Preliminary Volumetric Calculation of the Mucosal Surface in the Nares of Lambs Using a Segmentation of Computed Tomographic Images.

Intranasal vaccinations are becoming more important in both human and animal medicine to generate a localized IgA immune response not seen with parenteral vaccinations. This localized IgA response is more effective at reducing pathogen load on the mucosal surface of a potential host. One prerequisite for a successful nasal vaccination is the need to understand the distribution pattern of the nebulized vaccine, which requires an understanding the volume of the nares as well as the mucosal surface area. The exact mucosal surface area of ruminant nares has not yet been investigated. The aim of this concept study is to provide a detailed breakdown of a new method of volumetric rendering that can be used to calculate the volume and mucosal surface area of ruminant nares from computed tomographic images. The program Seg 3D was used to perform semi-automatic segmentation of a CT scan of a 9-month-old lamb head. Threshold segmentation and manual segmentation were used in combination to select the lamb's nasal cavity. The segmentation process yielded a volumetric rendering that was used to calculate the surface area and volume of the lamb's nasal cavity, with the segmentation process was repeated for each individual side of the lamb's nares. The surface area of the mucosal surface of each nostril is approximately 448 cm2, and the volume is approximately 45 cm3. The methodology described in this study successfully calculated the volume and surface area of a lamb's nares using volumetric rendering.

Effects of mesenchymal stromal cells versus serum on tendon healing in a controlled experimental trial in an equine model.

BACKGROUND: Mesenchymal stromal cells (MSC) have shown promising results in the treatment of tendinopathy in equine medicine, making this therapeutic approach seem favorable for translation to human medicine. Having demonstrated that MSC engraft within the tendon lesions after local injection in an equine model, we hypothesized that they would improve tendon healing superior to serum injection alone. METHODS: Quadrilateral tendon lesions were induced in six horses by mechanical tissue disruption combined with collagenase application 3 weeks before treatment. Adipose-derived MSC suspended in serum or serum alone were then injected intralesionally. Clinical examinations, ultrasound and magnetic resonance imaging were performed over 24 weeks. Tendon biopsies for histological assessment were taken from the hindlimbs 3 weeks after treatment. Horses were sacrificed after 24 weeks and forelimb tendons were subjected to macroscopic and histological examination as well as analysis of musculoskeletal marker expression. RESULTS: Tendons injected with MSC showed a transient increase in inflammation and lesion size, as indicated by clinical and imaging parameters between week 3 and 6 (p < 0.05). Thereafter, symptoms decreased in both groups and, except that in MSC-treated tendons, mean lesion signal intensity as seen in T2w magnetic resonance imaging and cellularity as seen in the histology (p < 0.05) were lower, no major differences could be found at week 24. CONCLUSIONS: These data suggest that MSC have influenced the inflammatory reaction in a way not described in tendinopathy studies before. However, at the endpoint of the current study, 24 weeks after treatment, no distinct improvement was observed in MSC-treated tendons compared to the serum-injected controls. Future studies are necessary to elucidate whether and under which conditions MSC are beneficial for tendon healing before translation into human medicine.

Sexual isolation promotes divergence between parapatric lake and stream stickleback.

Speciation can be initiated by adaptive divergence between populations in ecologically different habitats, but how sexually based reproductive barriers contribute to this process is less well understood. We here test for sexual isolation between ecotypes of threespine stickleback fish residing in adjacent lake and stream habitats in the Lake Constance basin, Central Europe. Mating trials exposing females to pairings of territorial lake and stream males in outdoor mesocosms allowing for natural reproductive behaviour reveal that mating occurs preferentially between partners of the same ecotype. Compared to random mating, this sexual barrier reduces gene flow between the ecotypes by some 36%. This relatively modest strength of sexual isolation is surprising because comparing the males between the two ecotypes shows striking differentiation in traits generally considered relevant to reproductive behaviour (body size, breeding coloration, nest size). Analysing size differences among the individuals in the mating trials further indicates that assortative mating is not related to ecotype differences in body size. Overall, we demonstrate that sexually based reproductive isolation promotes divergence in lake-stream stickleback along with other known reproductive barriers, but we also caution against inferring strong sexual isolation from the observation of strong population divergence in sexually relevant traits.

Fitness differences between parapatric lake and stream stickleback revealed by a field transplant.

