Controls on pathogen species richness in plants' introduced and native ranges: roles of residence time, range size and host traits

Introduced species escape many pathogens and other enemies, raising three questions. How quickly do introduced hosts accumulate pathogen species? What factors control pathogen species richness? Are these factors the same in the hosts' native and introduced ranges? We analysed fungal and viral pathogen species richness on 124 plant species in both their native European range and introduced North American range. Hosts introduced 400 years ago supported six times more pathogens than those introduced 40 years ago. In hosts' native range, pathogen richness was greater on hosts occurring in more habitat types, with a history of agricultural use and adapted to greater resource supplies. In hosts' introduced range, pathogen richness was correlated with host geographic range size, agricultural use and time since introduction, but not any measured biological traits. Introduced species have accumulated pathogens at rates that are slow relative to most ecological processes, and contingent on geographic and historic circumstance.     

Assessing Niche Separation among Coexisting Limnohabitans Strains through Interactions with a Competitor,Viruses, and a Bacterivore

We investigated potential niche separation in two closely related (99.1% 16S rRNA gene sequence similarity) syntopic bacterial strains affiliated with the R-BT065 cluster, which represents a subgroup of the genus Limnohabitans. The two strains, designated B4 and D5, were isolated concurrently from a freshwater reservoir. Differences between the strains were examined through monitoring interactions with a bacterial competitor, Flectobacillus sp. (FL), and virus- and predator-induced mortality. Batch-type cocultures, designated B4+FL and D5+FL, were initiated with a similar biomass ratio among the strains. The proportion of each cell type present in the cocultures was monitored based on clear differences in cell sizes. Following exponential growth for 28 h, the cocultures were amended by the addition of two different concentrations of live or heat-inactivated viruses concentrated from the reservoir. Half of virus-amended treatments were inoculated immediately with an axenic flagellate predator, Poterioochromonas sp. The presence of the predator, of live viruses, and of competition between the strains significantly affected their population dynamics in the experimentally manipulated treatments. While strains B4 and FL appeared vulnerable to environmental viruses, strain D5 did not. Predator-induced mortality had the greatest impact on FL, followed by that on D5 and then B4. The virus-vulnerable B4 strain had smaller cells and lower biomass yield, but it was less subject to grazing. In contrast, the seemingly virus-resistant D5, with slightly larger grazing-vulnerable cells, was competitive with FL. Overall, our data suggest contrasting ecophysiological capabilities and partial niche separation in two coexisting Limnohabitans strains.Bacterioplankton communities in freshwater systems frequently are characterized by the dominance of a relatively small number of clades as defined by 16S rRNA gene phylogenies (4, 50). These genus-like phylogenetic groups usually contribute large fractions to total prokaryotic numbers in typical stagnant freshwater systems (1, 34, 37, 39). While determining ecological differences between the organisms of these rather distantly related 16S rRNA clades is of enormous importance for understanding the mechanisms and factors shaping the diversity and composition of freshwater bacterioplankton (21), it is important to know if such clades actually represent ecologically coherent taxa, or if there is significant ecological variation within a single clade. Recent studies have uncovered ecological differences between closely related organisms within the same 16S rRNA clade (7, 13, 16, 18, 20, 26, 35). Several of these studies compared organisms affiliated with the same clade but originating from ecologically different habitats. To date only a few investigations have examined ecological differences between closely related and coexisting (syntopic) freshwater bacteria (7, 18, 35). For example, niche separation among Vibrionaceae strains coexisting in coastal bacterioplankton was demonstrated (16). Among a broad diversity of sympatric strains, Hunt and coworkers (16) identified phylogenetic subgroups differing in lifestyle (free living or association with different particle size classes) and seasonal preferences.Here, we investigate if two closely related strains coexisting in the same freshwater habitat differ significantly in their interactions with biological factors. We examined vulnerability to predation by a bacterivorous flagellate, sensitivity to virus assemblages recovered from their native habitat, and the interaction with a competitor of high growth potential. The two strains both were isolated from the same water sample taken from the meso-eutrophic Římov reservoir and belong to the recently proposed betaproteobacterial genus Limnohabitans (11). Within Limnohabitans, these two strains belong to a monophyletic group known as the R-BT065 cluster targeted by a homonymous fluorescent in situ hybridization (FISH) probe (41), which enabled intensive investigations of the ecology of this abundant bacterioplankton group in several different habitats (1, 15, 28, 29, 34, 37, 38). A recent survey on the distribution of this taxon revealed that it is a ubiquitous member of bacterioplankton communities in a broad variety of pH-neutral freshwater systems (102 systems inspected; see reference 39) that typically comprises ∼5 to 30% of total bacterial cell numbers. Previous investigations indicated a rather homogenous ecology of the R-BT065 group, characterized by the potential for a rapid response to changes of environmental conditions (34, 37, 38, 41) and a high sensitivity to flagellate predation (19, 42).We hypothesized that the two sympatric strains affiliated with the R-BT065 group would show ecological differences. These strains, designated B4 and D5, represent the most distantly (99.1% 16S rRNA gene sequence similarity) related pairing among the R-BT065 strains isolated so far (V. Kasalický, K. Šimek, J. Jezbera, and M. W. Hahn, unpublished data). To reveal ecological differences, the two strains were exposed to different manipulations in a laboratory experiment: (i) each of the R-BT065 strains were always separately cocultured with a fast-growing competitor of the genus Flectobacillus (strain MWH38; see reference 12), (ii) the cocultures were amended by additions of different amounts of a live or heat-killed virus concentrate collected from their home environment, and (iii) in parallel, these cocultures received an axenic bacterivore, Poterioochromonas sp., that was able to feed on all three bacterial strains involved in the study.     

