Evaluation of Major Ancestors of North American Soybean Cultivars for Resistance to Three Pythium Species that Cause Seedling Blight.

Pythium seedling blight, which can be caused by a number of Pythium spp., is a disease that affects soybean (Glycine max) in the United States and Canada. Pythium ultimum var. ultimum, one of the most common pathogenic species, is favored by cool, wet conditions in early spring and causes seed decay, root rot, and seedling damping-off. In all, 102 major ancestors of modern North American cultivars and "first progeny" cultivars developed directly from ancestral lines were evaluated for resistance to P. ultimum var. ultimum and two other species of Pythium in greenhouse assays. Several ancestors and first progeny cultivars, as well as the resistant check Archer, had varying levels of partial resistance to an Illinois isolate of P. ultimum var. ultimum. In a subsequent experiment, four of the most resistant lines (PI 84637, Maple Isle, Fiskeby III, and Fiskeby 840-7-3) and the susceptible cultivar Kanro were screened for resistance against isolates of P. irregulare and P. sylvaticum, and resistance to P. ultimum var. ultimum was confirmed. The lines that were partially resistant to P. ultimum var. ultimum in the first experiment were also partially resistant to P. irregulare and P. sylvaticum. The P. ultimum var. ultimum isolate was the most aggressive of the three isolates, followed by the P. irregulare and P. sylvaticum isolates. Modern cultivars descended from the soybean lines with partial resistance to these pathogens could be useful sources of resistance to Pythium seedling blight if they are found to have similar levels of resistance.

Comparison of Pathogenic Variation among Phakopsora pachyrhizi Isolates Collected from the United States and International Locations, and Identification of Soybean Genotypes Resistant to the U.S. Isolates.

A major constraint in breeding for resistance to soybean rust has been the virulence diversity in Phakopsora pachyrhizi populations. In greenhouse experiments, reactions of 18 soybean genotypes to 24 U.S. isolates from 2007 and 2008 and 4 foreign isolates were compared. Reactions of four differentials (Rpp1 to Rpp4) to these U.S. isolates were also compared with reactions to nine foreign isolates and three U.S. isolates from 2004. Principal component analysis (PCA) of the reaction types grouped the U.S. isolates into a single virulence group, whereas each of the foreign isolates had a unique virulence pattern. In another experiment, reactions of 11 differentials to the 24 U.S. isolates were compared and significant interactions (P < 0.001) were found between the isolates and host genotypes for rust severity and uredinia densities. PCA of these two measures of disease placed the 24 isolates into seven or six aggressiveness groups, respectively. In a third experiment, evaluation of 20 soybean genotypes for resistance to the previously established aggressive groups identified 10 genotypes resistant to isolates representing most of the groups. This study confirmed the pathogenic diversity in P. pachyrhizi populations and identified soybean germplasm with resistance to representative U.S. isolates that can be used in breeding.

First Report of Phakopsora pachyrhizi Adapting to Soybean Genotypes with Rpp1 or Rpp6 Rust Resistance Genes in Field Plots in the United States.

