2), phosphatidylglycerol phosphate methyl ester (PGP-Me) and mannose-2,6-dissulfate (1→2)-glucose glycerol diether (S(2)-DGD). Menaquinone MK-8 and MK-8(H(2)) were the major respiratory quinones. The DNA G+C content of strain MDB25(T) was 63.0%. The strains were facultatively anaerobic but grew better under aerobic conditions, nitrate served as electron acceptor. Analysis of the almost complete 16S rRNA gene sequence indicated that the strains MDB25(T) and MDB20 represented a member of the genus Natrinema in the family Halobacteriaceae. Both strains formed a distinct cluster and were most closely related to Natrinema ejinorense JCM 13890(T) and Haloterrigena longa JCM 13562(T) (98.0% and 97.9% sequence similarity, respectively). Based on 16S rRNA gene sequence analysis, DNA-DNA hybridization results, physiological and biochemical characteristics we describe a new species represented by strain MDB25(T) (=DSM 25055(T) =JCM 17869(T)) for which we propose the name Natrinema salaciae sp. nov.     

Aeromonas tecta sp. nov., isolated from clinical and environmental sources

Five Aeromonas strains, isolated from both clinical and environmental sources and characterized by a polyphasic approach, including phylogenetic analysis derived from gyrB, rpoD, and 16S rRNA gene sequencing, as well as DNA-DNA hybridization, extensive biochemical and antibiotic susceptibility tests, were recognized as members of an unknown, or undescribed, Aeromonas species. These "Aeromonas eucrenophila-like" strains were closely related to the species A. eucrenophila and Aeromonas encheleia, but they were negative for indole and acid from glycerol tests. Therefore, based on the results of the phylogenetic analyses and DNA-DNA pairing data of these strains, a novel species of the genus Aeromonas is described, for which the name Aeromonas tecta is proposed with isolate F518(T) (CECT7082(T), DSM17300(T), MDC91(T)) as the type strain.     

Purification and characterisation of a fructosyltransferase from Rhodotorula sp

The present work was devoted to investigations concerning the purification and characterisation of the fructooligosaccharide (FOS)-producing extracellular enzyme of Rhodotorula sp. LEB-V10. FOS are functional food ingredients showing prebiotic properties, meaning that it could stimulate selectively the growth and/or activity of probiotic bacteria in the gut. The purification of the enzyme was carried out according to the following sequential procedure: cell separation by centrifugation, recovering by ethanol precipitation and purification by anion exchange chromatography. The molecular weight was estimated to be 170 kDa by preparative gel filtration and 77 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, signifying that the native enzyme exists as a dimer. With sucrose as substrate, the data failed to fit the Michaelis-Menten behaviour, rather showing a sigmoid shape similar to that of the allosteric enzymes (cooperative behaviour), requiring high sucrose concentrations to obtain high reaction rates. The enzyme showed both fructofuranosidase (FA) and fructosyl-transferase (FTA) activities. The optimum pH and temperature for FA activity were found to be around 4.0 and 72-75 degrees C, respectively, while FTA showed optimum activity at pH 4.5 and 65-70 degrees C. Both activities were very stable at temperatures below 66 degrees C, while for FA, the enzyme was more stable at pH 4.0 and for FTA at pH 5.0.     

Photobacterium piscicola sp. nov., isolated from marine fish and spoiled packed cod

Five isolates from marine fish (W3^T, WM, W1S, S2 and S3) and three isolates misclassified as Photobacterium phosphoreum, originating from spoiled modified atmosphere packed stored cod (NCIMB 13482 and NCIMB 13483) and the intestine of skate (NCIMB 192), were subjected to a polyphasic taxonomic study. Phylogenetic analysis of 16S rRNA gene sequences showed that the isolates were members of the genus Photobacterium. Sequence analysis using the gapA, gyrB, pyrH, recA and rpoA loci showed that these isolates formed a distinct branch in the genus Photobacterium, and were most closely related to Photobacterium aquimaris, Photobacterium kishitanii, Photobacterium phosphoreum and Photobacterium iliopiscarium. The luxA gene was present in isolates W3^T, WM, W1S, S2 and S3 but not in NCIMB 13482, NCIMB 13483 and NCIMB 192. AFLP and (GTG)₅-PCR fingerprinting indicated that the eight isolates represented at least five distinct genotypes. DNA–DNA hybridizations revealed 89% relatedness between isolate W3^T and NCIMB 192, and values below 70% with the type strains of the phylogenetically closest species, P. iliopiscarium LMG 19543^T, P. kishitanii LMG 23890^T, P. aquimaris LMG 26951^T and P. phosphoreum LMG4233^T. The strains of this new taxon could also be distinguished from the latter species by phenotypic characteristics. Therefore, we propose to classify this new taxon as Photobacterium piscicola sp. nov., with W3^T (=NCCB 100098^T = LMG 27681^T) as the type strain.     

