Pesticide mixtures show potential toxicity to aquatic life in U.S. streams, water years 2013-2017.

During water years (WY) 2013-2017, the U.S. Geological Survey, National Water-Quality Assessment (NAWQA) Project, sampled the National Water Quality Network - Rivers and Streams (NWQN) year-round and reported on 221 pesticides at 72 sites across the United States in agricultural, developed, and mixed land use watersheds. The Pesticide Toxicity Index (PTI) was used to estimate the potential chronic and acute toxicity to three taxonomic groups - fish, cladocerans, and benthic invertebrates. For invertebrates (either cladocerans, benthic invertebrates, or both), the maximum PTI score exceeded the predicted acute toxicity screening level at 18 of the 72 sites (25%) at some point during WY 2013-2017. The predicted toxicity of a single pesticide compound was found to overwhelm the toxicity of other pesticides in the mixtures after concentrations were toxicity weighted. For this study, about 71%, 72%, and 92% of the Fish-, Cladoceran-, and Benthic Invertebrate-PTI scores, respectively, had one pesticide compound primarily contributing to sample potential toxicity (>50%). There were 17 (13 insecticides, 2 herbicides, 1 fungicide, and 1 synergist) of the 221 pesticide compounds analyzed that were the primary drivers of potential toxicity in each water sample in which the PTI and TUmax (toxic unit score for the pesticide that makes the single largest contribution to the PTI) scores were above predicted chronic (>0.1) or acute (>1) toxicity levels for one of the three taxa. For cladocerans and benthic invertebrates, the drivers of predicted chronic (>0.1) and acute (>1) PTIs were mostly insecticides. For cladocerans, the pesticide compounds driving the PTI scores were bifenthrin, carbaryl, chlorpyrifos, diazinon, dichlorvos, dicrotophos, diflubenzuron, flubendiamide, and tebupirimfos. For benthic invertebrates, atrazine (an herbicide), as well as the insecticides - bifenthrin, carbaryl, carbofuran, chlorpyrifos, diazinon, dichlorvos, fipronil, imidacloprid, and methamidophos - were the drivers of predicted toxicity. For fish, there were three pesticide types that contributed the most to predicted chronic (>0.1) PTIs - acetochlor, an herbicide; carbendazim, a fungicide degradate; and piperonylbutoxide, a synergist.

Production of Highly Polarized Positrons Using Polarized Electrons at MeV Energies.

The Polarized Electrons for Polarized Positrons experiment at the injector of the Continuous Electron Beam Accelerator Facility has demonstrated for the first time the efficient transfer of polarization from electrons to positrons produced by the polarized bremsstrahlung radiation induced by a polarized electron beam in a high-Z target. Positron polarization up to 82% have been measured for an initial electron beam momentum of 8.19 MeV/c, limited only by the electron beam polarization. This technique extends polarized positron capabilities from GeV to MeV electron beams, and opens access to polarized positron beam physics to a wide community.

Sexual reproduction in the soybean sudden death syndrome pathogen Fusarium tucumaniae.

We investigated the sexual reproductive mode of the two most important etiological agents of soybean sudden death syndrome, Fusarium tucumaniae and Fusarium virguliforme. F. tucumaniae sexual crosses often were highly fertile, making it possible to assign mating type and assess female fertility in 24 South American isolates. These crosses produced red perithecia and oblong-elliptical ascospores, as is typical for sexual members of the F. solani species complex. Genotyping of progeny from three F. tucumaniae crosses confirmed that sexual recombination had occurred. In contrast, pairings among 17 U.S. F. virguliforme isolates never produced perithecia. Inter-species crosses between F. tucumaniae and F. virguliforme, in which infertile perithecia were induced only in one of the two F. tucumaniae mating types, suggest that all U.S. F. virguliforme isolates are of a single mating type. We conclude that the F. tucumaniae life cycle in S. America includes a sexual reproductive mode, and thus this species has greater potential for rapid evolution than the F. virguliforme population in the U.S., which may be exclusively asexual.

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Minute supernumerary marker chromosomes identified in two patients with a related, larger pseudodicentric chromosome.

