Ca. Branchiomonas cysticola, Ca. Piscichlamydia salmonis and Salmon Gill Pox Virus transmit horizontally in Atlantic salmon held in fresh water.

Elucidation of the role of infectious agents putatively involved in gill disease is commonly hampered by the lack of culture systems for these organisms. In this study, a farmed population of Atlantic salmon pre-smolts, displaying proliferative gill disease with associated Candidatus Branchiomonas cysticola, Ca. Piscichlamydia salmonis and Atlantic salmon gill pox virus (SGPV) infections, was identified. A subpopulation of the diseased fish was used as a source of waterborne infection towards a population of naïve Atlantic salmon pre-smolts. Ca. B. cysticola infection became established in exposed naïve fish at high prevalence within the first month of exposure and the bacterial load increased over the study period. Ca. P. salmonis and SGPV infections were identified only at low prevalence in exposed fish during the trial. Although clinically healthy, at termination of the trial the exposed, naïve fish displayed histologically visible pathological changes typified by epithelial hyperplasia and subepithelial inflammation with associated bacterial inclusions, confirmed by fluorescent in situ hybridization to contain Ca. B. cysticola. The results strongly suggest that Ca. B. cysticola infections transmit directly from fish to fish and that the bacterium is directly associated with the pathological changes observed in the exposed, previously naïve fish.

Biofilm as an environment for dissemination of stx genes by transduction.

Dissemination of Shiga toxin (Stx)-encoding bacteriophages is the most likely mechanism for the spread of Stx-encoding genes and the emergence of new Stx-producing Escherichia coli (STEC). Biofilm has been reported to be a place where horizontal gene transfer by plasmid conjugation and DNA transformation may occur, and in this study, horizontal gene transfer by transduction has been demonstrated. Transfer of Stx-encoding bacteriophages to potentially pathogenic E. coli in biofilm was observed at both 20°C and 37°C. The infection rates were higher at 37°C than at 20°C. To our knowledge, this study is the first to show lateral gene transfer in biofilm mediated by a temperate bacteriophage. The study shows that the biofilm environment can be suitable for transduction events and can thereby be an environment for the emergence of new pathogenic E. coli.

Biogeographical patterns and determinants of invasion by forest pathogens in Europe.

A large database of invasive forest pathogens (IFPs) was developed to investigate the patterns and determinants of invasion in Europe. Detailed taxonomic and biological information on the invasive species was combined with country-specific data on land use, climate, and the time since invasion to identify the determinants of invasiveness, and to differentiate the class of environments which share territorial and climate features associated with a susceptibility to invasion. IFPs increased exponentially in the last four decades. Until 1919, IFPs already present moved across Europe. Then, new IFPs were introduced mainly from North America, and recently from Asia. Hybrid pathogens also appeared. Countries with a wider range of environments, higher human impact or international trade hosted more IFPs. Rainfall influenced the diffusion rates. Environmental conditions of the new and original ranges and systematic and ecological attributes affected invasiveness. Further spread of established IFPs is expected in countries that have experienced commercial isolation in the recent past. Densely populated countries with high environmental diversity may be the weakest links in attempts to prevent new arrivals. Tight coordination of actions against new arrivals is needed. Eradication seems impossible, and prevention seems the only reliable measure, although this will be difficult in the face of global mobility.

First Report of Mycosphaerella pini Causing Red Band Needle Blight on Scots Pine in Norway.