Molecular comparisons of populations diverging into ecologically different environments often reveal strong differentiation in localized genomic regions, with the remainder of the genome being weakly differentiated. This pattern of heterogeneous genomic divergence, however, is rarely connected to direct measurements of fitness differences among populations. We here do so by performing a field enclosure experiment in threespine stickleback fish residing in a lake and in three replicate adjoining streams, and displaying weak yet heterogeneous genomic divergence between these habitats. Tracking survival over 29 weeks, we consistently find that lake genotypes transplanted into the streams suffer greatly reduced viability relative to local stream genotypes and that the performance of F1 hybrid genotypes is intermediate. This observed selection against migrants and hybrids combines to a total reduction in gene flow from the lake into streams of around 80%. Overall, our study identifies a strong reproductive barrier between parapatric stickleback populations, and cautions against inferring weak fitness differences between populations exhibiting weak overall genomic differentiation.

How mechanisms of habitat preference evolve and promote divergence with gene flow.

Habitat preference may promote adaptive divergence and speciation, yet the conditions under which this is likely are insufficiently explored. We use individual-based simulations to study the evolution and consequence of habitat preference during divergence with gene flow, considering four different underlying genetically based behavioural mechanisms: natal habitat imprinting, phenotype-dependent, competition-dependent and direct genetic habitat preference. We find that the evolution of habitat preference generally requires initially high dispersal, is facilitated by asymmetry in population sizes between habitats, and is hindered by an increasing number of underlying genetic loci. Moreover, the probability of habitat preference to emerge and promote divergence differs greatly among the underlying mechanisms. Natal habitat imprinting evolves most easily and can allow full divergence in parameter ranges where no divergence is possible in the absence of habitat preference. The reason is that imprinting represents a one-allele mechanism of assortative mating linking dispersal behaviour very effectively to local selection. At the other extreme, direct genetic habitat preference, a two-allele mechanism, evolves under restricted conditions only, and even then facilitates divergence weakly. Overall, our results indicate that habitat preference can be a strong reproductive barrier promoting divergence with gene flow, but that this is highly contingent on the underlying preference mechanism.

Evidence for Systemic Infection by Puccinia horiana, Causal Agent of Chrysanthemum White Rust, in Chrysanthemum.

Puccinia horiana, causal agent of the disease commonly known as chrysanthemum white rust (CWR), is a quarantine-significant fungal pathogen of chrysanthemum in the United States and indigenous to Asia. The pathogen was believed to have been eradicated in the United States but recently reappeared on several occasions in northeastern United States. The objective of the study presented here was to determine whether P. horiana could systemically infect chrysanthemum plants, thus providing a means of survival through winters. Scanning and transmission electron microscopy revealed the development of P. horiana on the surface and within leaves, stems, or crowns of inoculated chrysanthemum plants artificially exposed to northeastern U.S. winter temperatures. P. horiana penetrated leaves directly through the cuticle and then colonized the mesophyll tissue both inter- and intracellularly. An electron-dense material formed at the interface between fungal and host mesophyll cells, suggesting that the pathogen adhered to the plant cells. P. horiana appeared to penetrate mesophyll cell walls by enzymatic digestion, as indicated by the absence of deformation lines in host cell walls at penetration sites. The fungus was common in vascular tissue within the infected crown, often nearly replacing the entire contents of tracheid cell walls. P. horiana frequently passed from one tracheid cell to an adjacent tracheid cell by penetration either through pit pairs or nonpitted areas of the cell walls. Individual, presumed, fungal cells in mature tracheid cells of the crown and stems arising from infected crowns suggested that the pathogen might have been moving at least partially by means of the transpiration stream. The demonstration that chrysanthemum plants can be systemically infected by P. horiana suggests that additional disease control measures are required to effectively control CWR.

Low condylectomy as the sole treatment for active condylar hyperplasia: facial, occlusal and skeletal changes. An observational study.

The purpose of this study was to measure the changes in facial, occlusal, and skeletal relationships in patients with active unilateral condylar hyperplasia whose sole treatment was a low condylectomy. A retrospective observational descriptive study was conducted. All patients had undergone a low condylectomy as the sole or initial surgical treatment. The size of the condylar segment removed was decided by matching the affected side with the healthy side, leaving them both like the healthy one. The length of the ramus was measured using panoramic X-ray (distance from the highest part of the condyle to the mandibular angle). Facial, occlusal, and skeletal changes were evaluated using clinical, photographic, and radiological records before and after surgery. Condylectomy as the sole treatment for patients with active condylar hyperplasia allowed improvements to the alterations produced by this pathology, such as chin deviation, tilted lip commissure plane, tilted occlusal plane, angle of facial convexity, unevenness of the mandibular angles, and length of the mandibular ramus. The occlusal relationship also improved with orthodontic and elastic therapy. To conclude, low condylectomy as a sole and aetiological treatment for patients with active condylar hyperplasia allowed improvements to alterations produced by this pathology.