We describe two cases in which a minute supernumerary marker chromosome (SMC) was identified in addition to a larger pseudodicentric chromosome. Case 1, a phenotypically normal male, had mosaicism for a psu dic(15;15)(q11.2;q11.2) chromosome and a minute SMC. Fluorescence in situ hybridization (FISH) showed that the minute SMC was D15Z1 positive, indicating a chromosome 15 origin. Case 2 was a 22-week fetus with mosaicism for a normal and two abnormal cell lines: one had a psu dic (22;22)(q11.2;q11.2) chromosome containing euchromatin, usually associated with cat eye syndrome; the other a minute SMC. The minute SMC was positive with the D14Z1/D22Z1 alpha-satellite probe, indicating a chromosome 14 or chromosome 22 origin. Deletion of centromeric material was proposed as one mechanism of centromere inactivation in dicentric chromosomes. The origin of these two minute SMC suggests that they were derived from one of the centromeres of the larger pseudodicentric chromosome. These stable minute SMC may be the by-product of a deletion event inactivating one centromere of a dicentric chromosome to generate a pseudodicentric chromosome. Alternatively, the minute SMC may originate from further rearrangement of the larger pseudodicentric chromosome. These cases suggest possible mechanisms for the origin of minute SMC.

The Neurospora crassa genome: cosmid libraries sorted by chromosome.

A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification.

Molecular mating type assay for Fusarium circinatum.

A rapid and reliable mating type assay for Fusarium circinatum was created by applying primers specific for the MAT1-1 and MAT1-2 mating type alleles to genomic DNA in a single PCR. A similar approach may be applied to fungi not previously shown to reproduce sexually, thus enabling studies of population structure and inheritance.

Physical map of a conditionally dispensable chromosome in Nectria haematococca mating population VI and location of chromosome breakpoints.

Certain isolates of the plant pathogenic fungus Nectria haematococca mating population (MP) VI contain a 1.6-Mb conditionally dispensable (CD) chromosome carrying the phytoalexin detoxification genes MAK1 and PDA6-1. This chromosome is structurally unstable during sexual reproduction. As a first step in our analysis of the mechanisms underlying this chromosomal instability, hybridization between overlapping cosmid clones was used to construct a map of the MAK1 PDA6-1 chromosome. The map consists of 33 probes that are linked by 199 cosmid clones. The polymerase chain reaction and Southern analysis of N. haematococca MP VI DNA digested with infrequently cutting restriction enzymes were used to close gaps and order the hybridization-derived contigs. Hybridization to a probe extended from telomeric repeats was used to anchor the ends of the map to the actual chromosome ends. The resulting map is estimated to cover 95% of the MAK1 PDA6-1 chromosome and is composed of two ordered contigs. Thirty-eight percent of the clones in the minimal map are known to contain repeated DNA sequences. Three dispersed repeats were cloned during map construction; each is present in five to seven copies on the chromosome. The cosmid clones representing the map were probed with deleted forms of the CD chromosome and the results were integrated into the map. This allowed the identification of chromosome breakpoints and deletions.

Partial MAT-2 gene structure and the influence of temperature on mating success in Gibberella circinata.

Genetic analysis of Gibberella circinata, the causative agent of pitch canker disease of pines, historically has been thwarted by a low frequency of mating success in the laboratory. We describe two findings that should facilitate genetic analysis of this fungus and related species. First, we determined that previously described degenerate primers could be used to amplify a portion of the MAT-2 mating type gene from G. circinata. This led to the cloning and sequencing of a fragment of the MAT-2 gene, which in turn made it possible to distinguish between G. circinata isolates of opposite mating types. Second, we discovered that of the 18 G. circinata field isolates in our collection, the 1 female fertile isolate expressed its fertility at 15 and 20 degrees C but not at 25 degrees C, the temperature used for crossing many Gibberella species. It is evident, therefore, that when sexual reproduction in other closely related species is initially being investigated, the crosses should be established at a variety of temperatures. Once we learned that female fertility in this G. circinata isolate was expressed at 20 degrees C, a high frequency of mating success was achieved.

Supernumerary chromosomes in filamentous fungi.

Within a fungal species, a subset of individuals may have more than the minimal complement of chromosomes. If the extra chromosomes are composed primarily of DNA not found in all representatives of the species, they are most appropriately referred to as supernumerary chromosomes. The patterns of repeated DNA sequences on certain supernumerary chromosomes suggest that they have a different evolutionary history from the essential chromosomes in the same genome. Supernumerary chromosomes can carry functional genes and, in at least two fungal species, genes on such chromosomes play important roles in host-pathogen interactions. Supernumerary chromosomes that confer an adaptive advantage in certain habitats, such as the ability to cause disease on a specific host, may be referred to as "conditionally dispensable" chromosomes in order to reflect their importance in some, but not all, growth conditions. In addition to describing the structural and functional characteristics of known supernumerary chromosomes in fungi, this review discusses the relative merits of the terms that have been used to describe them, and establishes experimental criteria for their identification.