During a survey conducted in August 2009 in northern Norway, symptoms typical for red band needle blight (1) were observed in four young Scots pine (Pinus sylvestris) stands. The stands, less than 15 years old, were located in humid sites near rivers in Bardu and Målselv municipalities, Troms County. Many of the oldest needles (2- to 3-yearsold) in the lower part of young trees were partially or completely brown, but still attached, and red bands could be observed. Aggregations of conidial stromata were often seen in the red bands. Conidia were hyaline, smooth, thin walled, and filiform, 1.9 to 2.6 µm wide and 12 to 36 µm long. Isolations were made from conidiomata and the identity of Mycosphaerella pini was confirmed by partial sequencing of the internal transcribed spacer (ITS) region of a sample from Målselv (69°00'N, 18°51'E) (GenBank Accession No. JF796109). In June 2010, a survey was done in southeastern Norway where nearly 100 stands with young Scots pine, up to 20 years old, were inspected and typical symptoms of red band needle blight were observed in 10 stands in three municipalities in Hedmark County (Eidskog, Kongsvinger, and Trysil), less than 25 km from the Swedish border, and in one stand in Buskerud County (Nedre Eiker). Lower branches of up to 5 m tall trees had symptoms of red band needle blight, mainly on the oldest needles. In the red bands, acervuli of Dothistroma septosporum, the anamorph of M. pini, appeared. Typical conidia from acervuli were germinated in water agar and resulting mycelium was transferred to modified orange serum agar. Two isolates were sequenced as above and both herbarium samples and isolates (Kongsvinger, 60°06'N, 12°04'E; GenBank Accession No. JF796108 and Trysil, 61°14'N, 12°22'E; GenBank Accession No. JF796107) were deposited at the Norwegian Forest and Landscape Institute. To fulfill Koch's postulates, symptoms were reproduced by artificial inoculation onto 1-year-old Scots pine seedlings. In June 2010, a pooled conidial suspension (2.4 to 3.8 × 103 conidia per ml) from two single-conidium cultures was sprayed to runoff onto 128 seedlings and 64 seedlings were mock sprayed with distilled water. First symptoms (brown segments and red bands) appeared on inoculated seedlings 1 month later and acervuli appeared after another 6 to 8 weeks. M. pini was reisolated from the acervuli. Three months after inoculation, 90% of inoculated seedlings showed symptoms while all uninoculated seedlings were healthy. Damage caused by M. pini has increased in the northern hemisphere during the last 15 to 20 years, possibly because of climate change (4). In 2006, the fungus was recorded in Estonia (2), in 2008 in Finland (3), and by 2009, the disease had spread over large areas in Finland including the region near the Norwegian border in the north. The disease has been in Sweden for a few years (J. Stenlid, personnel communication). In southern Norway, the disease has mainly been observed near the Swedish border so here the disease may originate from Sweden. So far, however, only small areas of Norway have been surveyed. Both natural spread and human transport dissemination may be occurring. References: (1) Anonymous. OEPP/EPPO Bull. 35, 303, 2005. (2) M. Hanso and R. Drenkhan. Plant Pathol. 57:170, 2008. (3) M. Müller et al. Plant Dis. 93:322, 2009. (4) A. Woods et al. BioScience 55:761, 2005.

Pathogenic potential and horizontal gene transfer in ovine gastrointestinal Escherichia coli.

AIMS: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer. METHODS AND RESULTS: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only. CONCLUSIONS: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA. SIGNIFICANCE AND IMPACT OF THE STUDY: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli.

Chalara fraxinea Isolated from Diseased Ash in Norway.

European ash (Fraxinus excelsior), also known as common ash, occurs naturally inland in lower areas of southeastern Norway and along the southern coast of the country. It is important both as a forest and ornamental tree. During the last decade, dieback has become a disastrous disease on F. excelsior in many European countries. The anamorphic fungus Chalara fraxinea T. Kowalski (1), described for the first time from dying ash trees in Poland, is now considered the cause of ash dieback (2). In May of 2008, C. fraxinea was isolated from 1.5 m high diseased F. excelsior in a nursery in Østfold County in southeastern Norway. Symptoms included wilting, necrotic lesions around leaf scars and side branches, and discoloration of the wood. From symptomatic branches, small pieces (approximately 1 cm3) were excised in the transition area between healthy and discolored wood. After surface sterilization (10 s in 70% ethanol + 90 s in NaOCl), the pieces were air dried for 1 min in a safety cabinet, cut into smaller pieces, and placed on media. The fungus was isolated on potato dextrose agar (PDA) and water agar (WA). On PDA, the cultures were tomentose, light orange, and grew slowly (21 mm mean colony diameter after 2 weeks at room temperature). Typical morphological features of C. fraxinea developed in culture. Brownish phialides (14.8 to 30.0 [19.5] × 2.5 to 5.0 [4.1] µm, n = 50) first appeared in the center of the colonies on the agar plugs that had been transferred. The agar plugs were 21 days old when phialides were observed. Abundant sporulation occurred 3 days later. Conidia (phialospores) extruded apically from the phialides and formed droplets. Conidia measured 2.1 to 4.0 (3.0) × 1.4 to 1.9 (1.7) µm (n = 50). The first-formed conidia from each phialide were different in size and shape from the rest by being longer (6 µm, n = 10) and more narrow in the end that first appeared at the opening of the phialide. Internal transcribed spacer sequencing confirmed that the morphological identification was correct (Accession No. EU848544 in GenBank). A pathogenicity test was carried out in June of 2008 by carefully removing one leaf per plant on 10 to 25 cm high F. excelsior trees (18 trees) and placing agar plugs from a 31-day-old C. fraxinea culture (isolate number 10636) on the leaf scars and covering with Parafilm. After 46 days, isolations were carried out as described above from discolored wood that had developed underneath necrotic lesions in the bark and subsequently caused wilting of leaves. All the inoculated plants showed symptoms, and C. fraxinea was successfully reisolated. No symptoms were seen on uninoculated control plants (eight trees) that had received the same treatment except that sterile PDA agar plugs had been used. References: (1) T. Kowalski. For. Pathol. 36:264, 2006. (2) T. Kowalski and O. Holdenrieder, For. Pathol. Online publication, doi: 10.1111/j.1439-0329.2008.00565.x, 2008.

