Shewanella cyperi sp. nov., a facultative anaerobic bacterium isolated from mangrove sediment.

Two Gram-stain-negative, motile, facultatively anaerobic, rod-shaped strains, FJAT-53720T and FJAT-53726, were isolated from rhizosphere sediment of plant Cyperus malaccensis. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that strains FJAT-53720T and FJAT-53726 were affiliated to the genus Shewanella (forming an independent cluster) with the highest sequence similarity to the type strain of Shewanella algae. Optimum growth of both strains was observed at 30 °C and pH 7. The respiratory quinones were Q-7, Q-8 and MK-7. The polar lipid profile included phosphatidylmethyl ethanolamine, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified aminophospholipid and four unknown phospholipids. The major fatty acids of strains FJAT-53720T and FJAT-53726 were iso-C15:0, C17 : 1 ω8c and summed feature 3. The genomic DNA G+C content of strain FJAT-53720T was 55.6 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between FJAT-53720T and FJAT-53726 were 97.4 and 77.9 %, confirming that they were similar species and hence FJAT-53720T was selected for further analysis. The ANI and dDDH values between FJAT-53720T and other members of the genus Shewanella were below the cut-off level (95-96 %; 70 %) for species delineation. Based on the above results, FJAT-53720T represents a novel species of the genus Shewanella, for which the name Shewanella cyperi sp. nov. is proposed. The type strain is FJAT-53720T (=KCTC 82444T=GDMCC 1.2207T).

[Effects of Mycorrhizal Fungi on Nitrification and Denitrification in the Rhizospheric Soil of Aquatic Plants and Its Microbial Mechanism].

To investigate the effect of mycorrhizal fungi inoculation on nitrification-denitrification in the rhizospheric soil of aquatic plants, Cyperus alternifolius and Acorus tatarinowii were inoculated with the strain MF-MD obtained from local soil. Uninoculated plants served as the control group. The plants were cultivated for 3 months under nitrogen eutrophication in water, and then the nitrification and denitrification activities in the rhizospheric soil were determined. The results showed that inoculation with MF-MD promoted nitrification in the rhizospheric soil of both plants. However, MF-MD inoculation promoted denitrification in the rhizospheric soil of Cyperus alternifolius but inhibited denitrification in the rhizospheric soil of Acorus tatarinowii. The mechanism of soil nitrification-denitrification activity was analyzed by measuring the changes in the community structure of nitrifying bacteria and denitrifying bacteria in the microbial biomass of the rhizospheric soil. It was found that the rhizospheric soil microbial biomass (SMB) of the experimental group was higher than that of the control group and that the changes in the microbial community structure related to soil nitrification and denitrification differed between groups. This study contributes to an understanding of how mycorrhizal fungi combined with aquatic plants can remove N content in eutrophic water.

Effects of ofloxacin on nitrogen removal and microbial community structure in constructed wetland.

Constructed wetlands (CWs) have emerged as a promising technology for the purification of micro-polluted water. However, their nitrogen removal performance can be significantly degraded by design, operational, and environmental factors. The present study investigates the effects of ofloxacin (OFL: 0.1, 10, and 1000 µg L-1) and plants (Cyperus alternifolius L. and Typha angustifolia L.) on nitrogen removal in a micro-polluted CW system over a duration of 12 weeks. The effects were evaluated by investigating NH4-N and NO3-N removal efficiency, nitrification genes (amoA-AOA and amoA-AOB), denitrification genes (nirK and nirS), fungal 18S rRNA gene and microorganism community structure. The results showed that in unplanted CWs, OFL increased the NH4-N removal efficiency (from 72.6% to 80.7-82.1%), the abundances of amoA-AOA, nirS, nirK and fungal 18S rRNA gene, and the bacterial diversity but decreased the abundance of both amoA-AOB and bacterial richness. In contrast, both the nitrogen removal efficiency (83.4-89.5% for NH4-N and 33.8-38.5% for NO3-N) and bacterial diversity/richness were not significantly affected by OFL in planted CWs. In planted systems, OFL increased the relative abundance of Arthrobacter, Pseudomonas, and Enterococcus, which are proven antibiotic-resistant bacteria. This study showed that CWs are able to remove nitrogen from antibiotic-contaminated micro-polluted water, which might primarily be attributed to the presence of plants that protect the microorganism community.