Inducing the loss of conditionally dispensable chromosomes in Nectria haematococca during vegetative growth.

A procedure for inducing and detecting the loss of conditionally dispensable (CD) chromosomes in filamentous fungi during vegetative growth was developed using Nectria haematococca mating population VI as a model. CD chromosomes in two different isolates of N. haematococca were tagged via integrative transformation with a gene conferring resistance to hygromycin B. In each case the transformation vector included chromosome-specific DNA in order to direct its homologous recombination with the desired chromosome. Chromosome loss was induced by exposing tagged isolates to inhibitory concentrations of benomyl either for protracted periods of time on solid medium or for short periods of time in liquid medium. After exposure to benomyl, isolates that lost the tagged chromosome were identified by their loss of resistance to hygromycin B. Electrophoretic karyotyping was used to verify that isolates which failed to grow on hygromycin B lacked an intact CD chromosome. Ten other chemicals known to interfere with mitotic events or cell development in other organisms did not induce CD chromosome loss in N. haematococca.

Radon measurements and mitigation at a fish hatchery.

Groundwater radon concentration of 83 Bq L(-1) generated indoor radon levels exceeding 3,300 Bq m(-3) at a commercial fish hatchery. Passive and active mitigation strategies to reduce the waterborne radon levels included a packed column, a waterfall through perforated grates, surface aeration, and bottom bubblers. Waterborne concentrations were reduced up to 83% using a combination of mitigation procedures, but a comparable reduction in indoor radon concentrations was not observed. A diurnal cycle showed that indoor radon levels peaked in early afternoon, probably as a result of warmer air being dissolved in the water during mitigation. Reduction of indoor radon levels below 148 Bq m(-3) was seldom achievable with both water mitigation and direct air ventilation at 23 room air changes hourly.

Nht1, a transposable element cloned from a dispensable chromosome in Nectria haematococca.

Certain isolates of the plant-pathogenic fungus Nectria haematococca mating population VI (MPVI) contain dispensable chromosomes that are unstable during sexual reproduction. Several of these chromosomes carry genes for phytoalexin detoxification and thus contribute to the pathogenic potential of this organism. A repeated DNA sequence, Nht1, was cloned from one of these dispensable chromosomes in N. haematococca MPVI. One copy of the repeated element (Nht1A) was completely sequenced. It is 2,198 bp long and it possesses incomplete inverted terminal repeats (ITRs) at each end. Nht1B, a partially sequenced copy of Nht1, has complete ITRs. Nht1A appears to contain 2 introns and encodes a protein of 550 amino acids that is highly similar to the protein encoded by the Fusarium oxysporum transposon, Fot1. Due to the presence of ITRs, its repeated nature, and its similarity to Fot1, we conclude that Nht1 is a transposable element. Within North American N. Haematococca MPVI populations, Nht1 is distributed discontinuously. Its copy number in different field isolates varies from zero to approximately 100 copies per genome. The Nht1A source isolate is estimated to contain nine to 11 copies of Nht1; at least six are on the chromosome from which Nht1A was cloned.

A gene for maackiain detoxification from a dispensable chromosome of Nectria haematococca.

