Resistance of a multiple-isolate marine culture to ultraviolet C irradiation: inactivation vs biofilm formation.

Ultraviolet (UV) irradiation is an emerging strategy for controlling the formation of undesired biofilms in water desalination facilities using reverse osmosis (RO). However, most studies examining these pretreatments are limited as they have been conducted on single-species cultures, while biofilms are composed of multiple-species communities. The goal of this study was to investigate the effect of UV-C irradiation on a model community composed of six environmental isolates from a marine biofilm formed in RO seawater desalination plant. There was a high variance in the susceptibility of the single-isolate cultures to UV-C, from no response (isolate Eryth23) to complete inactivation (isolate Vib3). The most active wavelength was around 260 nm, resulting in a loss of viability of single-isolate cultures and loss of vitality of the mixed-isolate cultures. With respect to biofilm formation, the activity of this wavelength was completely different compared to its activity on planktonic suspension. Irradiation with 260 nm did not inhibit the total biofilm formation by the six-isolate culture; moreover, isolates such as the resistant Eryth23 or the susceptible Pseudoalt17, even gained abundance in the mixed isolate biofilm. The only decrease in total biofilm was obtained from irradiation at 280 nm, which was less active against the planktonic culture. These results indicate that the complexity of the biofilm-forming microbial community may contribute to its resistance to UV-C irradiation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study examined the resistance of a multiple-isolate native marine culture to UV-C irradiation, in terms of viability, vitality and the ability to form biofilm. Results of this study showed that even though most of the cells were inactivated both in single-isolate and in multiple-isolate cultures, still the multiple-isolate cultures manages to form biofilms, surprisingly with higher biomass than without irradiation. The significance of the study is in its conclusion that studies on UV-C irradiation of biofilm-forming model micro-organisms are not always applicable to natural multiple-species communities.

First Report of Anthracnose Fruit Rot Caused by Colletotrichum acutatum on Pepper and Tomato in Bulgaria.

In the late summer of 2005, sporadic and unusual damage was observed on pepper (Capsicum annuum cv. Kurtovska kapia and local cv. Ribka) on two farms and tomato (Lycopersicon esculentum cv. Florida 47) fruits on one farm in the Plovdiv Region of Bulgaria. Dry, round, sunken zones (10 to 20 mm) were observed on pepper fruits that preserved their natural skin color even after black acervuli containing orange masses of conidia appeared. Eventually, the lesions turned brown, coalesced, and the fruits mummified on the plants. Tomato fruits developed similar symptoms, with less prominent discoloration and fewer acervuli. The pathogen was easily isolated from both hosts on potato dextrose agar where it formed white-to-gray colonies with salmon orange pigmentation on the reverse side of the plates. Conidia that formed were hyaline, fusiform, aseptate, and measured 13.3 to 17.4 × 3.5 to 5.5 µm and 11.6 to 15.5 × 4.1 to 5.0 µm for pepper and tomato isolates, respectively. Both cultural and morphological characteristics of the isolates were similar to those described for Colletotrichum acutatum (3). Koch's postulates were performed with two representative isolates from each host by artificial inoculation of healthy, green pepper and ripe tomato fruits from the respective cultivars. Fruits were wound inoculated with a sterile scalpel, and small agar plugs (3 to 4 mm) containing 7-day-old sporulating cultures were placed on each wound (five fruits per isolate), or by pipette tip-pricking and pipetting a 5-µl droplet of a conidial suspension (5 × 106 conidia ml-1) on each wound. The same number of wounded, noninoculated fruits was used as a control. Fruits were maintained in a humidity chamber at 22 to 25°C, and 4 days later, sunken necrotic zones were observed around the wounds of inoculated fruit, whereas control fruits remained symptomless. The pathogen was subsequently reisolated from the inoculated diseased tissues but not from the control fruits. Species-specific PCR (using primer pair CaInt2/ITS4) (2,4) of genomic DNA from three representative isolates (two from pepper and one from tomato) resulted in an amplification product of 490 bp, specific for C. acutatum, further confirming the identity of the pathogen. To our knowledge, this is the second report of C. acutatum in Bulgaria (1), and the first occurrence of that agent on tomato and pepper in this country. References: (1) S. G. Bobev et al. Plant Dis. 86:1178, 2002. (2) S. Freeman et al. Phytopathology 91:586, 2001. (3) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (4) M. L. Lewis Ivey et al. Plant Dis. 88:1198, 2004.

Influence of effluent irrigation on community composition and function of ammonia-oxidizing bacteria in soil.

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the alpha-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.

Genetic Diversity Within Colletotrichum acutatum sensu Simmonds.