Quantitative evaluation of bone scintigraphy of the spinous processes of the equine thoracic spine at different times after administering 99mTc-hydroxymethylene-diphosphonate.

Scintigraphic examination of the thoracic spine is well documented. However, there is limited information about the effects of time on image quality in the period following injection of radionuclide. This study aimed to determine the optimal time point after injection of (99m)Tc-HDP (hydroxymethylene-diphosphonate) to achieve scintigraphic images with the best possible contrast and adequate count rates. Scintigraphic images of the thoracic spine of 21 horses were acquired two, four and six hours after administering (99m)Tc-HDP. Eight regions of interest were drawn in the images, four in the spinous processes and four in the adjacent soft tissue. The bone uptake, soft tissue uptake and the bone-to-soft tissue ratio were determined and compared between the different time points. Total count rates decreased with time after injection, but were at least as high as 150,000 counts per image at every time point after injection. The bone-to-soft tissue ratio was significantly higher for the images acquired after six hours compared to those acquired after two and four hours (P<0.01). Delayed scintigraphic examinations of the spinous processes of the equine thoracic spine achieved images with high contrast and sufficient count rates. Therefore, the scintigraphic examination of the equine thoracic spine is recommended to be done four to six hours after injecting (99m)Tc-HDP. However, additional studies should be performed to determine the effect of delayed image acquisition compared to images taken after three hours on the detectability of lesions in other parts of the thoracic spine and the soft tissue.

Effects of frequency of "extreme" temperature highs on development of soybean rust.

Previously, we hypothesized that summer "extreme" diurnal temperature highs in the southeastern United States were responsible for the yearly absence or delay of soybean rust development until fall. Utilizing temperature-controlled growth chambers, a diurnal temperature pattern of 33°C high and 20°C low reduced urediniospore production by 81%. However, that study did not consider the influence of frequency of extreme temperatures on soybean rust. We now report that a temperature high of 35°C for 1 h on three consecutive days, initiated 15 days after inoculation, when lesions had formed, reduced urediniospore production by 50% and required 9 to 12 days for sporulation to resume once the extreme temperature highs ceased. Furthermore, three consecutive days in which the temperature high was 37°C, beginning immediately after inoculation and subsequent dew period, reduced lesion numbers by 60%. The combined effects of reduced numbers of lesions and urediniospores per lesion caused by extreme temperature highs can account for observed absence or delay of soybean rust development in the southeastern United States until fall. A comparison of frequency of extreme temperature highs with numbers of counties reporting presence of soybean rust from 2005 to 2012 verified that extreme temperature highs may be largely responsible for absence or delay of soybean rust development. This is the first report showing the effect of frequency of extreme temperature highs on development of soybean rust. Because the south-to-north progression of soybean rust is required for the disease to occur in the major soybean-production regions of the United States, temperatures in the southeastern United States have a major effect on the entire U.S. soybean industry.

Effects of daily temperature highs on development of Phakopsora pachyrhizi on soybean.

Although considerable information exists regarding the importance of moisture in the development of soybean rust, little is known about the influence of temperature. The purpose of our study was to determine whether temperature might be a significant limiting factor in the development of soybean rust in the southeastern United States. Soybean plants infected with Phakopsora pachyrhizi were incubated in temperature-controlled growth chambers simulating day and night diurnal temperature patterns representative of the southeastern United States during the growing season. At 3-day intervals beginning 12 days after inoculation, urediniospores were collected from each plant and counted. The highest numbers of urediniospores were produced when day temperatures peaked at 21 or 25°C and night temperatures dipped to 8 or 12°C. When day temperatures peaked at 29, 33, or 37°C for a minimum of 1 h/day, urediniospore production was reduced to 36, 19, and 0%, respectively, compared with urediniospore production at the optimum diurnal temperature conditions. Essentially, no lesions developed when the daily temperature high was 37°C or above. Temperature data obtained from the National Climatic Data Center showed that temperature highs during July and August in several southeastern states were too high for significant urediniospore production on 55 to 77% of days. The inhibition of temperature highs on soybean rust development in southeastern states not only limits disease locally but also has implications pertaining to spread of soybean rust into and development of disease in the major soybean-producing regions of the Midwestern and northern states. We concluded from our results that temperature highs common to southeastern states are a factor in the delay or absence of soybean rust in much of the United States.

Influence of head and neck position on radiographic measurement of intervertebral distances between thoracic dorsal spinous processes in clinically sound horses.

