Identification, pathogenicity and comparative virulence of Fusarium spp. associated with insect-damaged, diseased Centaurea spp. in Europe

Fusarium spp. isolated from insect-infested, diseased Centaurea diffusa and Centaurea maculosa in Europe were assessed for pathogenicity to North American plants of their respective original hosts: either C. diffusa or C. maculosa. Of the ten isolates of Fusarium spp. isolated from diseased Centaurea spp. in the Caucasus region of Russia and eastern Europe, all caused one or more disease symptoms or reductions in fresh weight of North American accessions of their original host species. In three instances, these reductions were statistically significant (p = 0.05). Symptoms included overall stunting, root lesions, and crown rot. Reductions in fresh weight of C. diffusa ranged from 17–78%, and C. maculosa exhibited reductions of 18–82%. The pathogenic cultures were identified as F. solani, F. tricinctum and F. oxysporum. Six of seven other cultures were identified as F. oxysporum, and one as F. tricinctum. It was concluded that further screening of a larger set of isolates of foreign Fusarium spp. under quarantine conditions stateside or in limited USDA-ARS overseas facilities is justified and promising.     

Identification, Pathogenicity and Comparative Virulence of Fusarium spp. Associated with Diseased Euphorbia spp. in Europe

Fusarium spp. isolated from diseased Euphorbia spp. in Europe were assessed for pathogenicity to North American accessions of leafy spurge ( Euphorbia esula/virgata ). Of the nine strains of Fusarium spp. isolated from diseased E. stepposa or E. virgata in the Caucasus region of Russia and E. esula/virgata in southern France, all were pathogenic to leafy spurge. There were significant differences in virulence among strains. Four strains, including the two that were most virulent, were identified as F. oxysporum . Four of the five other strains were identified as F. solani and one was identified as F. proliferatum . Three of the four most virulent strains to leafy spurge were isolated from E. stepposa . The most virulent strain was associated with root damage caused by insect biological control agents, as found earlier with domestic strains of Fusarium spp. pathogenic to leafy spurge. Two strains identified as F. solani were vegetatively compatible. It was concluded that further screening of a larger set of strains of foreign Fusarium spp. under quarantine conditions in the US or in limited overseas facilities would be justified, and could yield promising biological control agents for leafy spurge.     

Halophyte ion regulation traits support saline adaptation of Lepidium latifolium,L. draba,and L. alyssoides

Plant Ecology - Identification of saline resistance traits of invasive plants has received little study and could reveal how some plant species utilize salt to encroach upon salinized landscapes....     

Genetic Diversity Within and Among Lepidium draba Populations from Eastern Anatolia Based on RAPD Analysis

Genetic variation and structure of six natural populations of Lepidium draba L. from Eastern Anatolia were assessed using random amplified polymorphic DNA (RAPD) markers. For RAPD analysis, 12 primers generated 218 reproducible bands across the six populations analyzed, of which 73 bands (33.3%) were polymorphic. The mean Nei’s gene diversity value for all six populations was 0.1771. Shannon’s information index varied with population (0.2278–0.3082), averaging 0.2608. Analysis of molecular variance (AMOVA) showed that genetic diversity was greater within populations (58.66%) than among populations (30.68%). In addition, the variation between groups was 10.33%. The genetic differentiation among populations (G _ST) was 0.3210, indicating that most genetic diversity occurs within populations. Gene flow (Nm) was low, at only 0.5288.     

PCR detection, characterization, and distribution of virulence genes in Aeromonas spp

We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU (AHCYTOEN; GenBank accession no. M84709) against aerolysin genes of Aeromonas spp., suggesting the possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers from those genes. Amplicons obtained from Aeromonas sp. reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-Aeromonas reference strains, none were positive. PCR-restriction fragment length polymorphism (PCR-RFLP) with HpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HG8, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13. PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of Aeromonas spp. It overcomes the handicap of time-consuming biochemical and other DNA-based methods.     

Comparative survey of the phytophagous arthropod faunas associated with Lepidium draba in Europe and the western United States,and the potential for biological weed control

Hoary cress (Lepidium draba L.) is an invasive perennial mustard (Brassicaceae) introduced to North America from Eurasia. In 2001 we initiated a program to investigate the potential for classical biological control of this weed in the US. Literature and field surveys for the arthropods associated with L. draba were conducted on both continents. Field surveys began in Europe in 2001, and in 2002–2003 standardized surveys were conducted in both Europe and the western US. These field surveys resulted in 80 new host records for L. draba in Europe, and 37 new host records in the US. Although total species richness was nearly four times greater in Europe, there were approximately equal proportions of oligophagous and polyphagous species in each range. Monophagous species were only encountered in the European surveys. The literature surveys revealed that the arthropod fauna associated with L. draba is fairly well known in Europe (175 species), but not in the US (eight species), where the literature was virtually derelict of host records. In both the literature and field surveys, the order Coleoptera contained the most species (>50%) in Europe, whereas the order Hemiptera contained the most species (>40%) in the US. Nearly one-half of the species found in literature and field surveys are flower or seed feeders and the other half are primarily foliage or stem feeders. Ten potential biological control agents were discovered from the surveys, of which four are currently being investigated at the CABI Bioscience Centre in Switzerland. The phenologies of these four species are briefly described.     