Description of Dietzia lutea sp. nov., isolated from a desert soil in Egypt

An actinobacterial strain YIM 80766^T was isolated from a soil sample collected from the eastern desert of Egypt, and its taxonomic position was investigated by a polyphasic approach. The organism was found to have a range of chemical and morphological properties consistent with its classification in the genus Dietzia. Phylogenetic analysis indicated that the levels of 16S rRNA gene sequence similarity between strain YIM 80766^T and the other type strains of recognized members of the genus Dietzia were 97.0–98.9%. However, DNA–DNA hybridization values and phenotypic characteristics revealed that the strain differed from the currently recognized species of the genus Dietzia. Therefore, strain YIM 80766^T represents a novel species of the genus Dietzia, for which the name Dietzia lutea sp. nov. is proposed. The type strain is YIM 80766^T (=KCTC 19232^T=DSM 45074^T=CCTCC AA 207008^T). The 16S rRNA gene sequence of strain YIM 80766^T has been deposited in GenBank under the accession number EU821598.     

Pseudomonas aestusnigri sp. nov., isolated from crude oil-contaminated intertidal sand samples after the Prestige oil spill

Strains VGXO14^T and Vi1 were isolated from the Atlantic intertidal shore from Galicia, Spain, after the Prestige oil spill. Both strains were Gram-negative rod-shaped bacteria with one polar inserted flagellum, strictly aerobic, and able to grow at 18–37 °C, pH 6–10 and 2–10% NaCl. A preliminary analysis of the 16S rRNA and the partial rpoD gene sequences indicated that these strains belonged to the Pseudomonas genus but were distinct from any known Pseudomonas species. A polyphasic taxonomic approach including phylogenetic, chemotaxonomic, phenotypic and genotypic data confirmed that the strains belonged to the Pseudomonas pertucinogena group. In a multilocus sequence analysis, the similarity of VGXO14^T and Vi1 to the closest type strain of the group, Pseudomonas pachastrellae, was 90.4%, which was lower than the threshold of 97% established to discriminate species in the Pseudomonas genus. The DNA–DNA hybridisation similarity between strains VGXO14^T and Vi1 was 79.6%, but below 70% with the type strains in the P. pertucinogena group. Therefore, the strains should be classified within the genus Pseudomonas as a novel species, for which the name Pseudomonas aestusnigri is proposed. The type strain is VGXO14^T (=CCUG 64165^T = CECT 8317^T).     

Rhizobium nepotum sp. nov. isolated from tumors on different plant species

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different plant species in Hungary: strain 39/7(T) from Prunus cerasifera Myrobalan, strain 0 from grapevine var. Ezerjó, strain 7/1 from raspberry var. Findus and in Poland, strain C3.4.1 from Colt rootstock (Prunus avium × Prunus pseudocerasus) and strain CP17.2.2 from Prunus avium. Only one of these isolates, strain 0, is able to cause crown gall on different plant species. On the basis of 16S rRNA gene sequence similarity, the strains cluster together and belong to the genus Rhizobium and their closest relative is Rhizobium radiobacter (99.1%). Phylogenetic analysis of the novel strains using housekeeping genes atpD, glnA, gyrB, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to Rhizobium radiobacter. The major cellular fatty acids are 18:1 w7c, 16:0, 16:0 3OH, summed feature 2 (comprising 12:0 aldehyde, 16:1 iso I and/or 14:0 3OH) and summed feature 3 (comprising 16:1 w7c and/or 15 iso 2OH). DNA-DNA hybridization of strain 39/7(T) with the type strain of R. radiobacter LMG 140(T) revealed 45% DNA-DNA hybridization. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium nepotum sp. nov. (type strain 39/7(T)=LMG 26435(T)=CFBP 7436(T)) is proposed.     