In Nectria haematococca the MAK1 gene product converts a chick-pea (Cicer arietinum) phytoalexin, maackiain, into a less toxic compound. The presence of MAK1 in this fungal pathogen is also correlated with high virulence on chick-pea. Previous genetic analysis suggested that MAK1 is located on a meiotically unstable, dispensable chromosome. The unstable nature of this chromosome facilitated MAK1 cloning by allowing us to identify a subset of genomic cosmid clones likely to contain MAK1. Truncated forms of the chromosome, generated during meiosis, were isolated from strains either able (Mak+) or unable (Mak-) to metabolize maackiain and used to probe a chromosome-specific cosmid library. Only clones that hybridized exclusively to the chromosome from the Mak+ strain were then screened for their ability to transform a Mak- isolate to the Mak+ phenotype. A 2.7 kb HindIII-PstI fragment was subcloned from a cosmid conferring MAK1 activity, and its nucleotide sequence determined. Because MAK1 transcription is not induced strongly by maackiain, a reverse transcriptase-polymerase chain reaction was required to detect MAK1 transcription in a Mak+ strain, and to isolate MAK1 cDNA fragments. Comparison of the genomic and cDNA sequences of MAK1 revealed the presence of three introns and an open reading frame encoding a protein 460 amino acids in length. Two diagnostic domains in its deduced amino acid sequence suggest MAK1 encodes a flavin-containing mono-oxygenase. MAK1 is the first gene encoding maackiain detoxification to be cloned, and is the second functional gene cloned from this dispensable chromosome. Southern analysis of genomic DNA from ascospore isolates containing MAK2, MAK3, and MAK4 indicated that MAK1 is not homologous to other known maackianin-detoxifying genes.

Establishment of genetic linkage by allele-specific polymerase chain reaction: application to the lignin peroxidase gene family of Phanerochaete chrysosporium.

Determining linkage is problematic for genes lacking easily identifiable phenotypes and for organisms without well-defined genetic recombination systems. Phanerochaete chrysosporium with its lignin peroxidase (LiP) gene family typifies these difficulties. We describe an experimental approach whereby the segregation of specific alleles is directly monitored during sexual fruiting. The method establishes linkage relationships among genes for which there are no mutations, and it is applicable to a wide range of genes, gene families and organisms. Using this approach, five P. chrysosporium linkage groups were identified. Ten LiP genes were distributed among three of these groups. One co-segregating group contained eight closely linked LiP genes. Another LiP gene was linked to a cellobiohydrolase gene cluster. These genetic linkages were consistent with physical mapping by pulsed field gel electrophoresis. Based on the identification of allelic relationships, a uniform nomenclature for LiP genes is also described.

Myelinolysis after correction of hyponatremia in two dogs.

Two dogs developed delayed neurological deterioration after rapid correction of severe hyponatremia. Sequential magnetic resonance imaging showed the development of lesions in the thalamus. One dog was necropsied, and the lesions were characterized by myelinolysis with sparing of axons and neurons. The second dog gradually recovered with no detectable neurological deficits. The syndrome seems analogous to central pontine myelinolysis in human beings. Guidelines for correction of hyponatremia to prevent development of myelinolysis are given.

Identification of the gene encoding the major cellobiohydrolase of the white rot fungus Phanerochaete chrysosporium.

Previous studies have shown that the cellobiohydrolases of the white rot basidiomycete Phanerochaete chrysosporium are encoded by a family of structurally related genes. In this investigation, we identified and sequenced the most highly transcribed gene, cbh1-4. Evidence suggests that in this fungus the dominant isozyme, CBH1, is encoded by chb1-4.

Genomic organization of a cellulase gene family in Phanerochaete chrysosporium.

Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were found to hybridize to at least three different chromosomes, one of which is dimorphic. The organization of these genes was similar in another P. chrysosporium strain, ME 446. It is clear that, unlike T. reesei, the most well-studied cellulolytic fungus, P. chrysosporium contains a complex, cbh1-like gene family.

Structure, organization, and transcription of a cellobiohydrolase gene cluster from Phanerochaete chrysosporium.

Restriction mapping and sequence analysis of cosmid clones revealed a cluster of three cellobiohydrolase genes in Phanerochaete chrysosporium. P. chrysosporium cbh1-1 and cbh1-2 are separated by only 750 bp and are located approximately 14 kb upstream from a cellulase gene previously cloned from P. chrysosporium (P. Sims, C. James, and P. Broda, Gene 74:411-422, 1988). Within a well-conserved region, the deduced amino acid sequences of P. chrysosporium cbh1-1 and cbh1-2 are, respectively, 80 and 69% homologous to that of the Trichoderma reesei cellobiohydrolase I gene. The conserved cellulose-binding domain typical of microbial cellulases is absent from cbh1-1. Transcript levels of the three P. chrysosporium genes varied substantially, depending on culture conditions. cbh1-1 and cbh1-2 were not induced in the presence of cellulose, nor did they appear to be subject to glucose repression. Therefore, aspects of the chromosomal organization, structure, and transcription of these genes are unlike those of any previously described cellulase genes.