Pathogenicity islands and virulence evolution in Listeria.

As in other bacterial pathogens, the virulence determinants of Listeria species are clustered in genomic islands scattered along the chromosome. This review summarizes current knowledge about the structure, distribution and role in pathogenesis of Listeria virulence loci. Hypotheses about the mode of acquisition and evolution of these loci in this group of Gram-positive bacteria are presented and discussed.     

Distribution of antimicrobial resistance and virulence genes in Enterococcus spp. and characterization of isolates from broiler chickens

Enterococci are now frequent causative agents of nosocomial infections. In this study, we analyzed the frequency and distribution of antibiotic resistance and virulence genotypes of Enterococcus isolates from broiler chickens. Fecal and cecal samples from nine commercial poultry farms were collected to quantify total enterococci. Sixty-nine presumptive enterococci were isolated and identified by API 20 Strep, and their susceptibilities to antibiotics were determined. Genotypes were assessed through the use of a novel DNA microarray carrying 70 taxonomic, 17 virulence, and 174 antibiotic resistance gene probes. Total enterococcal counts were different from farm to farm and between sample sources (P < 0.01). Fifty-one (74%) of the isolates were identified as E. faecium, whereas nine (13%), seven (10%), and two (3%) isolates were identified as E. hirae, E. faecalis, and E. gallinarum, respectively. Multiple-antibiotic resistance was evident in E. faecium and E. faecalis isolates. The most common multiple-antibiotic resistance phenotype was Bac Ery Tyl Lin Str Gen Tet Cip. Genes conferring resistance to aminoglycoside (aac, aacA-aphD, aadB, aphA, sat4), macrolide (ermA, ermB, ermAM, msrC), tetracycline (tetL, tetM, tetO), streptogramin (satG_vatE8), bacitracin (bcrR), and lincosamide (linB) antibiotics were detected in corresponding phenotypes. A range of 9 to 12 different virulence genes was found in E. faecalis, including ace, agg, agrB(Efs) (agrB gene of E. faecalis), cad1, the cAM373 and cCF10 genes, cob, cpd1, cylAB, efaA(Efs), and gelE. All seven E. faecalis isolates were found to carry the gelE gene and to hydrolize gelatin and bile salts. Results from this study showed the presence of enterococci of public and environmental health concerns in broiler chicken farms and demonstrated the utility of a microarray to quickly and reliably analyze resistance and virulence genotypes of Enterococcus spp.     

Microtubules in statocytes from roots of cress (Lepidium sativum L.)

Summary Statocytes in root caps ofLepidium sativum L. were examined by means of ultrathin serial sections to evaluate the amount and distribution of cortical microtubules. The microtubules encircle the cell, oriented normal to the root length axis. In the distal cell edges, microtubules form a network, separating the distal complex of endoplasmic reticulum from the plasmalemma. Preprophase bands in meristem cells are observable rarely, structures which can be regarded as nucleating sites for microtubules are lacking. During ageing of the root cap cells, the number of microtubules increases in combination with a decrease of microtubule length. Development of the roots on a horizontal clinostat preserves a younger developmental stage of the microtubule system regarding amount and length of the individual microtubules. Evidence for an involvement of microtubules in graviperception is low, whereas their role in orienting cellulose microfibrils cannot be ruled out. Compression of the distal network of microtubules after centrifugation of the roots indicates that microtubules in statocytes ofLepidium sativum L. roots might function in stabilizing the distal complex of endoplasmic reticulum.     

Membrane-potential responses following gravistimulation in roots of Lepidium sativum L.

Membrane potentials were measured in lateral statocytes of vertically and nonvertically growing roots of Lepidium sativum L. using conventional glass-microelectrode techniques. Statocytes in vertically growing roots showed a stable resting potential of-118±5.9 mV without spontaneous fluctuations. Upon tilting the root 45° from the vertical, an electrical asymmetry was observed. Statocytes on the physically lower side of the root depolarized by approx. 25 mV. This depolarization occurred following a latent period of 8 s reaching a minimum (approx.-93 mV) after 170 s. This depolarization is the earliest event in graviperception ever recorded. After this depolarization, the cell repolarized within 60 s to a potential approx. 10 mV more positive than the original resting potential. Statocytes on the upper flank showed a slow hyperpolarization (t _1/2h=half time for hyperpolarization=168 s) reaching a final, stable potential at a level 10 mV more negative. These effects of gravistimulation were statenchyma-specific, since cells in the cortex and rhizodermis showed no similar effects. The gravi-electrical responses were observed in 25% of all roots tested. Roots which showed no gravi-electrical response had a reduced elongation growth, lacked gravity-induced bending and lacked the typical structural polarity in punctured statocytes. This observed transition from a symmetrical pattern of resting potential in the statenchyma to an asymmetrical pattern following gravistimulation supports the results observed with external current measurements (Behrens et al., Plant Physiol. 70, 1079–1083, 1982) and extends these results to the cellular level and to considerably improved temporal resolution. The asymmetry in the gravi-electrical response extends the graviperception model of Sievers and Volkmann (Planta 102, 160–172, 1972) which comprises an asymmetrical sedimentation of the amyloplasts on the distal endoplasmic reticulum of statocytes. This generates an intraorgan signal which then must be transmitted to the growth zone.Abbreviation ER endoplasmic reticulum Preliminary reports were presented at the 11^th International Conference on Plant Growth Substances, Aberystwyth, July 1982, and International Symposium, Membranes and Compartimentation in Regulation of Plant Functions, Toulouse, September 1983     

Sequence characterization and comparative analysis of three plasmids isolated from environmental Vibrio spp

The horizontal transfer of genes by mobile genetic elements such as plasmids and phages can accelerate genome diversification of Vibrio spp., affecting their physiology, pathogenicity, and ecological character. In this study, sequence analysis of three plasmids from Vibrio spp. previously isolated from salt marsh sediment revealed the remarkable diversity of these elements. Plasmids p0908 (81.4 kb), p23023 (52.5 kb), and p09022 (31.0 kb) had a predicted 99, 64, and 32 protein-coding sequences and G+C contents of 49.2%, 44.7%, and 42.4%, respectively. A phylogenetic tree based on concatenation of the host 16S rRNA and rpoA nucleotide sequences indicated p23023 and p09022 were isolated from strains most closely related to V. mediterranei and V. campbellii, respectively, while the host of p0908 forms a clade with V. fluvialis and V. furnissii. Many predicted proteins had amino acid identities to proteins of previously characterized phages and plasmids (24 to 94%). Predicted proteins with similarity to chromosomally encoded proteins included RecA, a nucleoid-associated protein (NdpA), a type IV helicase (UvrD), and multiple hypothetical proteins. Plasmid p0908 had striking similarity to enterobacteria phage P1, sharing genetic organization and amino acid identity for 23 predicted proteins. This study provides evidence of genetic exchange between Vibrio plasmids, phages, and chromosomes among diverse Vibrio spp.     

Morphological and molecular characterization of Tulasnella spp. fungi isolated from the roots of Epidendrum secundum,a widespread Brazilian orchid

Tulasnella spp. are the main fungal symbionts of Brazilian Epidendrum orchids. The taxonomy of these fungi is largely based on ITS rDNA similarity, but culture dependent techniques are still essential to establish the true biological entity of the mycobiont. The aim of this study was to characterize morphologically and molecularly 16 Tulasnella spp. fungi isolated from three different populations of E. secundum and to test the coincidences between morphological and molecular characterization. Two uninucleate rhizoctonia fungi, obtained from Oncidium barbaceniae, and two phytopathogenic isolates were included as outgroups. Qualitative and quantitative morphological characteristics were analyzed using multivariate statistics and were able to distinguish Ceratobasidium, Tulasnella and Thanatephorus genera and separate the isolates of Tulasnella spp. into two groups. Analysis of RAPD (Random Amplified Polymorphic DNA) and ITS rDNA sequences validated the morphological data. Symbionts of O. barbaceniae presented identity to ITS sequences of Ceratobasidium genus, while E. secundum isolates presented identity to two species of Tulasnella. We observed homogeneity among Tulasnella spp. obtained from a single population and from neighboring populations, but there was higher variability among isolates obtained from populations of regions that were farther apart. Morphological data associated with multivariate statistics proved to be a useful tool in the multi-level taxonomy of these orchid-associated fungi and in estimating the diversity of orchid mycorrhizal fungi.     

A role of microtubules in the polarity of statocytes from roots of Lepidium sativum L.

When roots of Lepidium sativum L. are immersed in a colchicine solution (10^-4 mol l^-1), the cortical microtubules of statocytes are affected such that the dense network ofmicrotubules at the distal cell edges, between the endoplasmic reticulum and the plasma membrane, disappears almost completely, whereas the microtubules, lining the anticlinal cell walls are reduced only to a limited extent. Upon inversion of colchicine-pretreated roots, the distal complex of endoplasmic reticulum sinks into the interior of the statocyte. Germination of seeds in the cold (3–4°C) leads to a retardation of statocyte development; the elaborated system of endoplasmic reticulum is lacking, and only a few microtubules are observable, lining the plasma membrane along the anticlinal cell walls. During an additional 4 h at 24°C, groups of microtubules develop near the plasma membrane in the distal one-third of the statocytes, coaligning with newly synthesized cisternae of the endoplasmic reticulum. It is proposed that, particularly at the distal statocyte pole, microtubules in coordination with cross-bridging structures, act in stabilizing the polar arrangement of the distal endoplasmic reticulum and, in turn, facilitate an integrated function of amyloplasts, endoplasmic reticulum and plasma membrane in graviperception.Abbreviations ER endoplasmic reticulum - MT microtubule     

Female mating frequencies in Bombus spp. from Central Europe

Summary: The mating frequency of females in social insects is particularly interesting, because polyandry reduces colony relatedness and increases within-colony genetic variance. It thereby affects a complex balance of benefits and costs that determine the degree of reproductive skew, sex allocation to offspring, or the opportunities for nepotism and policing strategies. Few systematic surveys of female mating frequencies exist and many are based on unreliable behavioural observation or sperm counts. Here, we report the results of a survey of mating frequencies in eight European Bombus spp. by means of highly polymorphic microsatellite loci. Only B. hypnorum was found to be multiply mated, while the pattern found in B. terrestris, B. lucorum, B. pratorum, B. lapidarius, B. sicheli, B. hortorum, and B. pascuorum was compatible with single mating. The findings are compatible with recent claims that, with some exceptions, mating frequencies of social insect females are generally low.     

Characterization and virulence of Beauveria spp. recovered from emerald ash borer in southwestern Ontario, Canada

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is an invasive wood boring beetle that is decimating North America's ash trees (Fraxinus spp.). To find effective and safe indigenous biocontrol agents to manage EAB, we conducted a survey in 2008-2009 of entomopathogenic fungi (EPF) infecting EAB in five outbreak sites in southwestern Ontario, Canada. A total of 78 Beauveria spp. isolates were retrieved from dead and mycosed EAB cadavers residing in the phloem tissues of dead ash barks, larval frass extracted from feeding galleries under the bark of dead trees. Molecular characterization using sequences of the ITS, 5' end of EF1-α and intergenic Bloc region fragments revealed that Beauveria bassiana and Beauveria pseudobassiana were commonly associated with EAB in the sampled sites. Based on phylogenetic analysis inferred from ITS sequences, 17 of these isolates clustered with B. bassiana, which further grouped into three different sub-clades. However, the combined EF1-α and Bloc sequences detected five genotypes among the three sub-clades. The remaining 61 isolates clustered with B. pseudobassiana, which had identical ITS sequences but were further subdivided into two genotypes by variation in the EF1-α and Bloc regions. Initial virulence screening against EAB adults of 23 isolates representing the different clades yielded 8 that produced more than 90% mortality in a single concentration assay. These isolates differed in virulence based on LC(50) values estimated from multiple concentration bioassay and based on mean survival times at a conidia concentration of 2×10(6) conidia/ml. B. bassiana isolate L49-1AA was significantly more virulent and produced more conidia on EAB cadavers compared to the other indigenous isolates and the commercial strain B. bassiana GHA suggesting that L49-1AA may have potential as a microbiological control agent against EAB.     

Variation in virulence to dry beans, soybeans and maize among isolates of Rhizoctonia solani from beans

The relative susceptibility of dry beans ( Phaseolus vulgaris ), soybeans and maize to anastomosis group 4 isolates of Rhizoctonia solani was determined in greenhouse experiments. Large variations in virulence were found among 30 field isolates. This variation was not due to differential reductions in isolate virulence during axenic culture. There was considerable variation among isolates from within the same field but variability within isolates was small. Twelve of 30 isolates of R. solani were highly virulent to dry beans and soybeans, while the others were of low virulence. Soybeans were more susceptible than dry beans to both pre-emergence mortality and hypocotyl disease. No isolates were highly virulent to maize. The importance of using isolates with a high level of virulence for testing soybean cultivars for resistance to Rhizoctonia disease is stressed.