Isolation and characterization of poly- and oligosaccharides from the red microalga Porphyridium sp.

The current study forms part of an ongoing research effort focusing on the elucidation of the chemical structure of the sulfated extracellular polysaccharide of the red microalga Porphyridium sp. (UTEX 637). We report here on the chemical structure of a fraction separated from an acidic crude extract of the polysaccharide, as investigated by methylation analysis, carboxyl reduction-methylation analysis, desulfation-methylation analysis, partial acid hydrolysis, Smith degradation, together with 1D and 2D ¹H and ¹³C NMR spectroscopy. This fraction with a molar mass of 2.39 × 10⁵ g mol⁻¹ comprised d- and l-Gal, d-Glc, d-Xyl, d-GlcA, and sulfate groups in a molar ratio of 1.0:1.1:2.1:0.2:0.7. The almost linear backbone of the fraction is composed of (1→2)- or (1→4)-linked d-xylopyranosyl, (1→3)-linked l-galactopyranosyl, (1→3)-linked d-glucopyranosyl, and (1→3)-linked d-glucopyranosyluronic acid and comprises a possible acidic building unit:

[(2 or 4)-β-d-Xylp-(l→3)]_m-α-d-Glcp-(1→3)-α-d-GlcpA-(1→3)-l-Galp(l→

Attached to the backbone are sulfate groups and nonreducing terminal d-xylopyranosyl and galactopyranosyl residues, which occur at the O-6 positions of Glc-derived moieties in the main chain.     

Methylobacterium bullatum sp. nov., a methylotrophic bacterium isolated from Funaria hygrometrica

A novel, pink-pigmented aerobic, facultatively methylotrophic bacterial strain (F3.2^T) isolated from the phyllosphere of Funaria hygrometrica, was analyzed using a polyphasic approach. Cells were Gram-negative, motile rods, strictly aerobic and non-spore-forming and exhibited surface structures varying in quantity, distribution and morphology. The isolate grew at 10–33 °C over a pH range of 5.5–8.0 and in the presence of less than 1.0% NaCl. Strain F3.2^T shared less than 70% DNA–DNA binding to the next type strain of the genus Methylobacterium (M. adhaesivum DSM 17169^T). In addition to the major cellular fatty acid C_18:1ω7c (81.7%), present in all Methylobacterium species (and also members of the genus Alphaproteobacteria), a high value (11.7%) of the fatty acids (summed feature) C_16:1ω7c and/or iso-C_15:02OH was determined. Phylogenetic analyses based on 16S rDNA and methanol dehydrogenase gene sequences, DNA–DNA hybridization values, chemotaxonomic and phenotypic characteristics indicate that the strain F3.2^T represents a novel species within the genus Methylobacterium. We propose the name Methylobacterium bullatum sp. nov. for this species. The type strain is the strain F3.2^T (DSM 21893^T = LMG 24788^T).     

Metabolites from the endophytic fungus Xylaria sp. PSU-D14

Glucoside derivatives, xylarosides A (1) and B (2), were isolated from the broth extract of the endophytic fungus Xylaria sp. PSU-D14 along with two known compounds, sordaricin (3) and 2,3-dihydro-5-hydroxy-2-methyl-4H-1-benzopyran-4-one (4). The structures were assigned by spectroscopic methods. Sordaricin (3), one of the known metabolites, exhibited moderate antifungal activity against Candida albicans ATCC90028 with a MIC value of 32 microg/ml.     

