University of Georgia Research Foundation

This article addresses the third leading cause of death among youths—adolescent suicide. Depression, stress, family, and peer factors that impinge on adolescents and predispose them to suicide are reviewed. The role of professionals in prevention efforts is examined and a practice paradigm for identifying helping suicidal adolescents is outlined.

Avian peritoneal exudate macrophages, when exposed to phagocytic stimuli, produced an appreciable oxidative burst as measured by production of chemiluminescence, superoxide anion, and hydrogen peroxide. Metabolic inhibitors of the oxidative burst and scavengers of oxygen radicals clearly inhibited macrophage chemiluminescence, but had no significant effect on macrophage bactericidal activity against Escherichia coli or fungistatic activity against Candida tropicalis. Therefore, avian macrophages were capable of oxygen-independent bactericidal and fungistatic activities.

A avaliacao do potencial tecnologico, da patentabilidade e do potencial comercial de invencoes desenvolvidas nas universidades e geralmente um processo muito dificil, muitas vezes sujeito a erros de avaliacao. O principal problema enfrentado pelos NITs academicos e o estagio muito incipiente das tecnologias desenvolvidas em universidades, que estao - normalmente - mais proximas do nivel de uma descoberta do que a de um produto acabado. Ao longo dos anos, NITs americanos desenvolveram ferramentas que facilitam e trazem um pouco mais de precisao ao processo de avaliacao. No Brasil, a INOVA-UniCamp foi a pioneira no desenvolvimento de tal ferramenta, apropriada a realidade nacional, aplicada ao seu portifolio tecnologico.   Estas ferramentas sao chamadas formularios de triagem, dois dos quais sao introduzidos aqui para a comunidade brasileira de transferencia de tecnologia.

Mycoplasma iowae (Mi) is a pathogenic avian mycoplasma which causes mortality in turkey embryos and as such has clinical and economic significance for the turkey breeder industry. Control of Mi infection is severely hampered by lack of adequate diagnostic tests, together with resistance to most antibiotics and resilience to environment. A markedly high degree of intra-species antigenic variation also contributes to difficulties in detection and control of infection. In this project we have designed an innovative gene-based diagnostic test based on specific amplification of the 16S rRNA gene of Mi. This reaction, designed Multi-species PCR-RFLP test, also amplifies the DNA of the pathogenic avian mycoplasmas M. gallisepticum (Mg) and M. synoviae (Ms). This test detects DNA equivalent to about 300 cfu Mi or either of the other two target mycoplasmas, individually or in mixed infection. It is a quick test, applicable to a wide variety of clinical samples, such as allantoic fluid or tracheal or cloacal swab suspensions. Differential diagnosis is carried out by gel electro-phoresis of the PCR amplicon digested with selected restriction enzymes (Restriction Fragment Length Polymorphism). This can also be readily accomplished by using a simple Dot-Blot hybridization assay with digoxigenin-labeled oligonucleotide probes reacting specifically with unique Mi, Mg or Ms sequences in the PCR amplicon. The PCR/OLIGO test increased sensitivity by at least 10-fold with a capacity for rapid testing of large numbers of samples. Experimental infection trials were carried out to evaluate the diagnostic tools and to study pathogenesis of Mi infection. Field studies and experimental infection of embryonated eggs indicated both synergistic and competitive interaction of mycoplasma pathogens in mixed infection. The value of the PCR diagnostic tests for following the time course of egg transmission was shown. A workable serological test (Dot Immunobinding Assay) was also developed but there was no clear-cut evidence that infected turkeys develop an immune response. Typing of a wide spectrum of Mi field isolates by a variety of gene-based molecular techniques indicated a higher degree of genetic homogeneity than predicted on the basis of the phenotypic variability. All known strains of Mi were detected by the method developed. Together with an M. meleagridis-PCR test based on the same gene, the Multi-species PCR test is a highly valuable tool for diagnosis of pathogenic mycoplasmas in single or mixed infection. The further application of this rapid and specific test as a part of Mi and overall mycoplasma control programs will be dependent on developments in the turkey industry.