Is lack of susceptible recipients in the intestinal environment the limiting factor for transduction of Shiga toxin-encoding phages?

AIM: To determine whether a Shiga toxin 2 (Stx2)-encoding phage from Escherichia coli O157:H7 could be transmitted to commensal E. coli in a ruminant host without adding a specific recipient strain. METHODS AND RESULTS: Sheep were inoculated with an E. coli O157:H7 strain containing an Stx2-encoding bacteriophage (Phi3538) in which a chloramphenicol-resistant gene, cat, is inserted into stx(2). A total of 149 faecal samples were sampled and analysed for detection and quantification of E. coli O157:H7 and presumptive transductants. Phage Phi3538 (Deltastx(2)::cat) was demonstrated to be transduced to an ovine E. coli O175:H16 at one occasion. CONCLUSIONS: The study demonstrates an in vivo transduction in sheep from an E. coli O157:H7 strain to an ovine E. coli O175:H16. A functional Stx2-encoding phage was incorporated into the host's DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first in vivo stx phage transduction study reported in which a recipient strain was not fed to the test animals. We suggest that the access to susceptible hosts is one main limiting factor for transduction to occur in the intestine.

Induced resistance to pathogenic fungi in norway spruce

Norway spruce (Picea abies) trees (approximately 16 m high) of a single clone were used to study the effects of fungal infection and wounding on induction of resistance to the bark beetle-associated bluestain fungus Ceratocystis polonica. A dose-response experiment was designed involving three different dosages of fungal (fungus and wound) and sterile agar (wound) pretreatment inoculations (10, 50, or 100 inoculations/m(2) on the stem between 0.8 and 2.0 m high). Three weeks after pretreatment, trees were challenged with a massive C. polonica inoculation (400 inoculations/m(2)). Control trees that received no pretreatment were heavily colonized and killed by the challenge inoculation. The high and medium fungal pretreatments reduced subsequent fungal colonization success by 76% to 97% relative to the control, and fungal pretreatments protected the trees much more efficiently than sterile agar pretreatments. The protection was demonstrated to be local and not systemic in a subsequent experiment, where trees were pretreated with the medium fungal dosage on the lower bole and challenge inoculated further up the stem. Protection was also demonstrated to be pathogen nonspecific, as trees that had been pretreated with a medium dosage of the root rot fungus Heterobasidion annosum showed enhanced resistance to challenge inoculation with C. polonica.

Transcriptional responses of Norway spruce (Picea abies) inner sapwood against Heterobasidion parviporum.

The white-rot fungus Heterobasidion parviporum Niemelä & Korhonen establishes a necrotrophic interaction with Norway spruce (Picea abies (L.) H.Karst.) causing root and butt rot and growth losses in living trees. The interaction occurs first with the bark and the outer sapwood, as the pathogen enters the tree via wounds or root-to-root contacts. Later, when the fungus reaches the heartwood, it spreads therein creating a decay column, and the interaction mainly occurs in the inner sapwood where the tree creates a reaction zone. While bark and outer sapwood interactions are well studied, little is known about the nature of the transcriptional responses leading to the creation of a reaction zone. In this study, we sampled bark and sapwood both proximal and distal to the reaction zone in artificially inoculated and naturally infected trees. We quantified gene expression levels of candidate genes in secondary metabolite, hormone biosynthesis and signalling pathways using quantitative polymerase chain reaction. An up-regulation of mainly the phenylpropanoid pathway and jasmonic acid biosynthesis was found at the inoculation site, when inoculations were compared with wounding. We found that transcriptional responses in inner sapwood were similar to those reported upon infection through the bark. Our data suggest that the defence mechanism is induced due to direct fungal contact irrespective of the tissue type. Understanding the nature of these interactions is important when considering tree breeding-based resistance strategies to reduce the spread of the pathogen between and within trees.