REASONS FOR PERFORMING STUDY: Reductions in distances between dorsal spinous processes on radiographs are used as criteria for the diagnosis of impingement of the thoracic dorsal spinous processes in horses but are potentially altered by spine motion and different head and neck positions. OBJECTIVES: To determine the influence of head and neck positions on intervertebral distances between dorsal spinous processes on radiographs of thoracic spines of clinically sound horses. METHODS: Lateral-lateral radiographs were obtained from 23 horses in 3 head and neck positions. The width of the thoracic dorsal spinous processes and intervertebral distances between adjacent thoracic dorsal spinous processes were measured at points perpendicular to a tangent between the dorsal spinous processes and the caudal extremity of the thoracic vertebrae. RESULTS: A low head and neck position increased intervertebral distances between adjacent thoracic dorsal spinous processes from the 8th to 15th dorsal spinous processes whereas a high head and neck position had the opposite effect (P < 0.05). Overall, intervertebral distances between adjacent thoracic dorsal spinous processes decreased from cranial to caudal in intermediate head and neck positions (P < 0.01). The 12th thoracic dorsal spinous process was readily identifiable due to its significant difference to the narrower cranial and broader caudal dorsal spinous process (P < 0.05). CONCLUSIONS: The head and neck position influences the distances between the dorsal spinous processes of the vertebrae of equine thoracic spine on radiography. POTENTIAL RELEVANCE: The measuring system reported here offers potential to improve and standardise radiographic evaluation of thoracic dorsal spinous processes.

First Report of Anthracnose of Mile-a-Minute (Persicaria perfoliata) Caused by Colletotrichum cf. gloeosporioides in Turkey.

Mile-a-minute (Persicaria perfoliata (L.) H. Gross; family: Polygonaceae) is an exotic annual barbed vine that has invaded the northeastern USA and Oregon (2). In July of 2010, in a search for potential biological control pathogens (3), diseased P. perfoliata plants were found along the Firtina River near Ardesen, Turkey. Symptoms were irregular dark necrotic lesions along leaf margins and smaller irregular reddish lesions on the lamellae of leaves. Symptomatic leaves were sent to the quarantine facility of FDWSRU, USDA, ARS in Ft. Detrick, MD, for pathogen isolation and testing. Symptomatic leaves were excised, surface disinfested in 0.615% NaOCl, and then incubated for 2 to 3 days in sterile moist chambers at 20 to 25°C. Numerous waxy sub-epidermal acervuli with 84-µm-long (mean) black setae were observed in all of the lesions after 2 to 3 days of incubation. Conidiophores within acervuli were simple, short, and erect. Conidia were one-celled, hyaline, guttulate, subcylindrical, straight, 12.3 to 18.9 × 3.0 to 4.6 µm (mean 14.3 × 3.7 µm). Pure cultures were obtained by transferring conidia onto 20% V-8 juice agar. Appressoria, formed 24 h after placing conidia on dialysis membrane over V-8 juice agar, were smooth, clavate, aseptate, regular in outline, and 6.4 to 10.0 × 5.1 to 7.2 µm (mean 7.5 × 6.6 µm). These characters conformed to the description of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (1). A voucher specimen was deposited in the U.S. National Fungus Collections (BPI 882461). Nucleotide sequences for the internal transcribed spacers (ITS 1 and 2), directly sequenced from ITS 1 and ITS 4 standard primers (4), were deposited in GenBank (JN887693). A comparison of these sequences with ITS 1 and 2 sequences of the C. gloeosporioides epitype IMI 356878 (GenBank EU 371022) (1) using BLAST found 479 of 482 identities with no gaps. Conidia from 14-day-old cultures, in an aqueous suspension of 1.0 × 106 conidia ml-1, were spray-inoculated onto healthy stems and leaves of twenty 30-day-old P. perfoliata plants. Another 10 plants were not inoculated. All plants were placed in a dew chamber at 25°C for 16 h with no lighting. They were then placed in a 20 to 25°C greenhouse with a 14-h photoperiod. Light was generated using 400W sodium vapor lights. Lesions developed on leaves and stems of all inoculated plants after 7 days, and symptoms were the same as observed in the field. Each plant was rated weekly for disease severity on a 0 to 10 rating scale where 0 = no disease symptoms and 10 = 100% symptomatic tissue. After 28 days, the average disease rating of inoculated plants was 3.95 ± 0.94. No disease developed on noninoculated plants. C. gloeosporioides was reisolated from all inoculated plants. Host range tests will determine the potential of this isolate as a biological control agent for P. perfoliata. To our knowledge, this is the first report of anthracnose caused by C. gloeosporioides on P. perfoliata. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) J. T. Kartesz and C. A. Meacham. Synthesis of the North American Flora, Version 1.0., North Carolina Botanical Garden, Chapel Hill, N.C. 1999. (3) D. L. Price et al. Environ. Entomol. 32:229, 2003. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Quantitative genetic inheritance of morphological divergence in a lake-stream stickleback ecotype pair: implications for reproductive isolation.