This report was prepared as an account of work sponsored by an agency of

In this project we focused on various aspects of biorefinery technology development including algal-biorefinery technology, thermochemical conversion of biomass to bio-oils and biochar; we tested characteristics and applications of biochars and evaluated nutrient cycling with wastewater treatment by the coupling of algal culture systems and anaerobic digestion. Key results include a method for reducing water content of bio-oil through atomized alcohol addition. The effect included increasing the pH and reducing the viscosity and cloud point of the bio-oil. Low input biochar production systems were evaluated via literature reviews and direct experimental work. Additionally, emissions were evaluated and three biochar systems were compared via a life cycle analysis. Attached growth systems for both algal cultivation and algal harvesting were found to be superior to suspended growth cultures. Nutrient requirements for algal cultivation could be obtained by the recycling of anaerobic digester effluents, thus experimentally showing that these two systems could be directly coupled. Twenty-two journal articles and six intellectual property applications resulted from the cumulative work that this project contributed to programmatically.

The main objectives of this research was to detect the specific polymorphisms responsible for observed quantitative trait loci and develop optimal strategies for genomic evaluations and selection for moderate (Israel) and large (US) dairy cattle populations. A joint evaluation using all phenotypic, pedigree, and genomic data is the optimal strategy. The specific objectives were: 1) to apply strategies for determination of the causative polymorphisms based on the “a posteriori granddaughter design” (APGD), 2) to develop methods to derive unbiased estimates of gene effects derived from SNP chips analyses, 3) to derive optimal single-stage methods to estimate breeding values of animals based on marker, phenotypic and pedigree data, 4) to extend these methods to multi-trait genetic evaluations and 5) to evaluate the results of long-term genomic selection, as compared to traditional selection. Nearly all of these objectives were met. The major achievements were: The APGD and the modified granddaughter designs were applied to the US Holstein population, and regions harboring segregating quantitative trait loci (QTL) were identified for all economic traits of interest. The APGD was able to find segregating QTL for all the economic traits analyzed, and confidence intervals for QTL location ranged from ~5 to 35 million base pairs. Genomic estimated breeding values (GEBV) for milk production traits in the Israeli Holstein population were computed by the single-step method and compared to results for the two-step method. The single-step method was extended to derive GEBV for multi-parity evaluation. Long-term analysis of genomic selection demonstrated that inclusion of pedigree data from previous generations may result in less accurate GEBV. Major conclusions are: Predictions using single-step genomic best linear unbiased prediction (GBLUP) were the least biased, and that method appears to be the best tool for genomic evaluation of a small population, as it automatically accounts for parental index and allows for inclusion of female genomic information without additional steps. None of the methods applied to the Israeli Holstein population were able to derive GEBV for young bulls that were significantly better than parent averages. Thus we confirm previous studies that the main limiting factor for the accuracy of GEBV is the number of bulls with genotypes and progeny tests. Although 36 of the grandsires included in the APGD were genotyped for the BovineHDBeadChip, which includes 777,000 SNPs, we were not able to determine the causative polymorphism for any of the detected QTL. The number of valid unique markers on the BovineHDBeadChip is not sufficient for a reasonable probability to find the causative polymorphisms. Complete resequencing of the genome of approximately 50 bulls will be required, but this could not be accomplished within the framework of the current project due to funding constraints. Inclusion of pedigree data from older generations in the derivation of GEBV may result is less accurate evaluations.

The objective of this report is to document the methodology used to calculate the three hydro-geomorphic indices: C Index, Nhot spot, and Interflow Contributing Area (IFC Area). These indices were applied in the Upper Four Mile Creek Watershed in order to better understand the potential mechanisms controlling retention time, path lengths, and potential for nutrient and solute metabolism and exchange associated with the geomorphic configurations of the upland contributing areas, groundwater, the riparian zone, and stream channels.

This final report presents the main activities and results of the project “A Carbon Flux Super Site: New Insights and Innovative Atmosphere-Terrestrial Carbon Exchange Measurements and Modeling” from 10/1/2006 to 9/30/2014. It describes the new AmeriFlux tower site (Aiken) at Savanna River Site (SC) and instrumentation, long term eddy-covariance, sodar, microbarograph, soil and other measurements at the site, and intensive field campaigns of tracer experiment at the Carbon Flux Super Site, SC, in 2009 and at ARM-CF site, Lamont, OK, and experiments in Plains, GA. The main results on tracer experiment and modeling, on low-level jet characteristics and their impact on fluxes, on gravity waves and their influence on eddy fluxes, and other results are briefly described in the report.