During the years following the first detection of soybean rust, caused by Phakopsora pachyrhizi Syd., in the continental United States in November, 2004, soybean (Glycine max [L.] Merr.) genotypes with the Rpp1 or Rpp6 resistance genes exhibited high levels of resistance there (1,2,3). When challenged with 72 different American isolates collected between 2006 and 2009, PI 200492 (source of Rpp1) produced no sporulating lesions (2). In 2011 and 2012, however, field populations of P. pachyrhizi from Gadsden County, FL, caused higher rust severity on plants with Rpp1 or Rpp6 than in previous years. To assess aggressiveness, sporulation ratings were made using a 1 to 5 scale (no sporulation to profuse sporulation) on leaflets collected from field plants at or near the R6 (full seed) stage of development. A dissecting microscope was used to examine 3 replications of 5 leaflets each in 2009 or 2 replications of 10 leaflets each in 2012. The sporulation ratings increased on PI 200492 (from 1.1 ± 0.1 in 2009 to 4.1 ± 0.4 in 2012), PI 567102B (Rpp6; from 1.1 ± 0.1 in 2009 to 2.4 ± 0.2 in 2012), and L85-2378, a 'Williams 82' isoline carrying the Rpp1 gene (from 1.0 ± 0 in 2009 to 4.0 ± 0.3 in 2012). The mean 2009 and 2012 sporulation ratings for susceptible control Williams 82 were 5.0 ± 0 and 4.2 ± 0.1, respectively. Single-uredinium-derived isolates were purified from bulk isolates collected from field plots in 2009 (FL-Q09-1), 2011 (FL-Q11-1), and 2012 (FL-Q12-1). Greenhouse and detached leaflet assays were then used to test the virulence of these isolates under controlled conditions. Detached leaflets from 3-week-old seedlings of Williams 82, PI 200492, PI 567102B, and L85-2378 were inoculated by pipetting 15-µl drops of a 30 to 40 urediniospore µl-1 suspension onto the abaxial side of 3 to 4 leaflets per genotype, which were then sealed in Petri plates and incubated in a growth chamber at 20 to 22°C. Plates were kept in the dark for 12 h following inoculation. For the greenhouse assay, the first trifoliolate leaves of at least 3 seedlings were each sprayed with 1.5 ml of a 40 urediniospore µl-1 suspension and incubated 24 h at 22 to 24°C in a dark mist chamber. The plants were then maintained at 22 to 24°C and 76 to 86% relative humidity in a greenhouse with 10 h of daylight on average. Two weeks after inoculation with FL-Q11-1 or FL-Q12-1, all of the genotypes had developed TAN lesions with abundant sporulation, indicating susceptibility. On leaves inoculated with FL-Q09-1, however, no visible reaction was observed on PI 200492, and PI 567102B developed reddish-brown (RB) lesions associated with incomplete resistance. Although the lesions on Rpp1 and Rpp6 greenhouse seedlings inoculated with the FL-Q11-1 and FL-Q12-1 isolates were slightly darker than those that developed on Williams 82 plants or on detached leaflets, the profuse sporulation that is characteristic of the TAN infection type was observed. The higher virulence of the 2011 and 2012 Florida isolates on two soybean genotypes with Rpp1 and one with Rpp6 confirmed the presence of a P. pachyrhizi pathotype in north-central Florida that is more virulent against these genes than earlier populations from the southeastern United States. References: (1) S. Li. Crop Sci. 49:887, 2009. (2) Twizeyimana and Hartman. Plant Dis. 96:75, 2012. (3) Walker et al. Crop Sci. 51:678, 2011.

High harmonic frequency combs for high resolution spectroscopy.

We generated a series of harmonics in a xenon gas jet inside a cavity seeded by pulses from a Ti:sapphire mode-locked laser with a repetition rate of 10.8 MHz. Harmonics up to 19th order at 43 nm were observed with plateau harmonics at the microW power level. An elaborate dispersion compensation scheme and the use of a moderate repetition rate allowed for this significant improvement in output power of the plateau harmonics of 4 orders of magnitude over previous results. With this power level and repetition rate, high-resolution spectroscopy in the extreme ultraviolet region becomes conceivable. An interesting target would be the 1S-2S transition in hydrogenlike He+ at 60 nm.

Effects of defoliating insect resistance QTLs and a cry1Ac transgene in soybean near-isogenic lines.