Pathogenicity of four blue-stain fungi associated with aggressive and nonaggressive bark beetles.

ABSTRACT The pathogenicity of two isolates of each of four bark beetle-associated blue-stain fungi was evaluated after mass inoculation of about 40-year-old Norway spruce trees (Picea abies). Trees were inoculated with a different isolate of each fungus in 1995 and 1996 at a density of 400 inoculations per m(2) in a 1.2-m-wide band on the lower bole (about 270 inoculations per tree). Trees were felled 15 weeks after inoculation. In 1995, Ceratocystis polonica was the only fungus that had stained the sapwood (56.3% of cross-sectional sapwood area). It induced five times longer phloem necroses, 21 times more dead cambium, and 11 times more dead phloem than any other fungus. In 1996, C. polonica induced less extensive host symptoms and an unidentified Ambrosiella sp. induced comparable symptoms to C. polonica in the phloem and cambium. No trees showed any foliar symptoms 15 weeks after inoculation, but six out of eight trees inoculated with C. polonica in 1995 had only 0 to 25% functional sapwood and probably would have died if felling had been delayed. This study confirms that C. polonica, an associate of the aggressive bark beetle Ips typographus, is pathogenic to Norway spruce. The pathogenicity of the Ambrosiella sp., which is associated with a nonaggressive bark beetle, seems moderate and varies between isolates. The two remaining fungi included in this study (Ophiostoma piceae and a dark fungus with sterile mycelium), which are associated with nonaggressive bark beetles, were nonpathogenic in both experiments. These results are consistent with the hypothesis that aggressive bark beetle species vector virulent fungi that may help them kill trees, but the results also show that some nonaggressive bark beetles may vector phytopathogenic fungi.

Ophiostoma denticiliatum sp. nov. and other Ophiostoma species associated with the birch bark beetle in southern Norway.

Ophiostomatoid fungi were isolated from Scolytus ratzeburgi infesting Betula pendula and B. pubescens in Norway. Fungi were identified based on morphology, DNA sequence comparison for two gene regions and phylogenetic analyses. The most abundant fungus was Ophiostoma karelicum, suggesting a specific relationship between the fungus, the vector insect and the host tree. Our results suggest that O. karelicum occurs across the geographic range of S. ratzeburgi and its close relatedness to the Dutch elm disease fungi suggests that it could be important if introduced into other parts of the world. Other fungi, only occasionally isolated from S. ratzeburgi, were identified as O. quercus and a novel taxon, described here as O. denticiliatum sp. nov.

Oral retention and discoloration tendency from a chlorhexidine mouth rinse.

Individual differences in tooth discoloration have been observed after using chlorhexidine mouth rinses. To study the correlation between retention of chlorhexidine and the discoloration tendency, two groups, 'stainers' and 'non-stainers', were selected. The following parameters were tested: initial retention of chlorhexidine in vivo, retention of chlorhexidine to saliva-coated hydroxyapatite in vitro, and prolonged release of chlorhexidine in vivo. The initial oral retention of chlorhexidine was identical for the two groups, whereas in vitro retention of hydroxyapatite and prolonged release of chlorhexidine in vivo were higher among 'stainers'.

Chemical plaque control and prevention of extrinsic tooth discoloration in vivo.

Extrinsic discoloration of teeth has frequently been observed in connection with the use of chemical plaque-preventive agents. Preliminary investigations have indicated that oxidizing peroxymonosulfate may effectively reduce this discoloration without interfering with the plaque-preventive capacity of chlorhexidine. In the present experiment a 0.2% chlorhexidine and a 1% peroxymonosulfate solution were used for oral rinsing in a group of 50 military recruits. A double-blind experimental design was followed. The plaque-preventive capacity of chlorhexidine was maintained, and a marked reduction in extrinsic tooth discoloration could be observed.

Chemical plaque control and extrinsic discoloration of teeth.

Extrinsic discoloration of teeth is an unwanted side-effect in connection with the use of the chemical plaque-preventive agent chlorhexidine. Individual differences in discoloration tendency have, however, been observed. In order to study the nature of these extrinsic discolorations a selection of 3 typical "stainers" and 3 "non-stainers" was made based on the individual tendency to develop extrinsic discolorations of teeth after a ten days rinsing with 0.2% chlorhexidine twice a day. The individual discoloration tendency was maintained through various plaque-preventive regimens. Interference with the retention of chlorhexidine by a cation demonstrated a close correlation between antiplaque activity and the discoloration tendency. A commercial oxidizing agent based on peroxy monosulfate showed the most promising properties in reducing this extrinsic discoloration without interfering with plaque-prevention.

Wound-induced traumatic resin duct development in stems of Norway spruce (Pinaceae): anatomy and cytochemical traits.

Wounding of Norway spruce by inoculation with sterile agar, or agar containing the pathogenic fungus Ceratocystis polonica, induced traumatic resin duct formation in the stem. Visible anatomical responses occurred in the cambium 6-9 d post-inoculation. Near the inoculation site cellular proliferation, polyphenolic accumulation, and lignification were induced as a wound reaction to seal the damaged area. Five centimetres from the inoculation site cells in the cambial zone swelled and divided to form clusters. By 18 d post-inoculation, these cells began to differentiate into resin duct epithelial cells surrounding incipient schizogenous lumens. Mature axial traumatic ducts appeared by 36 d as a row of ducts in the xylem centripetal to the cambium. The ducts formed an interconnected network continuous with radial resin ducts. Parenchyma cells surrounding the ducts accumulated polyphenols that disappeared as the cells differentiated into tracheids. These polyphenols appeared to contain fewer sugar residues compared to those accumulating in the secondary phloem, as indicated by the periodic acid-Schiff's staining. The epithelial cells did not accumulate polyphenols but contained immunologically detectable phenylalanine ammonia lyase (EC 4.3.1.5), indicating synthesis of phenolics as a possible resin component. These findings may represent a defense mechanism in Norway spruce against the pathogenic fungus Ceratocystis polonica.

Iron staining of the acquired enamel pellicle after exposure to tannic acid or chlorhexidine: preliminary report.

Extrinsic discoloration of teeth following a large consumption of tannin-containing beverages or a prolonged use of chlorhexidine mouthrinses is a well known observation. Tannins as well as chlorhexidine are denaturing agents. Based on preliminary studies revealing the presence of iron in chlorhexidine discolored pellicle material, the ability of iron to stain the integument after pretreatment with the two denaturants was studied in a human model. The denaturing effect of an acidic environment was also included. Enamel slabs fixed to acrylic appliances were carried in the oral cavity and alternately exposed to the test solutions in different sequences in vitro. Pretreatment with chlorhexidine or tannic acid led to marked discoloration upon iron application during 5-d tests, whereas the compounds individually had no such effect. A large content of the metal was found in the stained material. Stannous fluoride appeared to reduce the formation of the pigments, and strong oxidation completely bleached the established color. Possible mechanisms underlying the phenomena observed are discussed.

Mechanical injury and fungal infection induce acquired resistance in Norway spruce.

Norway spruce trees (Picea abies (L.) Karst.) pretreated by wounding and fungal infection showed highly enhanced resistance to a subsequent challenge inoculation with the pathogenic bluestain fungus Ceratocystis polonica (Siem.) C. Moreau. This is the first time the effectiveness of the constitutive and inducible defenses has been shown to depend on prior wounding and infection in conifers, although such acquired resistance has previously been found in several angiosperms. Trees that were pretreated with a combination of 12 bark wounds (1.6 x 10 cm), four fungal inoculations and four sterile inoculations 1-15 days before mass inoculation with C. polonica at 400 inoculations per square meter over a 0.8 m stem section had significantly shorter necroses in the phloem, less bluestained sapwood, and less dead cambium than untreated control trees. Pretreatment with four fungal or sterile inoculations alone did not lead to enhanced resistance. Pretreatment by bark wounding alone seemed to provide an intermediate degree of resistance compared to bark wounding, fungal inoculations and sterile inoculations combined. All trees had a marked increase in the number of resin ducts in the year of inoculation compared with previous years, suggesting that formation of traumatic resin ducts play an important role in the development and maintainance of enhanced resistance.