Anti-quorum Sensing and Anti-biofilm Activity of Delftia tsuruhatensis Extract by Attenuating the Quorum Sensing-Controlled Virulence Factor Production in Pseudomonas aeruginosa.

Multidrug-resistance bacteria commonly use cell-to-cell communication that leads to biofilm formation as one of the mechanisms for developing resistance. Quorum sensing inhibition (QSI) is an effective approach for the prevention of biofilm formation. A Gram-negative bacterium, Delftia tsuruhatensis SJ01, was isolated from the rhizosphere of a species of sedge (Cyperus laevigatus) grown along the coastal-saline area. The isolate SJ01 culture and bacterial crude extract showed QSI activity in the biosensor plate containing the reference strain Chromobacterium violaceum CV026. A decrease in the violacein production of approximately 98% was detected with the reference strain C. violaceum CV026. The bacterial extract (strain SJ01) exhibited anti-quorum sensing activity and inhibited the biofilm formation of clinical isolates wild-type Pseudomonas aeruginosa PAO1 and P. aeruginosa PAH. A non-toxic effect of the bacterial extract (SJ01) was detected on the cell growth of the reference strains as P. aeruginosa viable cells were present within the biofilm. It is hypothesized that the extract (SJ01) may change the topography of the biofilm and thus prevent bacterial adherence on the biofilm surface. The extract also inhibits the motility, virulence factors (pyocyanin and rhamnolipid) and activity (elastase and protease) in P. aeruginosa treated with SJ01 extract. The potential active compound present was identified as 1,2-benzenedicarboxylic acid, diisooctyl ester. Microarray and transcript expression analysis unveiled differential expression of quorum sensing regulatory genes. The key regulatory genes, LasI, LasR, RhlI, and RhlR were down-regulated in the P. aeruginosa analyzed by quantitative RT-PCR. A hypothetical model was generated of the transcriptional regulatory mechanism inferred in P. aeruginosa for quorum sensing, which will provide useful insight to develop preventive strategies against the biofilm formation. The potential active compound identified, 1,2-benzenedicarboxylic acid, diisooctyl ester, has the potential to be used as an anti-pathogenic drug for the treatment of biofilm-forming pathogenic bacteria. For that, a detailed study is needed to investigate the possible applications.

Cedrol, a malaria mosquito oviposition attractant is produced by fungi isolated from rhizomes of the grass Cyperus rotundus.

BACKGROUND: Cedrol, a sesquiterpene alcohol, is the first identified oviposition attractant for African malaria vectors. Finding the natural source of this compound might help to elucidate why Anopheles gambiae and Anopheles arabiensis prefer to lay eggs in habitats containing it. Previous studies suggest that cedrol may be a fungal metabolite and the essential oil of grass rhizomes have been described to contain a high amount of different sesquiterpenes. RESULTS: Rhizomes of the grass Cyperus rotundus were collected in a natural malaria mosquito breeding site. Two fungi were isolated from an aqueous infusion with these rhizomes. They were identified as Fusarium falciforme and a species in the Fusarium fujikuroi species complex. Volatile compounds were collected from the headspace above fungal cultures on Tenax traps which were analysed by gas chromatography-mass spectrometry (GCMS). Cedrol and a cedrol isomer were detected in the headspace above the F. fujikuroi culture, while only cedrol was detected above the F. falciforme culture. CONCLUSION: Cedrol an oviposition attractant for African malaria vectors is produced by two fungi species isolated from grass rhizomes collected from a natural mosquito breeding site.

[Advenella kashmirensis subsp. methylica PK1, a facultative methylotroph from carex rhizosphere].