Ecological selection against hybrids between populations occupying different habitats might be an important component of reproductive isolation during the initial stages of speciation. The strength and directionality of this barrier to gene flow depends on the genetic architecture underlying divergence in ecologically relevant phenotypes. We here present line cross analyses of inheritance for two key foraging-related morphological traits involved in adaptive divergence between stickleback ecotypes residing parapatrically in lake and stream habitats within the Misty Lake watershed (Vancouver Island, Canada). One main finding is the striking genetic dominance of the lake phenotype for body depth. Selection associated with this phenotype against first- and later-generation hybrids should therefore be asymmetric, hindering introgression from the lake to the stream population but not vice versa. Another main finding is that divergence in gill raker number is inherited additively and should therefore contribute symmetrically to reproductive isolation. Our study suggests that traits involved in adaptation might contribute to reproductive isolation qualitatively differently, depending on their mode of inheritance.

Characterizing Resistance to Phakopsora pachyrhizi in Soybean.

Resistance in soybean to Phakopsora pachyrhizi, the cause of soybean rust, is characterized by either reddish-brown (RB) lesions or an immune response. The RB type of resistance can be incomplete, as evidenced by the presence of sporulating uredinia within lesions. Susceptibility, on the other hand, is exemplified by tan-colored (TAN) lesions, and can be expressed in gradations of susceptibility or partial resistance that are less well defined. This study evaluated traits associated with incomplete or partial resistance to P. pachyrhizi in soybean by comparing 34 soybean accessions inoculated with four P. pachyrhizi isolates. Six accessions produced RB lesions to all four isolates, while 19 accessions produced TAN lesions, including plant introduction (PI) 200492 (Rpp1) and the susceptible check 'Williams'. Williams had among the largest area under the disease progress curve (AUDPC) values and area under the sporulating uredinia progress curve (AUSUPC) values, while eight accessions had lower AUSUPC values. Of the known sources of single-gene resistance, only PI 230970 (Rpp2), PI 459025B (Rpp4), and PI 594538A (Rpp1b) had lower AUDPC and AUSUPC values than Williams. PI 594538A and PI 561356 had RB lesions and had the lowest AUDPC and AUSUPC values. Of the known sources of single-gene resistance, only PI 230970 (Rpp2) and PI 594538A (Rpp1b) produced fewer and smaller-diameter uredinia than Williams. This study characterized reactions to P. pachyrhizi in 34 accessions based on lesion type and sporulation, and defined incomplete resistance and partial resistance in the soybean-P. pachyrhizi interaction.

Leaf Anthracnose, a New Disease of Swallow-Worts Caused by Colletotrichum lineola from Russia.

Black swallow-wort, Vincetoxicum nigrum (L.) Moench (= Cynanchum louiseae Kartesz & Gandhi), and pale swallow-wort, V. rossicum (Kleopow) Borhidi (= Cynanchum rossicum (Kleopow) Borhidi), are invasive plants belonging to the family Apocynaceae and are the targets of biological control efforts to control their spread in the United States. In 2010, a disease on a related species, V. scandens Sommier & Levier, was observed in the Krasnodar area of Russia. Disease symptoms were many small, dark red-to-purple leaf spots, approximately 2 to 5 mm in diameter, with white centers. Leaf spots were found on the upper leaf surface. Leaf tips and margins of leaves bearing many of these spots were necrotic. Symptomatic leaves were collected and sent to the BSL-3 containment facility at the Foreign Disease-Weed Science Research Unit (FDWSRU) of the USDA, ARS in Frederick MD. Surface-disinfested symptomatic leaves were incubated at 20 to 25°C in sterile moist chambers. After several days, acervuli and brown setae were observed inside the leaf spots. Pure cultures, designated FDWSRU 10-002, were obtained by transferring spore masses with sterile glass needles onto 20% V8 juice agar. Seeds of V. scandens, collected in Russia, were placed in a freezer at -20°C for 6 weeks and then germinated in sterile petri plates on moist filter paper. The seedlings were then transplanted and grown in a 20°C greenhouse under 12 h of light. Koch's postulates were fulfilled as follows: 2-month-old plants each of V. scandens, V. nigrum, and V. rossicum were inoculated with spores from 2-week-old cultures of isolate 10-002. Plants were inoculated by spraying an aqueous suspension of 106 spores per ml onto each plant until all leaves were wet. Plants were placed in 20 to 24°C dew chambers for 18 h and then placed in a 20°C greenhouse. Two weeks later, diseased leaves with the same symptoms observed in the field were harvested from each species, and the fungus was reisolated from seven of seven inoculated V. scandens plants, one of two V. nigrum plants, and four of four V. rossicum plants. Measurements of fungus fruiting structures were taken from cultures grown on synthetic nutrient-poor agar (SNA) (1). Conidiophores were brown, septate, and branched. Conidia were one-celled, hyaline, smooth walled, ovoid to oblong, falcate, and 20.1 to 26.2 × 1.7 to 3.6 µm (mean ± s.d. = 23.5 ± 1.3 × 2.6 ± 0.4 µm). Lengths of the conidia conformed to the description of Colletotrichum lineola Corda (1), but the conidia were slightly narrower than described. To induce appressoria formation, approximately 104 conidia were placed on sterile dialysis membranes on top of SNA in petri dishes that were wrapped in foil and incubated at 24°C for 24 h. After this time, appressoria were observed with a microscope at ×400 magnification. The appressoria were dark brown, smooth walled, ellipsoidal, and 5.5 to 25.5 × 3.6 to 12.1 µm (mean ± s.d. = 13.4 ± 4.0 × 7.3 ± 2.1 µm), which conformed to the description of appressoria of C. lineola Corda (1). DNA sequences of ITS1, 5.8S, and ITS2 were submitted to GenBank (No. HQ731491), and after BLAST analysis, aligned 100% to 15 previously identified isolates of C. lineola in GenBank. Voucher specimens of the fungus have been deposited in the U.S. National Fungus Collection and were designated as BPI 881105 and BPI 881106. Host range and efficacy tests are planned to determine the suitability of C. lineola for biological control of swallow-worts in the United States. Reference: (1) U. Damm et al. Fungal Divers. 39:45, 2009.

Testing for mating isolation between ecotypes: laboratory experiments with lake, stream and hybrid stickleback.

Mating isolation is a frequent contributor to ecological speciation - but how consistently does it evolve as a result of divergent selection? We tested for genetically based mating isolation between lake and stream threespine stickleback (Gasterosteus aculeatus L.) from the Misty watershed, Vancouver Island, British Columbia. We combined several design elements that are uncommon in the studies of stickleback mate choice: (i) we used second-generation laboratory-reared fish (to reduce environmental and maternal effects), (ii) we allowed for male-male competitive interactions (instead of the typical no-choice trials) and (iii) we included hybrids along with pure types. Males of different types (Lake, Inlet, hybrid) were paired in aquaria, allowed to build nests and then exposed sequentially to females of all three types. We found that Lake and Inlet males differed in behaviours thought to influence stickleback mate choice (inter- and intra-sexual aggression, display and nest activities), whereas hybrids were either intermediate or apparently 'inferior' in these behaviours. Despite these differences, Lake and Inlet fish did not mate assortatively and hybrid males did not have a mating disadvantage. Our study reinforces the noninevitability of mating isolation evolving in response to ecological differences and highlights the need to further investigate the factors promoting and constraining progress towards ecological speciation.

First Report of Leaf Spot on Horseweed (Conyza canadensis) Caused by Septoria erigerontis in Turkey.

Horseweed (Conyza canadensis (L).Cronq., Asteraceae) is an invasive exotic weed in Turkey and a problematic native weed in the United States where glyphosate-resistant populations of the weed have developed (2). These characteristics make horseweed a target for biological control efforts. In September 2009, small, brown leaf spots were observed on leaves of C. canadensis in Taflan, Turkey (41°25.398'N, 36°08.352'E). Globose, dark-walled pycnidia were also observed in brown spots on leaves. Diseased tissue was surface disinfested and placed on moist filter paper in petri plates. A fungus designated 09-Y-TR1 was isolated from the diseased leaves. Single-spore isolations were grown on potato dextrose agar (PDA). Cultures on PDA formed dark green-to-black colonies. Pycnidia matured after 3 to 4 weeks when plates were incubated at 23°C with a 12-h photoperiod (black light and cool white fluorescent light). Pycnidia were separate, immersed, and dark brown with a single apical ostiole. Matured conidia were one to three septate, filiform, straight to slightly curved, rounded at the apex, smooth walled, hyaline, and 22 to 40 × 1.4 to 2.5 µm. Morphology was consistent with Septoria erigerontis Peck (3). Comparison of the internal transcribed spacer (ITS) 1 and 2 sequence with available sequences of vouchered S. erigerontis specimens (GenBank EF535638.1, AY489273.1; KACC 42355, CBS 109094) showed 447 of 450 and 446 of 450 identities, respectively. Nucleotide sequences for the ribosomal ITS regions (ITS 1 and 2, including 5.8S rDNA) were deposited in GenBank (GU952666). For pathogenicity tests conidia were harvested from 3-week-old cultures grown on PDA, by brushing the surface of the colonies with a small paint brush, suspended in sterile distilled water, and filtered through cheese cloth. Conidia were then diluted in sterile distilled water plus 0.1% polysorbate 20 to a concentration of 5 × 106 conidia/ml. Stems and leaves of seven 5-month-old seedlings were spray inoculated with 10 ml of this aqueous suspension per plant. Inoculated plants and three noninoculated plants were placed in a dew chamber at 23°C in darkness and continuous dew, and after 48 h, plants were moved to a greenhouse bench. Symptoms were observed 2 days after inoculation. Disease severity was evaluated 2 weeks after inoculation by a rating system with a scale of 0 to 6 based on percentage of plant tissue necrosis, in which 0 = no symptoms, 1 = 1 to 5%, 2 = 6 to 25%, 3 = 26 to 75%, 4 = 76 to 95%, 5 = >95%, and 6 = dead plant. The average disease rating on inoculated plants was 3.55. No disease was observed on noninoculated plants. S. erigerontis was reisolated from all inoculated plants. To our knowledge, this is the first report of leaf spot on horseweed caused by S. erigerontis in Turkey where the fungus may have potential as a classical biological control agent. S. erigerontis has also been reported on C. canadensis in Korea and Portugal (1). In the United States, S. erigerontis has been reported on horseweed in several states (1) and these isolates may have potential as biological control agents of horseweed, particularly glyphosate-resistant horseweed, in the United States. References: (1) D. F. Farr et al. Fungal Databases. Systematic Mycology and Microbiology Laboratory, Online publication. ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , March 2010. (2) I. Heap. www.weedscience.org , 2006. (3) M. J. Priest. Fungi of Australia: Septoria. ABRS/CSIRO Publishing. Melbourne, 2006.

Correction of a bootstrap approach to testing for evolution along lines of least resistance.

Testing for an association between the leading vectors of multivariate trait (co)variation within populations (the 'line of least resistance') and among populations is an important tool for exploring variational bias in evolution. In a recent study of stickleback fish populations, a bootstrap-based test was introduced that takes into account estimation error in both vectors and hence improves the previously available bootstrap method. Because this test was implemented incorrectly, however, I here describe the correct test protocol and provide a reanalysis of the original data set. The application of this new test protocol should improve future investigations of evolution along lines of least resistance and other vector comparisons.

First Report of Leaf Spot Caused by Cercospora bizzozeriana on Lepidium draba in the United States.

The herbaceous perennial Lepidium draba L. is an invasive weed of rangelands and riparian areas in North America and Australia. As of 2002, it had infested 40,500 ha of rangeland in Oregon and large areas in Wyoming and Utah. Little is known of plant pathogens occurring on L. draba, especially in the United States, that could be useful for biological control of the weed. Leaf spots were first noted on a stand of L. draba near Shepherd, MT in 1997. The spots were mostly circular but sometimes irregularly shaped and whitish to pale yellow. The pathogen was erroneously assumed to be Cercospora beticola since its morphological traits closely resembled that species and the area had large fields of sugar beet with heavy Cercospora leaf spot incidence. Diseased leaves of L. draba were collected in 1997 and 2007. Conidia, borne singly on dark gray, unbranched conidiophores produced on dark stromata late in the season, were elongate, hyaline, multiseptate, 38 to 120 × 2 to 6 µm (mostly 38 to 50 × 2 to 5 µm) and had bluntly rounded tips and wider, truncate bases. These characteristics were consistent with the description of C. bizzozeriana Saccardo & Berlese (2). To isolate the fungus, spores were picked from fascicles of conidiophores with a fine-tipped glass rod, suspended in sterile water, and spread on plates of water agar. Germinated spores were transferred to potato dextrose agar (PDA). The ITS1, 5.8S, and ITS2 sequences of this fungus (GenBank Accession No. EU887131) were identical to sequences of an isolate of C. bizzozeriana from Tunisia (GenBank Accession No. DQ370428). However, these sequences were also identical to those of a number of Cercospora spp. in GenBank, including C. beticola. We also compared the actin gene sequences of the Montana isolate of C. bizzozeriana (GenBank Accession No. FJ205397) and an isolate of C. beticola from Montana (GenBank Accession No. AF443281); the sequences were 94.6% similar, an appreciable difference. For pathogenicity tests, cultures were grown on carrot leaf decoction agar. Aqueous suspensions of 104 spores per ml from cultures were sprayed on 6-week-old L. draba plants. Plants were covered with plastic bags and placed on the greenhouse bench at 20 to 25°C for 96 h. Koch's postulates were completed by reisolating the fungus from the circular leaf spots that appeared within 10 days, usually on lower leaves. Spores of C. bizzozeriana were also sprayed on seedlings of sugar beet, collard, mustard, radish, cabbage, and kale under conditions identical to those above. No symptoms occurred. After the discovery of the disease in 1997, plants of L. draba in eastern Montana, Wyoming, and Utah were surveyed from 1998 to 2003 for similar symptoms and signs, but none were found. This, to our knowledge, is the first report of C. bizzozeriana in the United States. The initial report of the fungus in North America was from Manitoba in 1938 (1). It has recently been reported as occurring on L. draba in Tunisia (4) and Russia (3) and is reported as common in Europe (2). A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI No. 878750A). References: (1) G. R. Bisby. The Fungi of Manitoba and Saskatchewan. Natl. Res. Council of Canada, Ottawa, 1938. (2) C. Chupp. A Monograph of the Fungus Genus Cercospora. C. Chupp, Ithaca, NY, 1953. (3) Z. Mukhina et al. Plant Dis. 92:316, 2008. (4) T. Souissi et al. Plant Dis. 89:206, 2005.