The specific objectives of this BARD proposal were: Use a comparative genomics approach to identify T3Es in group I, II and III strains of A. citrulli. Determine the bacterial genes contributing to host preference. Develop mutant strains that can be used for biological control of BFB. Background to the topic: Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovoraxcitrulli, is a devastating disease that affects watermelon (Citrulluslanatus) and melon (Cucumismelo) production worldwide, including both Israel and USA. Three major groups of A. citrullistrains have been classified based on their virulence on host plants, genetics and biochemical properties. The host selection could be one of the major factors that shape A. citrullivirulence. The differences in the repertoire of type III‐ secreted effectors (T3Es) among the three A. citrulligroups could play a major role in determining host preferential association. Currently, there are only 11 A. citrulliT3Es predicted by the annotation of the genome of the group II strain, AAC00‐1. We expect that new A. citrulliT3Es can be identified by a combination of bioinformatics and experimental approaches, which may help us to further define the relationship of T3Es and host preference of A. citrulli. Implications, both scientific and agricultural: Enriching the information on virulence and avirulence functions of T3Es will contribute to the understanding of basic aspects of A. citrulli‐cucurbit interactions. In the long term, it will contribute to the development of durable BFB resistance in commercial varieties. In the short term, identifying bacterial genes that contribute to virulence and host preference will allow the engineering of A. citrullimutants that can trigger SAR in a given host. If applied as seed treatments, these should significantly improve the effectiveness and efficacy of BFB management in melon and atermelon production.

The specific objectives of this BARD proposal were: (1) To determine whether Rxol can recognize AacavrRxo1 to trigger BFB disease resistance in stable transgenic watermelon plants. (2) To determine the distribution of Aac-avrRxo1 in a global population of Aae and to characterize the biological function of Aac-avrRxo1. (3) To characterize other TIS effectors of Aae and to identify plant R gene(s) that can recognize conserved TIS effectors of this pathogen. Background to the topic: Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax avenae subsp. citrulli (Aae), is a devastating disease that affects watermelon (Citrullus lanatus) and melon (Cucumis melo) production worldwide, including both Israel and USA. Two major groups of Aae strains have been classified based on their virulence on host plants, genetics and biochemical properties. Thus far, no effective resistance genes have been identified from cucurbit germplasm. In this project, we assessed the applicability of a non-host disease resistance gene, Rxol, to control BFB in watermelon. We also tried to identify Aae type III secreted (TIS) effectors that can be used as molecular probes to identify novel disease resistance genes in both cucurbits and Nieotianatabaeum. Major conclusions, solutions, achievements: We generated five independent transgenic watermelon (cv. Sugar Babay) plants expressing the Rxol gene. The transgenic plants were evaluated with Aae strains AAC001 and M6 under growth chamber conditions. All transgenic plants were found to be susceptible to both Aae strains. It is possible that watermelon is missing other signaling components that are required for Rxol-mediated disease resistance. In order to screen for novel BFB resistance genes, we inoculated two Aae strains on 60 Nieotiana species. Our disease assay revealed Nicotiana tabaeum is completely resistant to Aae, while its wild relative N. benthamiana is susceptible to Aae. We further demonstrated that Nieotiana benthamiana can be used as a surrogate host for studying the mechanisms of pathogenesis of Aae. We cloned 11 TIS effector genes including the avrRxolhomologues from the genomes of 22 Aae strains collected worldwide. Sequencing analysis revealed that functional avrRxol is conserved in group" but not group I Aae strains. Three effector genes- Aave_1548, Aave_2166 and Aave_2708- possessed the ability to trigger an HR response in N. tabacum when they were transiently expressed by Agrobaeterium. We conclude that N. tabacum carries at least three different non-host resistance genes that can specifically recognize AaeTIS effectors to trigger non-host resistance. Screening 522 cucurbits genotypes with two Aae strains led us to identify two germplasm (P1536473 and P1273650) that are partially resistant to Aae. Interestingly, transient expression of the TIS effector, Aave_1548, in the two germplasms also triggered HR-Iike cell death, which suggests the two lines may carry disease resistance genes that can recognize Aave_1548. Importantly, we also demonstrated that this effector contributes to the virulence of the bacterium in susceptible plants. Therefore, R genes that recognize effector Aave1548 have great potential for breeding for BFB resistance. To better understand the genome diversity of Aae strains, we generated a draft genome sequence of the Israeli Aae strain, M6 (Group I) using Iliumina technology. Comparative analysis of whole genomes of AAC001, and M6 allowed us to identify several effectors genes that differentiate groups I and II. Implications, both scientific and agricultural: The diversity of TIS effectors in group I and II strains of Aae suggests that a subset of effectors could contribute to the host range of group I and II Aae strains. Analysis of these key effectors in a larger Aae population may allow us to predict which cucurbit hosts may be at risk to BFB. Additionally, isolation of tobacco and cucurbit Rgenes that can recognize Aae type III effectors may offer new genetic resources for controlling BFB.