A strain (PK1) of facultative methylobacteria growing on methanol as a carbon and energy source was isolated from carex rhizosphere (Pamukkale National Park, Turkey). The cells were nonmotile gram-negative rods propagating by binary fission. The organism was a strict anaerobe, oxidase- and catalase-positive. Optimal growth occurred at 29°C, pH 8.0-8.5, and 0.5% NaCl; no growth occurred at 2% NaCl. The organism used the ribulose bisphosphate pathway of C1 assimilation. Predominant fatty acids were 11-octodecenoic (18:1ω7) and cis-hexadecenoic (16:1ω7c). Phosphatidylethanolamine and diphosphatidylglycerol were the dominant phospholipids. Q8 was the main ubiquinone. DNA G+C content was 55.4 mol % (mp). Sequencing of the 16S rRNA gene revealed that strain PK1 belonged to the genus Advenella with 98.8 and 99.2% similarity to the type strains A. incenata CCUG 45225T and A. kashmirensis WT001T, respectively. DNA-DNA homology of strain PK1 and A. kashmirensis WT001T was 70%. While MALDI analysis confirmed their close clusterization, RAPD analysis revealed the differences between strain PKI and other Advenella strains. Based on its geno- and phenotypic properties, the isolate PK1 was classified as A. kashmirensis subsp. methylica PK1 (VKM-B 2850 = DSM 27514), the first known methylotroph of the genus Advenella.

A survey of pathogens associated with Cyperus esculentus L (tiger nuts) tubers sold in a Ghanaian city.

BACKGROUND: Cyperus esculentus L, is a minor but important crop in Ghana. They are noted mostly by their aphrodisiac properties among others. The nuts are often eaten raw as an unprocessed snack due to its rich flavour and texture. Though eaten raw, the nuts are sometimes handled unhygienically, posing a public health threat. This study therefore aimed at determining the level and distribution of parasitic and bacterial contaminants associated with the crop as it is sold. RESULTS: Four types of intestinal parasites were identified, and the most prevalent was Cryptosporidium parvum (30.0%). Nuts contaminated with parasites were found only among street vendors. Bacteriological examination showed three different groups of bacterial isolates with the most prevalent being coliforms (54.2%). Unlike parasites, bacteria isolates were found among samples from both street vendors and market places. Multiple drug resistance was displayed by Proteus vulgaris. CONCLUSIONS: Buying and eating nuts as well as other fruits taken raw from street vendors and market places could pose a significant public health threat. There is a need for efficient monitoring systems for food borne pathogens in Ghana.

Characterization of Pseudomonas mendocina LR capable of removing nitrogen from various nitrogen-contaminated water samples when cultivated with Cyperus alternifolius L.

A new strain of denitrifying bacterium, Pseudomonas mendocina LR, was isolated from rhizosphere soil of aquatic plants living in a river contaminated with industrial wastewater and domestic sewage. The isolate was found to fully remove as much as 613.2 mg nitrate in 60 h under stationary culture conditions. The effects of carbon sources and nitrogen sources on nitrogen removal were investigated using a modified denitrification medium (DM). Sodium citrate was identified as the most effective source of carbon. The ability of LR to adapt to different nitrogen sources, including nitrite, indicated that LR could be used in the purification of wastewater containing different forms of nitrogen. The optimal C/N ratio was 7 for LR, and it was resistant to antibiotics Amp, Chl, Ery, and Str. Plant-microbe bioaugmentation was performed to remove nitrogen dissolved in Hoagland medium and natural wastewater. An increased rate of nitrogen removal was observed when root exudates of Cyperus alternifolius L. were added simultaneously with LR. LR was not able to survive in the natural wastewater unless root exudates from umbrella grass were added. LR cultured with umbrella grass exhibited a maximal nitrogen reduction rate of 95.9% and 97.3% in Hoagland medium and wastewater, respectively. This shows that bioaugmentation utilizing plant-microbe interactions can be an effective and exhaustive means of removing nitrogen and may be an attractive approach to nitrogen reduction in natural environments and wastewater treatment factories.

Polyphasic analysis of the bacterial community in the rhizosphere and roots of Cyperus rotundus L. grown in a petroleum-contaminated soil.

Cyperus rotundus L. is a perennial herb which was found prevailing in an area in the northeast of Brazil previously contaminated with petroleum. In order to enlarge the knowledge of microorganism-plant interaction in phytoremediation, the bacterial community present in the rhizosphere and roots of C. rotundus was evaluated by culture-dependent and molecular approaches. PCR-DGGE analyses based on the 16S rRNA gene showed that the profiles of bulk soil, rhizosphere and root samples had a high degree of similarity. A complex community of alkane utilizing-bacteria and a variable nitrogen-fixing community were observed when the PCR-DGGE analyses were based on the genes alkB and nifH, respectively. In addition, two clone libraries were generated from the alkB fragments of bulk and rhizosphere soils. Statistical analyses showed that the libraries were different concerning the alkB population composition. Using culture-dependent techniques, 209 bacterial strains were isolated from the rhizosphere and rhizoplane/roots of C. rotundus. Dot blotting analysis showed that the DNA from 17 strains hybridized, simultaneously, with the alkB and nifH probes. After partial 16S rRNA gene sequencing, these strains were affiliated with the genera Bosea, Cupriavidus, Enterobacter, Gordonia, Mycoplana, Pandoraea, Pseudomonas, Rhizobium and Rhodococcus. They can be considered of great potential for phytoremediation in this tropical soil area.