Effects of sap-feeding insect herbivores on growth and reproduction of woody plants: a meta-analysis of experimental studies

The majority of generalisations concerning plant responses to herbivory are based on studies of natural or simulated defoliation. However, effects caused by insects feeding on plant sap are likely to differ from the effects of folivory. We assessed the general patterns and sources of variation in the effects of sap feeding on growth, photosynthesis, and reproduction of woody plants through a meta-analysis of 272 effect sizes calculated from 52 papers. Sap-feeders significantly reduced growth (−29%), reproduction (−17%), and photosynthesis (−27%); seedlings suffered more than saplings and mature trees. Deciduous and evergreen woody plants did not differ in their abilities to tolerate damage imposed by sap-feeders. Different plant parts, in particular below- and above-ground organs, responded similarly to damage, indicating that sap-feeders did not change the resource allocation in plants. The strongest effects were caused by mesophyll and phloem feeders, and the weakest by xylem feeders. Generalist sap-feeders reduced plant performance to a greater extent than did specialists. Methodology substantially influenced the outcomes of the primary studies; experiments conducted in greenhouses yielded stronger negative effects than field experiments; shorter (<12 months) experiments showed bigger growth reduction in response to sap feeding than longer experiments; natural levels of herbivory caused weaker effects than infestation of experimental plants by sap-feeders. Studies conducted at higher temperatures yielded stronger detrimental effects of sap-feeders on their hosts. We conclude that sap-feeders impose a more severe overall negative impact on plant performance than do defoliators, mostly due to the lower abilities of woody plants to compensate for sap-feeders’ damage in terms of both growth and photosynthesis.     

Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.     

Occurrence of bifidobacteria and lactobacilli in digestive tract of some freshwater fishes

Distal parts of the fish intestine were analyzed for presence of bifidobacteria and lactobacilli using selective agars. Seventy seven samples from Cyprinus carpio, Oncorhynchus mykiss, Carassius auratus, Tinca tinca, Perca fluviatilis, Rutilus rutilus, Scardinius erythrophthalmus, Oreochromis niloticus, and Squalius cephalus were collected randomly throughout years 2008 and 2009. Bifidobacteria were detected in 5 samples from 4 fish species at counts 2.18–4.29 log CFU/g, lactobacilli were present in 6 fish species at counts 1.21–3.65 log CFU/g. Seven bifidobacterial isolates were identified to the species level using biochemical tests and by sequencing of 16S rRNA gene. Three strains belonged to species B. longum, two isolates were identified as B. dentium, one strain as B. asteroides and one isolate was not determined to the species level by employed methods. As identified bifidobacterial species are considered to be of human, animal or honeybee origin, they probably derived as contamination from sewage or other sources. After further more detail testing, the possible use of isolated bifidobacteria as probiotics is promising since they were able to pass through the digestive tract successfully.     

The influence of ploidy level on ultrastructure and motility of tench <Emphasis Type="Italic">Tinca tinca</Emphasis> (L.) spermatozoa

The ultrastructure of diploid and triploid tench Tinca tinca (L.) spermatozoa were examined using electron microscopy focusing on parameters that influence movement. Triploid tench were produced artificially using a cold shock. Spermatozoa of triploid males in comparison with diploids featured significantly larger head (P < 0.01), higher amount of mitochondria (P < 0.05), and, surprisingly larger widths of the peripheral doublets and central pair of microtubules and the single microtubule (P < 0.01). However, the diameters of the flagellum were without significant differences as well as the length of the flagellum and length and width of the midpiece. Also motility parameters of spermatozoa did not significantly differ between diploid and triploid males, but the total velocity (summary of spermatozoa velocity and duration of movement) positively correlated with the flagellum length and negatively with the head diameter of tench spermatozoa with a high significant influence (P < 0.01).     