The avian influenza virus, subtype H9N2 subtype, defined as having a low pathogenicity, causes extensive economical losses in commercial flocks, probably due to management and synergism with other pathogens. AIV H9N2 was first identified in Israel in the year 2000, and since then it became endemic and widespread in Israel. Control by vaccination of commercial flocks with an inactivated vaccine has been introduced since 2007. In face of the continuous H9N2 outbreaks, and the application of the vaccination policy, we aimed in the present study to provide a method of differentiating naturally infected from vaccinated animals (DIVA). The aim of the assay would be detect only antibodies created by a de-novo infection, since the inactivated vaccine virus is not reproducing, and might provide a simple tool for mass detection of novel infections of commercial flocks. To fulfill the overall aim, the project was designed to include four operational objectives: 1. Evaluation of the genetic evolution of AIV in Israel; 2. Assessment of the diagnostic value of an NS1 ELISA; 3. NS1 ELISA as evaluation criteria for measuring the efficacy of vaccination against H9N2 AIV; 4. Development of an AIV H9 subtype specific ELISA systems. Major conclusion and implications drawn from the project were: 1. A continuous genetic change occurred in the collection of H9N2 isolates, and new introductions were identified. It was shown thatthe differences between the HA proteins of viruses used for vaccine productionand local fieldisolatesincreasedin parallelwith the durationand intensity ofvaccine use, therefore, developing a differential assay for the vaccine and the wild type viruses was the project main aim. 2. To assess the diagnostic value of an NS1 ELISA we first performed experimental infection trials using representative viruses of all introductions, and used the sera and recombinant NS1 antigens of the same viruses in homologous and heterologous NS1 ELISA combination. The NS1 ELISA was evidently reactive in all combinations, and did not discriminate significantly between different groups. 3. However, several major drawbacks of the NS1 ELISA were recognized: a) The evaluation of the vaccination effect in challenged birds, showed that the level of the NS1 antibodies dropped due to the vaccination-dependent virus level drop; b) the applicability of the NS1-ELISA was verified on sera of commercial flocks and found to be unusable due to physico-chemical composition of the sera and the recombinant antigen, c) commercial sera showed non-reactivity that might be caused by many factors, including vaccination, uncertainty regarding the infection time, and possibly low antigen avidity, d) NS1 elevated antibody levels for less than 2 months in SPF chicks. Due to the above mentioned reasons we do not recommend the application of the DIVA NS1 ELISA assay for monitoring and differentiation AIV H9N2 naturally-infected from vaccinated commercial birds.

We sought to improve our understanding of the metabolic and regulatory networks that control hydrogen (H2) production in the hyperthermophilic marine archaeon Pyrococcus furiosus (Pf), a model microbial H2 producer. We used an approach based on genomic and transcriptomic analyses of stress response along with genetic and biochemical characterization of identified pathways to further define control checkpoints in H2 production that will be valuable in future metabolic engineering efforts.