Symptomatology and morphology of Claviceps cyperi on yellow nut sedge in South Africa.

Symptoms of ergot on yellow nut sedge, germination of sclerotia of the causal organism, Claviceps cyperi, and morphology of fresh specimens of the pathogen are described for the first time. The initial symptom of infection was a black sooty layer on inflorescences of infected plants due to colonization of the ergot honeydew by Cladosporium cladosporioides. Sclerotia of C. cyperi started to develop in March and April and could be discerned as small protuberances on inflorescences in the place of seed. Mature sclerotia were purplish-black. They generally remained viable for less than a year and germinated without prior cold treatment, although exposure for 21 d to 5 C before incubation significantly increased the germination rate. Under moist conditions at 24 C in the laboratory, germination commenced within 4-8 wk. Stromata took about 12 d to mature. Mature capitula were distinctly lobulate with a perithecium embedded in each lobe and a collar-like appendage around the base. Although dimensions of sclerotia, stipes, capitula, asci and ascospores were larger than in the original description, the general morphology supports treatment of C. cyperi as a distinct species.

Claviceps cyperi, a new cause of severe ergotism in dairy cattle consuming maize silage and teff hay contaminated with ergotised Cyperus esculentus (nut sedge) on the Highveld of South Africa.

During December/January 1996/97 typical summer syndrome (hyperthermia and a 30% drop in milk yield) occurred in succession in two Holstein dairy herds (n=240 and n=150 milking cows, respectively) on the South African Highveld. These farms are situated in the midst of the prime maize and dairy farming areas of South Africa where this condition had never been diagnosed before. The individual components of the concentrate on both farms were negative for ergot alkaloids. Endophytic fungi and/or ergot infestation of teff and other grasses fed to the cows were then suspected of being involved, but neither endophytes nor ergot alkaloids could be implicated from these sources. By measuring the serum prolactin levels of groups of sheep (n=5) fed the first farm's total mixed ration (TMR) or its three individual fibre components for a period of 11 days, the source of the ergot alkaloids was identified. A statistically significant decrease in the level of this hormone occurred only in the group on maize silage (which constituted 28% on dry matter base of the TMR). The involvement of the maize silage was further chemically confirmed by the high levels of total ergot alkaloids, predominantly ergocryptine, found by LC-MS in the silage as well as in the TMR (115-975 ppb and 65-300 ppb, respectively). The ergot alkaloid content (mainly ergocryptine) of the maize silage on the second affected farm was 875 ppb. Withdrawal of contaminated silage resulted in gradual recovery of stock on both farms. Nut sedge (Cyperus esculentus and Cyperus rotundus of the family Cyperaceae) has a world-wide distribution and is a common weed in annual crops, and can be parasitized by Claviceps cyperi. Careful examination of the maize silage from both farms revealed that it was heavily contaminated with nut sedge and that it contained minute sclerotia, identified as those of Claviceps cyperi, originating from the latter. Nut sedge was abundant on both farms and it is believed that late seasonal rain had resulted in mature, heavily ergotised nut sedge being cut with the silage. Claviceps cyperi sclerotia, collected on the affected fields in the following autumn contained 3600-4000 ppm ergocryptine. That the dominant alkaloid produced by this particular fungus was indeed ergocryptine, was confirmed by negative ion chemical ionization MS/MS. In one further outbreak in another Holstein herd, teff hay contaminated with ergotised nut sedge and containing 1200 ppb alkaloids, was incriminated as the cause of the condition. This is the first report of bovine ergotism not associated with the Poaceae infected with Claviceps purpureum or endophytes but with the family Cyperaceae and this particular fungal phytopathogen.

Improvement of the hydrocarbon phytoremediation rate by Cyperus laxus Lam. inoculated with a microbial consortium in a model system.

