Draft Genome Sequence of a Washington Isolate of "Candidatus Phytoplasma pruni".

Illumina sequencing of a Prunus avium tree with X-disease symptoms was performed to obtain a draft genome of "Candidatus Phytoplasma pruni." The genome consists of 14 contigs covering 588,767 bp. This is the first metagenome to be sequenced from the current X-disease epidemic in stone fruit in the Pacific Northwest.

Improved Detection of Little Cherry Virus-2 Using a Hydrolysis Probe to Manage the Pacific Northwest Little Cherry Disease Epidemic.

Little cherry virus-2 (LChV-2) is a viral pathogen that is reaching epidemic levels in Washington State. This virus is insect vectored and has significant impacts on sweet cherry production. To aid growers in making informed management decisions, we sought to develop a diagnostic assay to better detect isolates of LChV-2 currently found in Washington, allowing more accurate estimations of disease occurrence. This study showed that there were two distinct genotypes of LChV-2 present in Washington State. This information was used to develop an up-to-date reverse transcription real-time quantitative PCR assay, which was then optimized, validated, and compared with four previously published assays of a panel of field samples. This comparison demonstrated that the newly developed assay provided greater sensitivity, accurately detecting <10 copies per reaction and could detect both LChV-2 genotypes. Finally, we examined the effect of potential inhibitors in various tissue types from cherry, finding that young leaf tissue affected sensitivity of detection less than root tissues.

Titer and distribution of little cherry virus 2 in Prunus avium.

Little cherry virus 2 (LChV-2) is a causal agent of little cherry disease, which produces small, misshapen fruit with poor color and taste. As LChV-2 symptoms are only present near harvest, molecular detection is essential for effective control. Therefore, we determined the titer and distribution of this virus in infected trees over time. While initial infections were found to be basipetal, in field trees, early-stage infection was characterized by uneven distribution and low titer, concentrated in woody stems. In contrast, established infections were systemic, and detection was consistent across tissues. These data provide improved sampling recommendations for the detection of LChV-2.

A bushel of viruses: Identification of seventeen novel putative viruses by RNA-seq in six apple trees.

Apple decline in Washington state has been increasing in incidence, particularly on Honeycrisp trees grown on G.935 rootstock. In this disease the trees exhibit dieback with necrosis at the graft union and in the rootstock. The cause of this disease remains unknown. To identify viral candidates, RNA-seq was performed on six trees: four trees exhibiting decline and two healthy trees. Across the samples, eight known viruses and Apple hammerhead viroid were detected, however none appear to be specifically associated with the disease. A BLASTx analysis of the RNA-seq data was performed to identify novel viruses that might be associated with apple decline. Seventeen novel putative viruses were detected, including an ilarvirus, two tombus-like viruses, a barna-like virus, a picorna-like virus, three ourmia-like viruses, three partiti-like viruses, and two narna-like viruses. Four additional viruses could not be classified. Three of the viruses appeared to be missing key genes, suggesting they may be dependent upon helper viruses for their function. Others showed a specific tropism, being detected only in the roots or only in the leaves. While, like the known apple viruses, none were consistently associated with diseased trees, it is possible these viruses may have a synergistic effect when co-infecting that could contribute to disease. Or the presence of these viruses may weaken the trees for some other factor that ultimately causes decline. Additional research will be needed to determine how these novel viruses contribute to apple decline.

Survey of the genomic landscape surrounding the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant Amaranthus palmeri from geographically distant populations in the USA.

BACKGROUND: Glyphosate resistance in Amaranthus palmeri, one of the most prevalent herbicide-resistant weeds in the USA, is attributable to amplification and increased expression of the gene encoding the target site of glyphosate, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The EPSPS gene and the surrounding 287 kilobases (kb) of amplified sequence are unique to glyphosate-resistant plants and termed the EPSPS cassette. It has only been sequenced in one A. palmeri population from Mississippi. This research compares EPSPS cassettes in seven resistant and five sensitive populations from geographically distant locations within the USA, including Mississippi, Arizona, Kansas, Maryland, Delaware and Georgia. RESULTS: Polymerase chain reaction (PCR) products from 40 primer pairs specific to the cassette were similar in size and sequence in resistant populations. Several primer pairs failed to generate PCR products in sensitive populations. Regions of the cassette sequenced in the resistant populations were found to be nearly identical to those from Mississippi. Gene expression analysis showed that both EPSPS and another gene in the cassette, a reverse transcriptase, were elevated in all resistant populations tested relative to the sensitive populations. CONCLUSION: EPSPS cassettes from distant resistant populations were nearly homologous. Considering the complexity of the cassette, and the degree of similarity among some cassette sequences, the results are consistent with the hypothesis that glyphosate resistance probably evolved once and then rapidly spread across the USA. © 2017 Society of Chemical Industry.

Transcriptomic changes in Echinochloa colona in response to treatment with the herbicide imazamox.

MAIN CONCLUSION: Presented here is the first Echinochloa colona leaf transcriptome. Analysis of gene expression before and after herbicide treatment reveals that E. colona mounts a stress response upon exposure to herbicide. Herbicides are the most frequently used means of controlling weeds. For many herbicides, the target site is known; however, it is considerably less clear how plant gene expression changes in response to herbicide exposure. In this study, changes in gene expression in response to herbicide exposure in imazamox-sensitive (S) and- resistant (R) junglerice (Echinochloa colona L.) biotypes was examined. As no reference genome is available for this weed, a reference leaf transcriptome was generated. Messenger RNA was isolated from imazamox-treated- and untreated R and S plants and the resulting cDNA libraries were sequenced on an Illumina HiSeq2000. The transcriptome was assembled, annotated, and differential gene expression analysis was performed to identify transcripts that were upregulated or downregulated in response to herbicide exposure for both biotypes. Differentially expressed transcripts included transcription factors, protein-modifying enzymes, and enzymes involved in metabolism and signaling. A literature search revealed that members of the families represented in this analysis were known to be involved in abiotic stress response in other plants, suggesting that imazamox exposure induced a stress response. A time course study examining a subset of transcripts showed that expression peaked within 4-12 h and then returned to untreated levels within 48 h of exposure. Testing of plants from two additional biotypes showed a similar change in gene expression 4 h after herbicide exposure compared to the resistant and sensitive biotypes. This study shows that within 48 h junglerice mounts a stress response to imazamox exposure.