The involvement of glycans in microbial adherence, recognition and signaling is often a critical determinant of pathogenesis. Although the major glycan components of fungal cell walls have been identified there is limited information available on its ‘minor sugar components’ and how these change during different stages of fungal development. Our aim was to define the role of Rhacontaining-glycans in the gray mold disease caused by the necrotrophic fungus B. cinerea. The research was built on the discovery of two genes, Bcdhand bcer, that are involved in formation of UDP-KDG and UDP-Rha, two UDP- sugars that may serve as donors for the synthesis of cell surface glycans. Objectives of the proposed research included: 1) To determine the function of B. cinereaBcDh and BcEr in glycan biosynthesis and in pathogenesis, 2) To determine the expression pattern of BcDH and BcERand cellular localization of their encoded proteins, 3) Characterize the structure and distribution of Rha- containing glycans, 4) Characterization of the UDP-sugar enzymes and potential of GTs involved in glycanrhamnosylation. To address these objectives we generated a series of B. cinereamutants with modifications in the bchdhand bcergenes and the phenotype and sugar metabolism in the resulting strains were characterized. Analysis of sugar metabolites showed that changes in the genes caused changes in primary and secondary sugars, including abolishment of rhamnose, however abolishment of rhamnose synthesis did not cause changes in the fungal phenotype. In contrast, we found that deletion of the second gene, bcer, leads to accumulation of the intermediate sugar – UDP- KDG, and that such mutants suffer from a range of defects including reduced virulence. Further analyses confirmed that UDP-KDG is toxic to the fungus. Studies on mode of action suggested that UDP-KDG might affect integrity of the fungal cell wall, possibly by inhibiting UDP-sugars metabolic enzymes. Our results confirm that bcdhand bcerrepresent a single pathway of rhamnose synthesis in B. cinerea, that rhamnose does not affect in vitro development or virulence of the fungus. We also concluded that UDP-KDG is toxic to B. cinereaand hence UDP-KDG or compounds that inhibit Er enzymes and lead to accumulation of UDP-KDG might have antifungal activity. This toxicity is likely the case with other fungi, this became apparent in a collaborative work with Prof. Bart Thomma of Wageningen University, NETHERLANDS . We have shown the deletion of ER mutant in Verticillium dahlia gave plants resistance to the fungal infection.

Pod-filling, an important yield-determining stage is strongly influenced by water stress. This is particularly true for peanut (Arachishypogaea), wherein pods are developed underground and are directly affected by the water condition. Pod-filling in peanut has a significant genetic component as well, since genotypes are considerably varied in their pod-fill (PF) and seed-fill (SF) potential. The goals of this research were to: Examine the effects of genotype, irrigation, and genotype X irrigation on PF and SF. Detect global changes in mRNA and metabolites levels that accompany PF and SF. Explore the response of the duplicate peanut pod transcriptome to drought stress. Study how entire duplicated PF regulatory processes are networked within a polyploid organism. Discover locus-specific SNP markers and map pod quality traits under different environments. The research included genotypes and segregating populations from Israel and US that are varied in PF, SF and their tolerance to water deficit. Initially, an extensive field trial was conducted to investigate the effects of genotype, irrigation, and genotype X irrigation on PF and SF. Significant irrigation and genotypic effect was observed for the two main PF related traits, "seed ratio" and "dead-end ratio", demonstrating that reduction in irrigation directly influences the developing pods as a result of low water potential. Although the Irrigation × Genotype interaction was not statistically significant, one genotype (line 53) was found to be more sensitive to low irrigation treatments. Two RNAseq studies were simultaneously conducted in IL and the USA to characterize expression changes that accompany shell ("source") and seed ("sink") biogenesis in peanut. Both studies showed that SF and PF processes are very dynamic and undergo very rapid change in the accumulation of RNA, nutrients, and oil. Some genotypes differ in transcript accumulation rates, which can explain their difference in SF and PF potential; like cvHanoch that was found to be more enriched than line 53 in processes involving the generation of metabolites and energy at the beginning of seed development. Interestingly, an opposite situation was found in pericarp development, wherein rapid cell wall maturation processes were up-regulated in line 53. Although no significant effect was found for the irrigation level on seed transcriptome in general, and particularly on subgenomic assignment (that was found almost comparable to a 1:1 for A- and B- subgenomes), more specific homoeologous expression changes associated with particular biosynthesis pathways were found. For example, some significant A- and B- biases were observed in particular parts of the oil related gene expression network and several candidate genes with potential influence on oil content and SF were further examined. Substation achievement of the current program was the development and application of new SNP detection and mapping methods for peanut. Two major efforts on this direction were performed. In IL, a GBS approach was developed to map pod quality traits on Hanoch X 53 F2/F3 generations. Although the GBS approach was found to be less effective for our genetic system, it still succeeded to find significant mapping locations for several traits like testa color (linkage A10), number of seeds/pods (A5) and pod wart resistance (B7). In the USA, a SNP array was developed and applied for peanut, which is based on whole genome re-sequencing of 20 genotypes. This chip was used to map pod quality related traits in a Tifrunner x NC3033 RIL population. It was phenotyped for three years, including a new x-ray method to phenotype seed-fill and seed density. The total map size was 1229.7 cM with 1320 markers assigned. Based on this linkage map, 21 QTLs were identified for the traits 16/64 weight, kernel percentage, seed and pod weight, double pod and pod area. Collectively, this research serves as the first fundamental effort in peanut for understanding the PF and SF components, as a whole, and as influenced by the irrigation level. Results of the proposed study will also generate information and materials that will benefit peanut breeding by facilitating selection for reduced linkage drag during introgression of disease resistance traits into elite cultivars. BARD Report - Project4540 Page 2 of 10

Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.

In September 2006 an outbreak of 'Bluetongue like' disease struck the cattle herds in Israel. Over 100 dairy and beef cattle herds were affected. Epizootic hemorrhagic disease virus (EHDV) (an Orbivirusclosely related to bluetongue virus (BTV)), was isolated from samples collected from several herds during the outbreaks. Following are the aims of the study and summary of the results: which up until now were published in 6 articles in peer-reviewed journals. Three more articles are still under preparation: 1. To identify the origin of the virus: The virus identified was fully sequenced and compared with the sequences available in the GenBank. It appeared that while gene segment L2 was clustered with EHDV-7 isolated in Australia, most of the other segments were clustered with EHDV-6 isolates from South-Africa and Bahrain. This may suggest that the strain which affected Israel on 2006 may have been related to similar outbreaks which occurred in north-Africa at the same year and could also be a result of reassortment with an Australian strain (Wilson et al. article in preparation). Analysis of the serological results from Israel demonstrated that cows and calves were similarly positive as opposed to BTV for which seropositivity in cows was significantly higher than in calves. This finding also supports the hypothesis that the 2006 EHD outbreak in Israel was an incursive event and the virus was not present in Israel before this outbreak (Kedmi et al. Veterinary Journal, 2011) 2. To identify the vectors of this virus: In the US, Culicoides sonorensis was found as an efficient vector of EHDV as the virus was transmitted by midges fed on infected white tailed deer (WTD; Odocoileusvirginianus) to susceptible WTD (Ruder et al. Parasites and Vectors, 2012). We also examined the effect of temperature on replication of EHDV-7 in C. sonorensis and demonstrated that the time to detection of potentially competent midges decreased with increasing temperature (Ruder et al. in preparation). Although multiple attempts were made, we failed to evaluate wild-caught Culicoidesinsignisas a potential vector for EHDV-7; however, our finding that C. sonorensis is a competent vector is far more significant because this species is widespread in the U.S. As for Israeli Culicoides spp. the main species caught near farms affected during the outbreaks were C. imicolaand C. oxystoma. The vector competence studies performed in Israel were in a smaller scale than in the US due to lack of a laboratory colony of these species and due to lack of facilities to infect animals with vector borne diseases. However, we found both species to be susceptible for infection by EHDV. For C. oxystoma, 1/3 of the Culicoidesinfected were positive 11 days post feeding. 3. To identify the host and environmental factors influencing the level of exposure to EHDV, its spread and its associated morbidity: Analysis of the cattle morbidity in Israel showed that the disease resulted in an average loss of over 200 kg milk per cow in herds affected during September 2006 and 1.42% excess mortality in heavily infected herds (Kedmi et al. Journal of Dairy Science, 2010). Outbreak investigation showed that winds played a significant role in virus spread during the 2006 outbreak (Kedmi et al. Preventive Veterinary Medicine, 2010). Further studies showed that both sheep (Kedmi et al. Veterinary Microbiology, 2011) and wild ruminants did not play a significant role in virus spread in Israel (Kedmi et al. article in preparation). Clinical studies in WTD showed that this species is highly susceptibile to EHDV-7 infection and disease (Ruder et al. Journal of Wildlife Diseases, 2012). Experimental infection of Holstein cattle (cows and calves) yielded subclinical viremia (Ruder et al. in preparation). The findings of this study, which resulted in 6 articles, published in peer reviewed journals and 4 more articles which are in preparation, contributed to the dairy industry in Israel by defining the main factors associated with disease spread and assessment of disease impact. In the US, we demonstrated that sufficient conditions exist for potential virus establishment if EHDV-7 were introduced. The significant knowledge gained through this study will enable better decision making regarding prevention and control measures for EHDV and similar viruses, such as BTV.