ABSTRACT Isolates of Colletotrichum acutatum from several hosts were characterized by various molecular methods in comparison with morphological identification. Species-specific primer analysis was reliable for grouping C. acutatum isolates to their designated species. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses identified four subgroups within C. acutatum. Subgroup I contained U.S. isolates from almond, apple, peach, and pecan, subgroup II contained isolates from anemone, olive, and strawberry, subgroup III contained isolates from almond (Israel) and strawberry (Spain), and subgroup IV contained a single isolate from anemone (the Netherlands). Likewise, sequence analysis of the internal transcribed spacer (ITS) 2 region alone or the complete ITS (ITS 1-5.8S-ITS 2) region grouped the isolates into the same four subgroups. Percent similarity of the complete ITS region within each cluster ranged from 99.6 to 100.0, 99.8 to 100.0, and 98.6% among subgroups I, II, and III, respectively. DNA sequence analysis of the ITS 2 region alone or the entire ITS 1-2 region was more informative than that of the ITS 1 region, which could only group the isolates into two main clusters. The molecular methods employed for studying genetic variation in populations of C. acutatum determined that this species is diverse, indicating that isolates within populations of each subgroup are not host specific.

Distribution and diversity of archaea corresponding to the limnological cycle of a hypersaline stratified lake (Solar lake, Sinai, Egypt).

The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing. Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis. The 165 clones studied were grouped into four phylogenetically different clusters. Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to the Halobacteriaceae family. The three additional clusters were all isolated from the anaerobic hypolimnion. Cluster II is phylogenetically located between the genera Methanobacterium and Methanococcus. Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genus Thermoplasma. No crenarchaeota were found in the water column of the Solar Lake. The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10%. During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion.

Prey range characterization, ribotyping, and diversity of soil and rhizosphere Bdellovibrio spp. isolated on phytopathogenic bacteria.

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 x 10(2) to 6 x 10(3) and 2.8 x 10(2) to 2.3 x 10(4) PFU g(-1). A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.

Culture-Independent Detection of Changes in Root-Associated Bacterial Populations of Common Bean (Phaseolus vulgaris L.) Following Nitrogen Depletion.

The structure of root-associated bacterial populations in the legume common bean (Phaseolus vulgaris L.), was studied in plants grown under nitrogen sufficiency and under conditions inducing nitrogen deficiency. Similar cell numbers were obtained in the rhizosphere of nitrogen-amended plants as compared to nitrogen-deficient plants and between various root parts-tip, elongation and branching zones-using DAPI staining. In contrast, a higher proportion of DAPI-stained cells from the nitrogen-amended plants hybridized with a fluorescence-labeled EUB338 probe for the Bacteria domain than cells originating from nitrogen-deficient plants. Shifts in the percentages of EUB338-reactive cells-as well as in absolute cell number-hybridizing to fluorescent rRNA-directed probes specific for the a and g Proteobacteria and for high GC content gram-positive bacteria in separated root segments were detected between the treatments. No such differences were found using b and d Proteobacteria or rRNA group I pseudomonad targeted probes. Denaturating gradient gel electrophoresis profiles of PCR products obtained from the same samples and amplified with Bacteria-domain targeted primers supported the results obtained with the whole cell hybridizations. The advantages and drawbacks of the techniques applied are discussed.

Molecular analyses of colletotrichum species from almond and other fruits.

ABSTRACT Isolates of Colletotrichum spp. from almond, avocado, and strawberry from Israel and isolates of the pink subpopulation from almond from the United States were characterized by various molecular methods and compared with morphological identification. Taxon-specific primer analysis grouped the avocado isolates within the species C. gloeosporioides and the U.S. almond and Israeli strawberry isolates within the species C. acutatum. However, the Israeli almond isolates, previously identified morphologically as C. gloeosporioides, reacted with C. acutatum-specific primers. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses determined that each population from almond and strawberry was distinct and clonal. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS 1-5.8S-ITS 2) revealed a similarity of between 97.03 and 98.72% among almond isolates from Israel, C. acutatum almond isolates from the United States, and C. acutatum strawberry isolates from Israel. Similarity of the above populations to that of C. gloeosporioides of avocado was between 92.42 and 92.86%. DNA sequence analysis of the entire ITS region supported the phylogeny inferred from the ITS 1 tree of 14 different Colletotrichum species. Although morphological criteria indicated that the Israeli isolates from almond are unique, this population was grouped within the C. acutatum species according to molecular analyses.

[Suicidal Tachmalcor poisoning--a case report]. / Suizidale Tachmalcor-Intoxikation--Ein Fallbericht.