First Report of Stem Canker of Salsola tragus Caused by Diaporthe eres in Russia.

Salsola tragus L. (Russian thistle) is a problematic invasive weed in the western United States and a target of biological control efforts. In September of 2007, dying S. tragus plants were found along the Azov Sea at Chushka, Russia. Dying plants had irregular, necrotic, canker-like lesions near the base of the stems and most stems showed girdling and cracking. Stem lesions were dark brown and contained brown pycnidia within and extending along lesion-free sections of the stems and basal portions of leaves. Diseased stems were cut into 3- to 5-mm pieces and disinfested in 70% ethyl alcohol. After drying, stem pieces were placed into petri dishes on the surface of potato glucose agar. Numerous, dark, immersed erumpent pycnidia with a single ostiole were observed in all lesions after 2 to 3 days. Axenic cultures were sent to the Foreign Disease-Weed Science Research Unit, USDA, ARS, Ft. Detrick, MD for testing in quarantine. Conidiophores were simple, cylindrical, and 5 to 25 × 2 µm (mean 12 × 2 µm). Alpha conidia were biguttulate, one-celled, hyaline, nonseptate, ovoid, and 6.3 to 11.5 × 1.3 to 2.9 µm (mean 8.8 × 2.0 µm). Beta conidia were one-celled, filiform, hamate, hyaline, and 11.1 to 24.9 × 0.3 to 2.5 µm (mean 17.7 × 1.2 µm). The isolate was morphologically identified as a species of Phomopsis, the conidial state of Diaporthe (1). The teleomorph was not observed. A comparison with available sequences in GenBank using BLAST found 528 of 529 identities with the internal transcribed spacer (ITS) sequence of an authentic and vouchered Diaporthe eres Nitschke (GenBank DQ491514; BPI 748435; CBS 109767). Morphology is consistent with that of Phomopsis oblonga (Desm.) Traverso, the anamorph of D. eres (2). Healthy stems and leaves of 10 30-day-old plants of S. tragus were spray inoculated with an aqueous suspension of conidia (1.0 × 106 alpha conidia/ml plus 0.1% v/v polysorbate 20) harvested from 14-day-old cultures grown on 20% V8 juice agar. Another 10 control plants were sprayed with water and surfactant without conidia. Plants were placed in an environmental chamber at 100% humidity (rh) for 16 h with no lighting at 25°C. After approximately 24 h, plants were transferred to a greenhouse at 20 to 25°C, 30 to 50% rh, and natural light. Stem lesions developed on three inoculated plants after 14 days and another three plants after 21 days. After 70 days, all inoculated plants were diseased, four were dead, and three had more than 75% diseased tissue. No symptoms occurred on control plants. The Phomopsis state was recovered from all diseased plants. This isolate of D. eres is a potential biological control agent of S. tragus in the United States. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 878717). Nucleotide sequences for the ribosomal ITS regions (ITS 1 and 2) were deposited in GenBank (Accession No. EU805539). To our knowledge, this is the first report of stem canker on S. tragus caused by D. eres. References: (1) B. C. Sutton. Page 569 in: The Coelomycetes. CMI, Kew, Surrey, UK, 1980. (2) L. E. Wehmeyer. The Genus Diaporthe Nitschke and its Segregates. University of Michigan Press, Ann Arbor, 1933.