Oroginal Objectives: (i) identify DNA markers linked to the avirulence (Avr) locus and locate the Avr locus through genetic mapping with an inter-race Orobanche cumana population; (ii) develop high-throughput fingerprint DNA markers for genotypingO. cumana races; (iii) identify nucleotide binding domain leucine rich repeat (NB-LRR) genes encoding R proteins conferring resistance to O. cumana in sunflower; (iv) increase the resolution of the chromosomal segment harboring Or₅ and related R genes through genetic and physical mapping in previously and newly developed mapping populations of sunflower; and (v) develop high-throughput DNA markers for rapidly and efficiently identifying and transferring sunflower R genes through marker-assisted selection. Revisions made during the course of project: Following changes in O. cumana race distribution in Israel, the newly arrived virulent race H was chosen for further analysis. HA412-HO, which was primarily chosen as a susceptible sunflower cultivar, was more resistant to the new parasite populations than var. Shemesh, thus we shifted sunflower research into analyzing the resistance of HA412-HO. We exceeded the deliverables for Objectives #3-5 by securing funding for complete physical and high-density genetic mapping of the sunflower genome, in addition to producing a complete draft sequence of the sunflower genome. We discovered limited diversity between the parents of the O. cumana population developed for the mapping study. Hence, the developed DNA marker resources were insufficient to support genetic map construction. This objective was beyond the scale and scope of the funding. This objective is challenging enough to be the entire focus of follow up studies. Background to the topic: O. cumana, an obligate parasitic weed, is one of the most economically important and damaging diseases of sunflower, causes significant yield losses in susceptible genotypes, and threatens production in Israel and many other countries. Breeding for resistance has been crucial for protecting sunflower from O. cumana, and problematic because new races of the pathogen continually emerge, necessitating discovery and deployment of new R genes. The process is challenging because of the uncertainty in identifying races in a genetically diverse parasite. Major conclusions, solutions, achievements: We developed a small collection of SSR markers for genetic mapping in O. cumana and completed a diversity study to lay the ground for objective #1. Because DNA sequencing and SNPgenotyping technology dramatically advanced during the course of the study, we recommend shifting future work to SNP discovery and mapping using array-based approaches, instead of SSR markers. We completed a pilot study using a 96-SNP array, but it was not large enough to support genetic mapping in O.cumana. The development of further SNPs was beyond the scope of the grant. However, the collection of SSR markers was ideal for genetic diversity analysis, which indicated that O. cumanapopulations in Israel considerably differ frompopulations in other Mediterranean countries. We supplied physical and genetic mapping resources for identifying R-genes in sunflower responsible for resistance to O. cumana. Several thousand mapped SNP markers and a complete draft of the sunflower genome sequence are powerful tools for identifying additional candidate genes and understanding the genomic architecture of O. cumana-resistanceanddisease-resistance genes. Implications: The OrobancheSSR markers have utility in sunflower breeding and genetics programs, as well as a tool for understanding the heterogeneity of races in the field and for geographically mapping of pathotypes.The segregating populations of both Orobanche and sunflower hybrids are now available for QTL analyses.