Kytococcus aerolatus sp. nov., isolated from indoor air in a room colonized with moulds

A Gram-positive, coccoid bacterial isolate (02-St-019/1^T), forming beige pigmented colonies was obtained from an indoor air sample. Based on 16S rRNA gene sequence similarity studies it was determined that this isolate 02-St-019/1^T belonged to the genus Kytococcus, showing sequence similarties of 98.6% to Kytococcus schroeteri DSM 13884^T and 98.3% to Kytococcus sedentarius DSM 20547^T, respectively. The diagnostic diaminoacid of the peptidoglycan was lysine, cell wall sugars were ribose and xylose. The major menaquinones detected were MK-7 and MK-8. The polar lipid profile consisted of the major phospholipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and phosphatidylinositol mannoside. Fatty acid patterns were composed of major amounts of the iso- and anteiso-branched fatty acids anteiso C_17:0, iso C_15:0 and iso C_17:0 and unsaturated fatty acids (C_17:1 ω8c, iso C_17:1 ω9c, and C_17:1 ω8c) with smaller amounts of the straight-chain fatty acids C_15:0, C_16:0 and C_17:0. The results of DNA–DNA hybridizations and physiological and biochemical tests clearly allowed a genotypic and phenotypic differentiation of strain 02-St-019/1^T from the two described Kytococcus species. On the basis of these results a novel species to be named Kytococcus aerolatus sp. nov., is proposed, with the type strain 02-St-019/1^T (=DSM 22179^T=CCM 7639^T).     

Saccharopolyspora endophytica sp. nov., an endophytic actinomycete isolated from the root of Maytenus austroyunnanensis

A Gram-positive, aerobic, non-motile, non-acid-alcohol-fast strain, designated YIM 61095(T), was isolated from the root of Maytenus austroyunnanensis collected from a tropical rainforest of Xishuangbanna in Yunnan Province, south-west China. Strain YIM 61095(T) exhibited chemotaxonomic and morphological characteristics that were consistent with members of the genus Saccharopolyspora. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 61095(T) was a member of the genus Saccharopolyspora and was most closely related to Saccharopolyspora flava AS 4.1520(T) (97.7% sequence similarity). The major fatty acids were iso-C(15:0), iso-C(16:0), iso-C(17:0) and anteiso-C(17:0). The predominant quinone detected was MK-9(H(4)). The DNA G+C content was 66.2 mol%. The phenotypic characteristics and DNA-DNA hybridization relatedness data indicated that strain YIM 61095(T) should be distinguished from Saccharopolyspora flava AS 4.1520(T). On the basis of the evidence presented in this study, strain YIM 61095(T) represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora endophytica sp. nov. is proposed. The type strain is YIM 61095(T) (=KCTC 19397(T)=CCTCC AA 208003(T)).     

Toxic polyketides produced by Fusarium sp., an endophytic fungus isolated from Melia azedarach

A new isocoumarin derivative named fusariumin (1), together with two known related resorcylic acid lactones aigialomycin D (2) and pochonin N (3), has been isolated from the cultures of Fusarium sp. LN-10, an endophytic fungus originated from the leaves of Melia azedarach. Their structures were established on the basis of extensive spectroscopic analyzes including 1D- and 2D- NMR (¹H-¹H COSY, HSQC, HMBC, and NOESY) experiments. Compounds 1-3 displayed significant growth inhibitory activity against the brine shrimp (Artemia salina).     

The impact of mixed infection of three species of microsporidia isolated from the gypsy moth,Lymantria dispar L. (Lepidoptera: Lymantriidae)

The outcome of mixed infection by three species of microsporidia in the genera Endoreticulatus, Nosema, and Vairimorpha, isolated from different populations of Lymantria dispar in Bulgaria, was evaluated in the laboratory. All possible combinations of two species were administered either simultaneously or sequentially to larvae, and mortality, duration of development, and larval weight at 20 days post-infection (simultaneous inoculation) or 23 days post-infection (sequential inoculation) were chosen as the outcome variables. Larvae were also dissected and the presence of each species of microsporidia and the tissues infected were recorded for each treatment. Effects of infection were dependent on both host sex and the type of exposure. Infected larvae were more likely to die than uninfected larvae, but there were no differences in mortality between single and mixed infections. Addition of Endoreticulatus to infections of Nosema or Vairimorpha significantly increased duration of development to the fourth ecdysis; this effect was additive. Addition of Nosema or Vairimorpha to an existing infection had no such effect. When Nosema was administered simultaneously with Endoreticulatus or Vairimorpha, infected larvae weighed more than larvae that had single infections with either pathogen. Nosema was displaced from the silk glands by Vairimorpha and Nosema suppressed octospore formation by Vairimorpha in fat body. The histological evidence combined with the data on larval weight supports the hypothesis that competition occurred in mixed infections.