The crystal proteins coded by transgenes from Bacillus thuringiensis (Bt) have shown considerable value in providing effective insect resistance in a number of crop species, including soybean, Glycine max (L.) Merr. Additional sources of soybean insect resistance would be desirable to manage the development of tolerance/resistance to crystal proteins by defoliating insects and to sustain the deployment of Bt crops. The objective of this study was to evaluate the effects and interactions of three insect resistance quantitative trait loci (QTLs; QTL-M, QTL-H, and QTL-G) originating from Japanese soybean PI 229358 and a cry1Ac gene in a "Benning" genetic background. A set of 16 BC(6)F(2)-derived near isogenic lines (NILs) was developed using marker-assisted backcrosses and evaluated for resistance to soybean looper [SBL, Pseudoplusia includens (Walker)] and corn earworm [CEW, Helicoverpa zea (Boddie)] in field cage, greenhouse, and detached leaf assays. Both Bt and QTL-M had significantly reduced defoliation by both SBL and CEW and reduced larval weight of CEW. The antibiosis QTL-G had a significant effect on reducing CEW larval weight and also a significant effect on reducing defoliation by SBL and CEW in some assays. The antixenosis QTL-H had no main effect, but it appeared to function through interaction with QTL-M and QTL-G. Adding QTL-H and QTL-G further enhanced the resistance of the Bt and QTL-M combination to CEW in the field cage assay. These results should help guide the development of strategies for effective management of insect pests and for sustainable deployment of Bt genes.

Measurement of Fourier-synthesized optical waveforms.

Following the experiments of Shverdin and colleagues [Phys. Rev. Lett. 94, 033904 (2005)], we describe a technique for determining the temporal envelope of an optical beam whose spectrum consists of n discrete, equally spaced frequency components. Four-wave mixing is employed to generate n-1 higher-frequency sidebands. The relative intensities of these sidebands, together with the intensities of the incident side-bands, determine the unknown relative phases of the incident beam.

Generation of a single-cycle optical pulse.

We make use of coherent control of four-wave mixing to the ultraviolet as a diagnostic and describe the generation of a periodic optical waveform where the spectrum is sufficiently broad that the envelope is approximately a single-cycle in length, and where the temporal shape of this envelope may be synthesized by varying the coefficients of a Fourier series. Specifically, using seven sidebands, we report the generation of a train of single-cycle optical pulses with a pulse width of 1.6 fs, a pulse separation of 11 fs, and a peak power of 1 MW.

Comparison of four flow cytometric SNP detection assays and their use in plant improvement.

Single nucleotide polymorphisms (SNPs) are attractive DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed, but it is unclear which assays are best suited and most efficient for various types of plant improvement research. The objective of this study was to compare the accuracy, efficiency, and cost of four SNP genotyping assays: single-base extension (SBE), allele-specific primer extension (ASPE), oligonucleotide ligation (OL), and direct hybridization (DH). All four assay methods used the same Luminex 100 flow cytometer platform. Fifty-eight F(2)-derived soybean [Glycine max (L.) Merr.] lines from a cross between inbred lines G99-G725 and N00-3350 were genotyped at four SNPs. SBE and ASPE clearly differentiated between the two homozygotes and the heterozygote at each SNP. Results were in agreement with those identified using the SNaPshot minisequencing assay as a control. In contrast, the OL and DH assays were unable to differentiate between genotypes at some of the SNPs. However, when the cost per data point for the four different assays was compared, the cost of OL and DH was only about 70% of that for SBE, with DH requiring the least time of the four assays. On the basis of cost and labor, ASPE is more cost-effective and simpler than SBE, and would therefore be a good method for genetic mapping and diversity studies which require a large number of markers and a high level of multiplexing. DH appears to be the most economical assay for marker-assisted selection, though optimization for DH would be required for some SNP markers.

Quasiperiodic Raman technique for ultrashort pulse generation.

We report the experimental demonstration of a new Raman technique that produces 200 sidebands, ranging in wavelength from 3 microm to 195 nm. By studying multiphoton ionization of nitric oxide (NO) molecules, we show mutual phase coherence among 15 visible sidebands covering 0.63 octaves of bandwidth.

Rotational Raman generation with near-unity conversion efficiency.

We demonstrate collinear generation of equidistant rotational sidebands in low-pressure molecular hydrogen with near-unity conversion efficiency. The spectrum consists of 37 coherent sidebands covering over 20, 000 cm(-1) of spectral bandwidth and ranging from 1.37mum to 352 nm in wavelength.

Raman self-focusing at maximum coherence.

We demonstrate a type of Raman self-focusing and -defocusing that is inherent in operation at maximum coherence. In this regime the two-photon detuning from the Raman resonance controls the refractive index of the medium.