In a case report, a Tachmalcor intoxication with a dose of 18 mg/kg body weight is described. This dose caused a ventricular flutter in the patient which lasted for a total of 10 hours, despite intensive treatment. The treatment began approximately three hours after the intoxication and concentrated on therapy of the ventricular tachycardia. The use of Xylocitin 2%, defibrillation, glucagon and sodium chloride is recommended with such symptoms. Additionally, we used hemoperfusion for drug elimination. Despite the cardiac rhythm disorder of such duration, no neurological deficiencies were observed in the patient. Intoxications caused by these drugs in normal intensive therapies are extremely rare and for this reason treatment can often be very problematic. The following article reports on the successful therapy of one such patient.

Unexpected population distribution in a microbial mat community: sulfate-reducing bacteria localized to the highly oxic chemocline in contrast to a eukaryotic preference for anoxia.

The distribution and abundance of sulfate-reducing bacteria (SRB) and eukaryotes within the upper 4 mm of a hypersaline cyanobacterial mat community were characterized at high resolution with group-specific hybridization probes to quantify 16S rRNA extracted from 100-microm depth intervals. This revealed a preferential localization of SRB within the region defined by the oxygen chemocline. Among the different groups of SRB quantified, including members of the provisional families "Desulfovibrionaceae" and "Desulfobacteriaceae," Desulfonema-like populations dominated and accounted for up to 30% of total rRNA extracted from certain depth intervals of the chemocline. These data suggest that recognized genera of SRB are not necessarily restricted by high levels of oxygen in this mat community and the possibility of significant sulfur cycling within the chemocline. In marked contrast, eukaryotic populations in this community demonstrated a preference for regions of anoxia.

Diversity of sulfate-reducing bacteria in oxic and anoxic regions of a microbial mat characterized by comparative analysis of dissimilatory sulfite reductase genes.

Sequence analysis of genes encoding dissimilatory sulfite reductase (DSR) was used to identify sulfate-reducing bacteria in a hypersaline microbial mat and to evaluate their distribution in relation to levels of oxygen. The most highly diverse DSR sequences, most related to those of the Desulfonema-like organisms within the delta-proteobacteria, were recovered from oxic regions of the mat. This observation extends those of previous studies by us and others associating Desulfonema-like organisms with oxic habitats.

Anaerobic oxidation of o-xylene, m-xylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria.

Various alkylbenzenes were depleted during growth of an anaerobic, sulfate-reducing enrichment culture with crude oil as the only source of organic substrates. From this culture, two new types of mesophilic, rod-shaped sulfate-reducing bacteria, strains oXyS1 and mXyS1, were isolated with o-xylene and m-xylene, respectively, as organic substrates. Sequence analyses of 16S rRNA genes revealed that the isolates affiliated with known completely oxidizing sulfate-reducing bacteria of the delta subclass of the class Proteobacteria. Strain oXyS1 showed the highest similarities to Desulfobacterium cetonicum and Desulfosarcina variabilis (similarity values, 98.4 and 98.7%, respectively). Strain mXyS1 was less closely related to known species, the closest relative being Desulfococcus multivorans (similarity value, 86.9%). Complete mineralization of o-xylene and m-xylene was demonstrated in quantitative growth experiments. Strain oXyS1 was able to utilize toluene, o-ethyltoluene, benzoate, and o-methylbenzoate in addition to o-xylene. Strain mXyS1 oxidized toluene, m-ethyltoluene, m-isoproyltoluene, benzoate, and m-methylbenzoate in addition to m-xylene. Strain oXyS1 did not utilize m-alkyltoluenes, whereas strain mXyS1 did not utilize o-alkyltoluenes. Like the enrichment culture, both isolates grew anaerobically on crude oil with concomitant reduction of sulfate to sulfide.

Anaerobic degradation of naphthalene by a pure culture of a novel type of marine sulphate-reducing bacterium.

Incubation of marine sediment in anoxic, sulphate-rich medium in the presence of naphthalene resulted in the enrichment of sulphate-reducing bacteria. Pure cultures with short, oval cells (1.3 by 1.3-1.9 microm) were isolated that grew with naphthalene as the only organic carbon source and electron donor for sulphate reduction to sulphide. One strain, NaphS2, was characterized. It affiliated with completely oxidizing sulphate-reducing bacteria of the delta-subclass of the Proteobacteria, as revealed by 16S rRNA sequence analysis. 2-Naphthoate, benzoate, pyruvate and acetate were used in addition to naphthalene. Quantification of substrate consumption, sulphide formation and formed cell mass revealed that naphthalene was completely oxidized with sulphate as the electron acceptor.

Cadmium binding by bacteria: screening and characterization of new isolates and mutants.

A fast and simple methodology was developed that enables screening of microbial strains for their ability to bind cadmium. It is based on the use of a radioisotope of cadmium (109Cd) for screening colonies and for evaluation of cadmium binding. The methods described here can be used to screen new environmental isolates or to obtain mutants with altered ability to bind cadmium. Examples for the two uses are described in the paper.