Alga-derived substrates select for distinct Betaproteobacterial lineages and contribute to niche separation in Limnohabitans strains

We examined the proportions of major Betaproteobacteria subgroups within bacterial communities in diverse nonaxenic, monospecific cultures of algae or cyanobacteria: four species of cryptophyta (genera Cryptomonas and Rhodomonas), four species of chlorophyta (genera Pediastrum, Staurastrum, and Chlamydomonas), and two species of cyanobacteria (genera Dolichospermum and Aphanizomenon). In the cryptophyta cultures, Betaproteobacteria represented 48 to 71% of total bacteria, the genus Limnohabitans represented 18 to 26%, and the Polynucleobacter B subcluster represented 5 to 16%. In the taxonomically diverse chlorophyta group, the genus Limnohabitans accounted for 7 to 45% of total bacteria. In contrast, cyanobacterial cultures contained significantly lower proportions of the Limnohabitans bacteria (1 to 3% of the total) than the cryptophyta and chlorophyta cultures. Notably, largely absent in all of the cultures was Polynucleobacter necessarius (Polynucleobacter C subcluster). Subsequently, we examined the growth of Limnohabitans strains in the presence of different algae or their extracellular products (EPP). Two strains, affiliated with Limnohabitans planktonicus and Limnohabitans parvus, were separately inoculated into axenic cultures of three algal species growing in an inorganic medium: Cryptomonas sp., Chlamydomonas noctigama, and Pediastrum boryanum. The Limnohabitans strains cocultured with these algae or inoculated into their EPP consistently showed (i) pronounced population growth compared to the control without the algae or EPP and (ii) stronger growth stimulation of L. planktonicus than of L. parvus. Overall, growth responses of the Limnohabitans strains cultured with algae were highly species specific, which suggests a pronounced niche separation between two closely related Limnohabitans species likely mediated by different abilities to utilize the substrates produced by different algal species.     

Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture

The in vitro study was carried out for detection of the cisplatin in free form and in culture medium, depending on various conditions of sonodynamic human ovarian cancer cells A2780 treatment by differential pulse polarography (DPP). For sonodynamic treatment, we used cisplatin alone and combined cisplatin/ultrasound treatments. The ultrasound exposure intensity of 1.0 and 2.0 W x cm(-2) in far field for incubation periods 1, 24 and 48 h was used. The parameters of DPP measurements were--1 s drop time, 5 mV x s(-1) voltage scan rate, 50 mV modulation amplitude and negative scanning direction; platinum wire served as counter electrode and Ag/AgCl/3 M KCl as reference electrode. The results showed the dependence of free platinum quantities in culture medium on incubation time and treatment protocol. We found difference in concentration of free cisplatin between conventional application of cisplatin and sonodynamic treatment. The sonodynamic combined treatment of cisplatin and ultrasound field showed a higher cisplatin content in the culture medium than cisplatin treatment alone; a difference of 20% was observed for incubation time 48 h. The results also showed the influence of a time sequence of ultrasound and cytostatics in the sonodynamic treatment. The highest amount of free cisplatin in the solution was found for primary application of cisplatin and the subsequent ultrasound exposure. The quantity of free cisplatin increased with time, namely for time intervals 1-24 h. There was no difference between the DPP signal of cisplatin in reaction mixture containing cells in small quantities and micro-filtered mixture without cells. Thus, the DPP method is suitable for the detection and quantification of free cisplatin in the culture medium of cell suspension. Ultrasound field can be important factor during cytostatic therapy.     

The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.     

Wild populations as a source of germplasm for black poplar (Populus nigra L.) breeding programmes

The aims were to explore the use of wild populations of Populus nigra for breeding and to compare crosses made between genotypes from different areas with different climates. Genetic distances ranged from 0.175 (populations growing under optimal conditions) up to 0.875 (populations growing under marginal conditions). A total of 39 trees were chosen from these populations, and in four different years 58 different parental crosses were made, from which a total of 2,277 individuals were produced for assessment as potential biomass producers. The progenies from each cross were rated according to a selection index (SI) based on three characteristics (height, leading shoot diameter and resistance to the rust Melampsora larici-populina). Sixteen of such crosses had significantly higher SI compared to the lowest SI values observed. In 19 of the crosses, it was shown that certain individual trees had made a significant contribution to lower susceptibility to rust, and in particular, two male trees used in the parental crosses were implicated as being the source of rust resistance. Selected individuals were cloned and used in a field trial with NE-42, as a control. It was found that six clones had significantly higher SI than NE-42. In crosses between trees originating from contrasting conditions, there was a higher average SI than in crosses between trees from the same conditions. However, these differences were not statistically significant. The successful clones are recommended for use in those areas where the native populations of pure black poplar are threatened by cross-pollination with allochthonous species.