Femtosecond light source for phase-controlled multiphoton ionization.

We describe a femtosecond Raman light source with more than an octave of optical bandwidth. We use this source to demonstrate phase control of multiphoton ionization under conditions where ionization requires eleven photons of the lowest frequency of the spectrum or five photons of the highest frequency. The nonlinearity of the photoionization process allows us to characterize the light source. Experiment-to-theory comparison implies generation of a near single-cycle waveform.

Optical frequency conversion by a rotating molecular wave plate.

We demonstrate efficient four-wave mixing in low-pressure molecular deuterium without the need for phase matching. We use two laser fields with opposite circular polarizations to produce a strong excitation of a rovibrational transition at a frequency of 3167 cm(-1) . The coherent molecular motion, in turn, modulates a third laser field (also circularly polarized) and results in highly efficient single-sideband conversion.

Raman generation by phased and antiphased molecular states.

We use molecular deuterium and two driving lasers to demonstrate collinear generation of mutually coherent equidistant sidebands, covering 50 000 cm(-1) of spectral bandwidth and ranging from 2.94 microm to 195 nm in wavelength. The essential idea is the adiabatic preparation of a single, highly coherent (|rho(ab)| = 0.33) molecular eigenstate.

Field evaluation of soybean engineered with a synthetic cry1Ac transgene for resistance to corn earworm, soybean looper, velvetbean caterpillar (Lepidoptera: Noctuidae), and lesser cornstalk borer (Lepidoptera: Pyralidae).

A transgenic line of the soybean 'Jack', Glycine max (L.) Merrill, expressing a synthetic cry1Ac gene from Bacillus thuringiensis variety kurstaki (Jack-Bt), was evaluated for resistance to four lepidopteran pests in the field. Jack-Bt and genotypes serving as susceptible and resistant controls were planted in field cages and artificially infested with larvae of corn earworm, Helicoverpa zea (Boddie), and velvetbean caterpillar, Anticarsia gemmatalis (Hübner), in 1996, 1997, and 1998, and also with soybean looper, Pseudoplusia includens (Walker), in 1996. Susceptible controls included Jack (1996-1998), 'Cobb' (1996), and Jack-HPH (1996). GatIR 81-296 was used as the resistant control in all 3 yr. Compared with untransformed Jack, Jack-Bt showed three to five times less defoliation from corn earworm and eight to nine times less damage from velvetbean caterpillar. Defoliation of GatIR 81-296 was intermediate between that of Jack and Jack-Bt for corn earworm, and similar to that of Jack for velveltbean caterpillar. Jack-Bt exhibited significant, but lower resistance to soybean looper. Jack-Bt also showed four times greater resistance than Jack to natural infestations of lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), in conventional field plots at two locations in 1998. Data from these experiments suggest that expression of this cry1Ac construct in soybean should provide adequate levels of resistance to several lepidopteran pests under field conditions.

A deviation bar chart for detecting dengue outbreaks in Puerto Rico.

OBJECTIVES: A Centers for Disease Control and Prevention deviation bar chart (Statistical Software for Public Health Surveillance) and laboratory-based surveillance data were evaluated for their utility in detecting dengue outbreaks in Puerto Rico. METHODS: A significant increase in dengue incidence was defined as an excess of suspected cases of more than 2 SDs beyond the mean for all 4-week periods from April through June (the period of lowest seasonal incidence), 1989 through 1993. An outbreak was defined as a cumulative annual rate of reported dengue greater than 3 per 1000 population. RESULTS: Retrospective application of the system to 1994 data showed agreement with previous analyses. In 1995 and 1996, 36.4% and 27.3%, respectively, of municipalities with a significant increase in reports for 2 or more consecutive weeks before the first week of September had an outbreak, compared with 9.0% (in 1995, P = .042) and 6.0% (in 1996, P = .054) of towns without a significant increase. The system showed sensitivity near 40%, specificity near 89%, and accuracy in classifying municipalities near 84%. CONCLUSIONS: This method provides a statistically based, visually striking, specific, and timely signal for dengue control efforts.

Evolutionary conservation and somatic mutation hotspot maps of p53: correlation with p53 protein structural and functional features.

Missense mutations in p53 frequently occur at 'hotspot' amino acids which are highly conserved and represent regions of structural or functional importance. Using the p53 mutation database and the p53 DNA sequences for 11 species, we more precisely defined the relationships among conservation, mutation frequency and protein structure. We aligned the p53 sequences codon-by-codon and determined the degree of substitution among them. As a whole, p53 is evolving at an average rate for a mammalian protein-coding gene. As expected, the DNA binding domain is evolving more slowly than the carboxy and amino termini. A detailed map of evolutionary conservation shows that within the DNA binding domain there are repeating peaks and valleys of higher and lower evolutionary constraint. Mutation hotspots were identified by comparing the observed distribution of mutations to the pattern expected from a random multinomial distribution. Seventy-three hotspots were identified; these 19% of codons account for 88% of all reported p53 mutations. Both high evolutionary constraint and mutation hotspots are noted at amino acids close to the protein-DNA interface and at others more distant from DNA, often buried within the core of the folded protein but sometimes on its surface. The results indicate that targeting highly conserved regions for mutational and functional analysis may be efficient strategies for the study of cancer-related genes.

Conserved domains in DNA repair proteins and evolution of repair systems.

A detailed analysis of protein domains involved in DNA repair was performed by comparing the sequences of the repair proteins from two well-studied model organisms, the bacterium Escherichia coli and yeast Saccharomyces cerevisiae, to the entire sets of protein sequences encoded in completely sequenced genomes of bacteria, archaea and eukaryotes. Previously uncharacterized conserved domains involved in repair were identified, namely four families of nucleases and a family of eukaryotic repair proteins related to the proliferating cell nuclear antigen. In addition, a number of previously undetected occurrences of known conserved domains were detected; for example, a modified helix-hairpin-helix nucleic acid-binding domain in archaeal and eukaryotic RecA homologs. There is a limited repertoire of conserved domains, primarily ATPases and nucleases, nucleic acid-binding domains and adaptor (protein-protein interaction) domains that comprise the repair machinery in all cells, but very few of the repair proteins are represented by orthologs with conserved domain architecture across the three superkingdoms of life. Both the external environment of an organism and the internal environment of the cell, such as the chromatin superstructure in eukaryotes, seem to have a profound effect on the layout of the repair systems. Another factor that apparently has made a major contribution to the composition of the repair machinery is horizontal gene transfer, particularly the invasion of eukaryotic genomes by organellar genes, but also a number of likely transfer events between bacteria and archaea. Several additional general trends in the evolution of repair proteins were noticed; in particular, multiple, independent fusions of helicase and nuclease domains, and independent inactivation of enzymatic domains that apparently retain adaptor or regulatory functions.

Analogous enzymes: independent inventions in enzyme evolution.

It is known that the same reaction may be catalyzed by structurally unrelated enzymes. We performed a systematic search for such analogous (as opposed to homologous) enzymes by evaluating sequence conservation among enzymes with the same enzyme classification (EC) number using sensitive, iterative sequence database search methods. Enzymes without detectable sequence similarity to each other were found for 105 EC numbers (a total of 243 distinct proteins). In 34 cases, independent evolutionary origin of the suspected analogous enzymes was corroborated by showing that they possess different structural folds. Analogous enzymes were found in each class of enzymes, but their overall distribution on the map of biochemical pathways is patchy, suggesting multiple events of gene transfer and selective loss in evolution, rather than acquisition of entire pathways catalyzed by a set of unrelated enzymes. Recruitment of enzymes that catalyze a similar but distinct reaction seems to be a major scenario for the evolution of analogous enzymes, which should be taken into account for functional annotation of genomes. For many analogous enzymes, the bacterial form of the enzyme is different from the eukaryotic one; such enzymes may be promising targets for the development of new antibacterial drugs.