Control of Avian Influenza (AI) infection is a highly topical subject of major economicimportance for the worldwide poultry industry at the national level and for international trade.H9N2 viruses are endemic in poultry throughout Asia and the Middle East, causing major losses inproduction. Moreover, these viruses pose wider threats since they have been isolated from bothswine and humans. At the same time, study of the AI viruses affords an opportunity to explore anumber of problems of intriguing scientific interest. The overall goal of this project was to developa sound control strategy for avian influenza subtype H9N2 viruses (AI H9N2) in commercialpoultry in Israel. The one-year feasibility study focused on two main goals, namely: to study themolecular characteristics of AI H9N2 circulating during the last seven years in Israel and todevelop tools enabling differentiation between the immune response to vaccination and infectionwith H9N2.Genetic and phylogenetic characterization of 29 selected AI H9N2 isolates (2000-2006)was performed by complete sequencing of hemagglutinin (HA), neuraminidase (NA), and all sixinternal genes [nucleoprotein (NP), polymerase basic 1 (PB1), polymerase basic 2 (PB2),polymerase acid (PA), matrix (M), and nonstructural (NS) genes]; comparative phylogenetic andgenetic analyses of these sequences; and comparative genetic analyses of deduced amino acidsequences of the HA, NA, NS1, and NS2 proteins. The major conclusions of the molecularanalyses were: (1) Israeli isolates, together with other H9N2 viruses isolated in Middle Eastcountries, comprise a single regional sublineage related to the G1-lineage. In addition, Israeliisolates subdivided into three different subgroups. Genetic analysis of these viruses suggests thatthey underwent divergent evolution paths.

The research problem. Understanding of insect reproduction has been critical to the design of insect pest control strategies including disruptions of mate-finding, courtship and sperm transfer by male insects. It is well known that males transfer proteins to females during mating that profoundly affect female reproductive physiology, but little is known about the molecular basis of female mating response and no attempts have yet been made to interfere with female post-mating responses that directly bear on the efficacy of fertilization. The female reproductive tract provides a crucial environment for the events of fertilization yet thus far those events and the role of the female tract in influencing them are poorly understood. For this project, we have chosen to focus on the lower reproductive tract because it is the site of two processes critical to reproduction: sperm management (storage, maintenance, and release from storage) and fertilization. E,fforts during this project period centered on the elucidation of mating responses in the female lower reproductive tract The central goals of this project were: 1. To identify mating-responsive genes in the female lower reproductive tract using DNA microarray technology. 2. In parallel, to identify mating-responsive genes in these tissues using proteomic assays (2D gels and LC-MS/MS techniques). 3. To integrate proteomic and genomic analyses of reproductive tract gene expression to identify significant genes for functional analysis. Our main achievements were: 1. Identification of mating-responsive genes in the female lower reproductive tract. We identified 539 mating-responsive genes using genomic and proteomic approaches. This analysis revealed a shift from gene silencing to gene activation soon after mating and a peak in differential gene expression at 6 hours post-mating. In addition, comparison of the two datasets revealed an expression pattern consistent with the model that important reproductive proteins are pre-programmed for synthesis prior to mating. This work was published in Mack et al. (2006). Validation experiments using real-time PCR techniques suggest that microarray assays provide a conservativestimate of the true transcriptional activity in reproductive tissues. 2.lntegration of proteomics and genomics data sets. We compared the expression profiles from DNA microarray data with the proteins identified in our proteomic experiments. Although comparing the two data sets poses analyical challenges, it provides a more complete view of gene expression as well as insights into how specific genes may be regulated. This work was published in Mack et al. (2006). 3. Development of primary reproductive tract cell cultures. We developed primary cell cultures of dispersed reproductive tract cell types and determined conditions for organ culture of the entire reproductive tract. This work will allow us to rapidly screen mating-responsive genes for a variety of reproductive-tract specifi c functions. Scientific and agricultural significance. Together, these studies have defined the genetic response to mating in a part of the female reproductive tract that is critical for successful fertllization and have identified alarge set of mating-responsive genes. This work is the first to combine both genomic and proteomic approaches in determining female mating response in these tissues and has provided important insights into insect reproductive behavior.