Hydrocarbon phytoremediation by Cyperus laxus Lam. growing on perlite and inoculated with hydrocarbon-degrading microorganisms was evaluated. Total petroleum hydrocarbons (TPH) were extracted from weathered soil (60.7 g of TPH kg(-1) of dry soil) and spiked on perlite at initial concentration of 5 g of TPH kg(-1) of dry perlite. Phenological characteristics, total microbial viable counts, hydrocarbon degraders and residual hydrocarbons were determined through 180 days of culture. Phenological characteristics of inoculated plants were improved as compared with non-inoculated plants: root biomass was 1.6 times greater, flowering time was reduced (13%), and the number of inflorescences was 1.5 times higher. The rhizospheric bacterial and fungi counts were higher for planted treatments (inoculated and not inoculated) than for unplanted pots. The maximum phytoremediation rate (0.51 mg of TPH g(-1) of dry plant d(-1)) for inoculated plants was reached at 60 days of culture, and was two times higher than for non-inoculated plants (55% TPH removal). Similar hydrocarbon phytoremediation extent values for inoculated (90%) and non-inoculated (85%) plants were obtained at 180 days of culture. The present study demonstrated that mutual benefits between C. laxus and inoculated hydrocarbon-degrading microorganisms are improved during phytoremediation. It is pertinent to note that this is the first report of hydrocarbon phytoremediation by Cyperus laxus Lam., a native plant growing in highly contaminated swamps.

Biodegradation of Maya crude oil fractions by bacterial strains and a defined mixed culture isolated from Cyperus laxus rhizosphere soil in a contaminated site.

Ten bacterial strains were isolated by enrichment culture, using as carbon sources either aliphatics or an aromatic-polar mixture. Oxygen uptake rate was used as a criterion to determine culture transfer timing at each enrichment stage. Biodegradation of aliphatics (10,000 mg L(-1)) and an aromatic-polar mixture (5000 mg L(-1), 2:1) was evaluated for each of the bacterial strains and for a defined culture made up with a standardized mixture of the isolated strains. Degradation of total hydrocarbons (10,000 mg L(-1)) was also determined for the defined mixed culture. Five bacterial strains were able to degrade more than 50% of the aliphatic fraction. The most extensive biodegradation (74%) was obtained with strain Bs 9A, while strains Ps 2AP and UAM 10AP were able to degrade up to 15% of the aromatic-polar mixture. The defined mixed culture degraded 47% of the aliphatics and 6% of the aromatic-polar mixture. The defined mixed culture was able to degrade about 40% of the aliphatic fraction and 26% of the aromatic fraction when grown in the presence of total hydrocarbons, while these microorganisms did not consume the polar hydrocarbons fraction. The proposed strategy that combines enrichment culture together with oxygen uptake rate allowed the isolation of bacterial strains that are able to degrade specific hydrocarbons fractions at high consumption rates.

Investigations on rhizoplane Actinobacteria communities of papyrus (Cyperus papyrus) from an Egyptian wetland.

Wetlands have important global ecological functions, which include carbon storage and water interception. Wetland contributes to the maintenance of regional and global biodiversity. Though many important wetland ecological functions are based on microbial metabolism, we have scanty knowledge on microbial diversity in wetlands. Plant rhizoplane habitats are considered to harbor highly diverse bacterial communities. Most of the floating mats on river Nile are dominated by papyrus (Cyperus papyrus). Papyrus root samples were collected from a floating mat at the "Gold Island" inside the Nile River at Cairo, Egypt in February 1996 and May 1997 in order to investigate the rhizoplane actinobacteria communities. The root-tip regions were cut off, repeatedly washed, macerated and plated. Using the plate-count technique with three actinobacteria media, an average of 2.1 x 10(4) CFUg-1 root actinobacteria were obtained. All actinobacteria colonies were isolated, purified and investigated by classical and molecular methods. In the papyrus rhizoplane Streptomyces anulatus, Micromonospora sp., Rhodococcus luteus, Verrucosispora gifhornensis and Aureobacterium liquefaciens dominated, moreover Actinoplanes utahensis, and Str. diastaticus were also present. The physiological traits of the members of dominant groups revealed that these bacteria might be active in the rhizoplane and can be present there is their vegetative forms.