The unique genomic landscape surrounding the EPSPS gene in glyphosate resistant Amaranthus palmeri: a repetitive path to resistance.

BACKGROUND: The expanding number and global distributions of herbicide resistant weedy species threaten food, fuel, fiber and bioproduct sustainability and agroecosystem longevity. Amongst the most competitive weeds, Amaranthus palmeri S. Wats has rapidly evolved resistance to glyphosate primarily through massive amplification and insertion of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene across the genome. Increased EPSPS gene copy numbers results in higher titers of the EPSPS enzyme, the target of glyphosate, and confers resistance to glyphosate treatment. To understand the genomic unit and mechanism of EPSPS gene copy number proliferation, we developed and used a bacterial artificial chromosome (BAC) library from a highly resistant biotype to sequence the local genomic landscape flanking the EPSPS gene. RESULTS: By sequencing overlapping BACs, a 297 kb sequence was generated, hereafter referred to as the "EPSPS cassette." This region included several putative genes, dense clusters of tandem and inverted repeats, putative helitron and autonomous replication sequences, and regulatory elements. Whole genome shotgun sequencing (WGS) of two biotypes exhibiting high and no resistance to glyphosate was performed to compare genomic representation across the EPSPS cassette. Mapping of sequences for both biotypes to the reference EPSPS cassette revealed significant differences in upstream and downstream sequences relative to EPSPS with regard to both repetitive units and coding content between these biotypes. The differences in sequence may have resulted from a compounded-building mechanism such as repetitive transpositional events. The association of putative helitron sequences with the cassette suggests a possible amplification and distribution mechanism. Flow cytometry revealed that the EPSPS cassette added measurable genomic content. CONCLUSIONS: The adoption of glyphosate resistant cropping systems in major crops such as corn, soybean, cotton and canola coupled with excessive use of glyphosate herbicide has led to evolved glyphosate resistance in several important weeds. In Amaranthus palmeri, the amplification of the EPSPS cassette, characterized by a complex array of repetitive elements and putative helitron sequences, suggests an adaptive structural genomic mechanism that drives amplification and distribution around the genome. The added genomic content not found in glyphosate sensitive plants may be driving evolution through genome expansion.

Distinguishing between weedy Amaranthus species based on intron 1 sequences from the 5-enolpyruvylshikimate-3-phosphate synthase gene.

BACKGROUND: Hybridization between Amaranthus species and the potential for herbicide resistance to be transferred by hybridization are of growing concern in the weed science community. Early detection of evolved herbicide resistance and hybrids expressing resistance to single or multiple herbicides is important to develop an effective control strategy. RESULTS: A PCR test was developed for quick identification of weedy amaranths and any hybrids. The sequences of intron 1 for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) gene were determined for Amaranthus palmeri, A. spinosus, A. retroflexus, A. blitoides, A. viridis, A. tuberculatus and A. hybridus. These sequences were aligned and primers were developed in areas where the sequence differed between species. Species-specific primers and cycle conditions were successfully developed. These primers produce a single robust band only for the species for which they were designed. CONCLUSION: The PCR techniques described here allow identification of a weedy amaranth or suspect hybrid in a few hours. Using a similar target, it may be possible to design simple PCR tests to identify even more difficult to distinguish weed species or weeds prone to interspecific hybridization. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

EPSPS amplification in glyphosate-resistant spiny amaranth (Amaranthus spinosus): a case of gene transfer via interspecific hybridization from glyphosate-resistant Palmer amaranth (Amaranthus palmeri).

BACKGROUND: Amaranthus spinosus, a common weed of pastures, is a close relative of Amaranthus palmeri, a problematic agricultural weed with widespread glyphosate resistance. These two species have been known to hybridize, allowing for transfer of glyphosate resistance. Glyphosate-resistant A. spinosus was recently suspected in a cotton field in Mississippi. RESULTS: Glyphosate-resistant A. spinosus biotypes exhibited a fivefold increase in resistance compared with a glyphosate-susceptible biotype. EPSPS was amplified 33-37 times and expressed 37 times more in glyphosate-resistant A. spinosus biotypes than in a susceptible biotype. The EPSPS sequence in resistant A. spinosus plants was identical to the EPSPS in glyphosate-resistant A. palmeri, but differed at 29 nucleotides from the EPSPS in susceptible A. spinosus plants. PCR analysis revealed similarities between the glyphosate-resistant A. palmeri amplicon and glyphosate-resistant A. spinosus. CONCLUSIONS: Glyphosate resistance in A. spinosus is caused by amplification of the EPSPS gene. Evidence suggests that part of the EPSPS amplicon from resistant A. palmeri is present in glyphosate-resistant A. spinosus. This is likely due to a hybridization event between A. spinosus and glyphosate-resistant A. palmeri somewhere in the lineage of the glyphosate-resistant A. spinosus plants. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Identification of genetic elements associated with EPSPs gene amplification.

Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world's most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S) A. palmeri, and that only one of these was amplified in glyphosate-resistant (R) A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs) were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac) transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene.