Winter rye cover crops shelter competent squash phyllosphere bacteria to reduce Pseudomonas syringae pv. lachrymans growth and angular leaf spot symptoms.

Cover crops, a soil conservation practice, can contribute to reducing disease pressure caused by Pseudomonas syringae, considered one of the most important bacterial plant pathogens. We recently demonstrated that phyllosphere (leaf surface) bacterial community structure changed when squash (Cucurbita pepo) was grown with a rye (Secale cereale) cover crop treatment, followed by a decrease of angular leaf spot (ALS) disease symptoms on squash caused by P. syringae pv. lachrymans. Application of biocontrol agents is a known agricultural practice to mitigate crop losses due to microbial disease. In this study, we tested the hypothesis that some phyllosphere bacteria promoted when squash are grown on cover crops could be isolated and used as a biocontrol agent to decrease ALS symptoms. We grew squash during a two-year field experiment using four agricultural practices: bare soil, cover crops, chemically terminated cover crops, and plastic cover. We sampled squash leaves at 3 different dates each year and constructed a collection of cultivable bacterial strains isolated from squash leaves and rye cover crop material. Each isolated strain was identified by 16S rRNA gene sequencing and used in in vitro (Petri dish) pathogen growth and in vivo (greenhouse) symptom control assays. Four bacterial isolates belonging to the genera Pseudarthrobacter, Pseudomonas, Delftia and Rhizobium were shown to inhibit P. syringae pv. lachrymans growth and ALS symptom development. Strikingly, the symptom control efficacy of all strains was stronger on older leaves. This study sheds light on the importance of bacterial isolation from cover crops sources to promote disease control.

The ancestral karyotype of the Heliantheae Alliance, herbicide resistance, and human allergens: Insights from the genomes of common and giant ragweed.

Ambrosia artemisiifolia and Ambrosia trifida (Asteraceae) are important pest species and the two greatest sources of aeroallergens globally. Here, we took advantage of a hybrid to simplify genome assembly and present chromosome-level assemblies for both species. These assemblies show high levels of completeness with Benchmarking Universal Single-Copy Ortholog (BUSCO) scores of 94.5% for A. artemisiifolia and 96.1% for A. trifida and long terminal repeat (LTR) Assembly Index values of 26.6 and 23.6, respectively. The genomes were annotated using RNA data identifying 41,642 genes in A. artemisiifolia and 50,203 in A. trifida. More than half of the genome is composed of repetitive elements, with 62% in A. artemisiifolia and 69% in A. trifida. Single copies of herbicide resistance-associated genes PPX2L, HPPD, and ALS were found, while two copies of the EPSPS gene were identified; this latter observation may reveal a possible mechanism of resistance to the herbicide glyphosate. Ten of the 12 main allergenicity genes were also localized, some forming clusters with several copies, especially in A. artemisiifolia. The evolution of genome structure has differed among these two species. The genome of A. trifida has undergone greater rearrangement, possibly the result of chromoplexy. In contrast, the genome of A. artemisiifolia retains a structure that makes the allotetraploidization of the most recent common ancestor of the Heliantheae Alliance the clearest feature of its genome. When compared to other Heliantheae Alliance species, this allowed us to reconstruct the common ancestor's karyotype-a key step for furthering of our understanding of the evolution and diversification of this economically and allergenically important group.

Distribution and genetic characterization of bird rape mustard (Brassica rapa) populations and analysis of glyphosate resistance introgression.

BACKGROUND: The introgression of a transgene conferring glyphosate resistance from Brassica napus (rapeseed, canola) to Brassica rapa weeds (bird rape) was documented at a single location in 2007. In 2015, several cases of glyphosate resistant mustard were reported by growers in areas where rapeseed was seldom grown. RESULTS: Survey result indicated glyphosate resistant bird rape mustard is present in areas where glyphosate tolerant corn and soybean are often grown in rotation. Genetic analyses reveal that hybridization followed by introgression and progressive loss of chromosome is the likely mechanism for the horizontal gene transfer (HGT) of glyphosate resistance. CONCLUSION: Introgression of the glyphosate-resistance conferring transgene in the populations studied appears to have occurred several times, consistent with the ease for B. rapa to form hybrids with B. napus. The introduction of a transgene into a crop should therefore take into account the weediness of the species that share a common genome and their ability to form hybrids. We provide here such an example between B. napus and B. rapa, and potentially between B. napus and Raphanistrum raphanistrum. © 2022 Her Majesty the Queen in Right of Canada. Pest Management Science © 2022 Society of Chemical Industry. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

Insights from the genomes of 4 diploid Camelina spp.

Plant evolution has been a complex process involving hybridization and polyploidization making understanding the origin and evolution of a plant's genome challenging even once a published genome is available. The oilseed crop, Camelina sativa (Brassicaceae), has a fully sequenced allohexaploid genome with 3 unknown ancestors. To better understand which extant species best represent the ancestral genomes that contributed to C. sativa's formation, we sequenced and assembled chromosome level draft genomes for 4 diploid members of Camelina: C. neglecta C. hispida var. hispida, C. hispida var. grandiflora, and C. laxa using long and short read data scaffolded with proximity data. We then conducted phylogenetic analyses on regions of synteny and on genes described for Arabidopsis thaliana, from across each nuclear genome and the chloroplasts to examine evolutionary relationships within Camelina and Camelineae. We conclude that C. neglecta is closely related to C. sativa's sub-genome 1 and that C. hispida var. hispida and C. hispida var. grandiflora are most closely related to C. sativa's sub-genome 3. Further, the abundance and density of transposable elements, specifically Helitrons, suggest that the progenitor genome that contributed C. sativa's sub-genome 3 maybe more similar to the genome of C. hispida var. hispida than that of C. hispida var. grandiflora. These diploid genomes show few structural differences when compared to C. sativa's genome indicating little change to chromosome structure following allopolyploidization. This work also indicates that C. neglecta and C. hispida are important resources for understanding the genetics of C. sativa and potential resources for crop improvement.

Differential expression of genes associated with non-target site resistance in Poa annua with target site resistance to acetolactate synthase inhibitors.

BACKGROUND: Poa annua is a pervasive grassy, self-pollinating, weed that has evolved resistance to 10 different herbicide modes-of-action, third most of all weed species. We investigated constitutive overexpression of genes associated with non-target site resistance (NTSR) in POAAN-R3 and the response of those genes when treated with trifloxysulfuron despite the biotype having a known target site mutation in acetolactate synthase (ALS). RESULTS: Despite having an ALS target site mutation, POAAN-R3 still had a transcriptomic response to herbicide application that differed from a susceptible biotype. We observed differential expression of genes associated with transmembrane transport and oxidation-reduction activities, with differences being most pronounced prior to herbicide treatment. CONCLUSIONS: In the P. annua biotype we studied with confirmed target site resistance to ALS inhibitors, we also observed constitutive expression of genes regulating transmembrane transport, as well as differential expression of genes associated with oxidative stress after treatment with trifloxysulfuron. This accumulation of mechanisms, in addition to the manifestation of target site resistance, could potentially increase the chance of survival when plants are challenged by different modes of action.

Patients who failed to conceive following an in vitro fertilization cycle can be clustered into different failure causes using gene expression hierarchical analysis†.

The patient's response to an IVF stimulation protocol is highly variable and thus difficult to predict. When a cycle fails, there are often no apparent or obvious reasons to explain the failure. Having clues on what went wrong during stimulation could serve as a basis to improve and personalize the next protocol. This exploratory study aimed to investigate if it is possible to distinguish different failure causes or different follicular responses in a population of nonpregnant IVF patients. Using qRT-PCR, we analyzed a panel of genes indicative of different failure causes in patients who did not achieve pregnancy following an IVF cycle. For each patient, a pool of follicular cells from all aspirated follicles was used as a sample which gives a global picture of the patient's ovary and not a specific picture of each follicle. We performed hierarchical clustering analysis to split the patients according to the gene expression pattern. Hierarchical analysis showed that the population of nonpregnant IVF patients could be divided into three clusters. Gene expression was significantly different, and each cluster displayed a particular gene expression pattern. Follicular cells from patients in clusters 1, 2 and 3 displayed respectively a pattern of gene expression related to large incompetent follicles with a higher apoptosis (over matured), to follicles not ready to ovulate (under mature) and to an excess of inflammation with no visible symptoms. This study reinforces the idea that women often have different response to the same protocol and would benefit from more personalized treatments.

A chromosome-scale draft sequence of the Canada fleabane genome.

BACKGROUND: Due to the accessibility of underlying technologies the 'Omics', in particular genomics, are becoming commonplace in several fields of research, including the study of agricultural pests. The weed community is starting to embrace these approaches; genome sequences have been made available in the past years, with several other sequencing projects underway, as promoted by the International Weed Genome Consortium. Chromosome-scale sequences are essential to fully exploit the power of genetics and genomics. RESULTS: We report such an assembly for Conyza canadensis, an important agricultural weed. Third-generation sequencing technology was used to create a genome assembly of 426 megabases, of which nine chromosome-scale scaffolds cover more than 98% of the entire assembled sequence. As this weed was the first to be identified with glyphosate resistance, and since we do not have a firm handle on the genetic mechanisms responsible for several herbicide resistances in the species, the genome sequence was annotated with genes known to be associated with herbicide resistance. A high number of ABC-type transporters, cytochrome P450 and glycosyltransferases (159, 352 and 181, respectively) were identified among the list of ab initio predicted genes. CONCLUSION: As C. canadensis has a small genome that is syntenic with other Asteraceaes, has a short life cycle and is relatively easy to cross, it has the potential to become a model weed species and, with the chromosome-scale genome sequence, contribute to a paradigm shift in the way non-target site resistance is studied. © 2020 Her Majesty the Queen in Right of CanadaPest Management Science © 2020 Society of Chemical Industry.

Population Genomic Approaches for Weed Science.

Genomic approaches are opening avenues for understanding all aspects of biological life, especially as they begin to be applied to multiple individuals and populations. However, these approaches typically depend on the availability of a sequenced genome for the species of interest. While the number of genomes being sequenced is exploding, one group that has lagged behind are weeds. Although the power of genomic approaches for weed science has been recognized, what is needed to implement these approaches is unfamiliar to many weed scientists. In this review we attempt to address this problem by providing a primer on genome sequencing and provide examples of how genomics can help answer key questions in weed science such as: (1) Where do agricultural weeds come from; (2) what genes underlie herbicide resistance; and, more speculatively, (3) can we alter weed populations to make them easier to control? This review is intended as an introduction to orient weed scientists who are thinking about initiating genome sequencing projects to better understand weed populations, to highlight recent publications that illustrate the potential for these methods, and to provide direction to key tools and literature that will facilitate the development and execution of weed genomic projects.

Rapid identification of Erwinia amylovora and Pseudomonas syringae species and characterization of E. amylovora streptomycin resistance using quantitative PCR assays.

Erwinia amylovora and Pseudomonas syringae are bacterial phytopathogens responsible for considerable yield losses in commercial pome fruit production. The pathogens, if left untreated, can compromise tree health and economically impact entire commercial fruit productions. Historically, the choice of effective control methods has been limited. The use of antibiotics was proposed as an effective control method. The identification of these pathogens and screening for the presence of antibiotic resistance is paramount in the adoption and implementation of disease control methods. Molecular tests have been developed and accepted for identification and characterization of these disease-causing organisms. We improved existing molecular tests by developing methods that are equal or superior in robustness for identifying E. amylovora or P. syringae while being faster to execute. In addition, the real-time PCR-based detection method for E. amylovora provided complementary information on the susceptibility or resistance to streptomycin of individual isolates. Finally, we describe a methodology and results that compare the aggressiveness of the different bacterial isolates on four apple cultivars. We show that bacterial isolates exhibit different behaviors when brought into contact with various apple varieties and that the hierarchical clustering of symptom severity indicates a population structure, suggesting a genetic basis for host cultivar specificity.

Acetyl-CoA carboxylase overexpression in herbicide-resistant large crabgrass (Digitaria sanguinalis).

BACKGROUND: The occurrence of herbicide-resistant weed biotypes is increasing and this report of an acetyl-CoA carboxylase (ACCase) inhibitor-resistant Digitaria sanguinalis L. Scop. from southwestern Ontario is another example. The identified weed escaped control in an onion and carrot rotation in which graminicides were used for several consecutive years. Our goal was to characterize the level and mechanism of resistance of the biotype. RESULTS: The biotype was resistant to all five ACCase inhibitor herbicides tested. Gene-expression profiling was performed because none of the mutations known to confer resistance in the ACCase gene were detected. RNASeq and quantitative reverse-transcriptase PCR (qRT-PCR) results indicated that transcription of ACCase was 3.4-9.3 times higher in the resistant biotype than the susceptible biotype. ACCase gene copy number was determined by qPCR to be five to seven times higher in the resistant compared with the susceptible biotype. ACCase gene overexpression was directly related to the increase of the ACCase gene copy number. CONCLUSION: Our results are consistent with the hypothesis that overexpression of the herbicide target gene ACCase confers resistance to the herbicide. This is the first reported case of target gene duplication conferring resistance to a herbicide other than glyphosate. © 2017 Society of Chemical Industry See related Article.

A new amino acid substitution (Ala-205-Phe) in acetolactate synthase (ALS) confers broad spectrum resistance to ALS-inhibiting herbicides.

MAIN CONCLUSION: This is a first report of an Ala-205-Phe substitution in acetolactate synthase conferring resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl-triazolinones, and pyrimidinyl (thio) benzoate herbicides. Resistance to acetolactate synthase (ALS) and photosystem II inhibiting herbicides was confirmed in a population of allotetraploid annual bluegrass (Poa annua L.; POAAN-R3) selected from golf course turf in Tennessee. Genetic sequencing revealed that seven of eight POAAN-R3 plants had a point mutation in the psbA gene resulting in a known Ser-264-Gly substitution on the D1 protein. Whole plant testing confirmed that this substitution conferred resistance to simazine in POAAN-R3. Two homeologous forms of the ALS gene (ALSa and ALSb) were detected and expressed in all POAAN-R3 plants sequenced. The seven plants possessing the Ser-264-Gly mutation conferring resistance to simazine also had a homozygous Ala-205-Phe substitution on ALSb, caused by two nucleic acid substitutions in one codon. In vitro ALS activity assays with recombinant protein and whole plant testing confirmed that this Ala-205-Phe substitution conferred resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl- triazolinones, and pyrimidinyl (thio) benzoate herbicides. This is the first report of Ala-205-Phe mutation conferring wide spectrum resistance to ALS inhibiting herbicides.

Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian physiology and to the development of new oocyte culture medium.

High-resolution mapping of the Brassica napus Rfp restorer locus using Arabidopsis-derived molecular markers.

The two forms of cytoplasmic male sterility (CMS) native to the oilseed rape or canola species Brassica napus, nap and pol, have novel features that may provide insight into the molecular mechanisms through which CMS/nuclear restorer systems evolve. One such feature is the finding that the distinct nuclear restorer genes for the two systems represent different alleles or haplotypes of the same nuclear locus. Improved understanding of how these systems have evolved will require molecular cloning and characterization of this novel locus. We have employed an approach that exploits the regional co-linearity between the Arabidopsis and Brassica genomes to construct a high-resolution genetic map of the nuclear restorer for the pol system, Rfp. Specifically, Arabidopsis-derived sequences have been used as a set of ordered RFLP probes to localize Rfp to a region of the B. napus genome equivalent to a 115 kb interval on Arabidopsis chromosome 1. Based on the known relationship of physical distances between orthologous segments of Arabidopsis and Brassica chromosomes, it is anticipated that the B. napus restorer locus is now mapped to sufficient resolution to permit its isolation and characterization.

Symmetrized characterization of noisy quantum processes.

A major goal of developing high-precision control of many-body quantum systems is to realize their potential as quantum computers. A substantial obstacle to this is the extreme fragility of quantum systems to "decoherence" from environmental noise and other control limitations. Although quantum computation is possible if the noise affecting the quantum system satisfies certain conditions, existing methods for noise characterization are intractable for present multibody systems. We introduce a technique based on symmetrization that enables direct experimental measurement of some key properties of the decoherence affecting a quantum system. Our method reduces the number of experiments required from exponential to polynomial in the number of subsystems. The technique is demonstrated for the optimization of control over nuclear spins in the solid state.

Adenosine 5'-monophosphate kinase-activated protein kinase (PRKA) activators delay meiotic resumption in porcine oocytes.

Adenosine monophosphate-activated kinase (PRKA) is a serine/threonine kinase that functions as a metabolic switch in a number of physiological functions. The present study was undertaken to assess the role of this kinase in nuclear maturation of porcine oocytes. RT-PCR and immunoblotting revealed the expression of the PRKAA1 subunit in granulosa cells, cumulus-oocyte complexes (COC), and denuded oocytes (DO). Porcine COC and DO contained transcripts that corresponded to the expected sizes of the designed primers for PRKAB1 and PRKAG1. The PRKAA2 subunit was detected in granulosa cells and COC, whereas the PRKAG3 subunit was not detected in granulosa cells, COC or DO, whereas it was detected in the heart. The PRKAA1 protein was detected in granulosa cells, COC, DO, and zona pellucida (ZP). In the presence of the pharmacological activator of PRKA 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP), COC were transiently maintained in meiotic arrest in a fully reversible manner. This inhibitory effect was not observed in DO. Other known PRKA activators, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and metformin, also blocked meiotic resumption in COC. In contrast to mouse oocytes, in which PRKA activators reverse the inhibitory effect of PDE3 inhibitors, this combination still blocked meiotic resumption in porcine COC. These results demonstrate that the meiotic resumption of porcine COC is transiently blocked by PRKA activators in a dose-dependent manner, and that this effect is dependent on PRKA activity in cumulus cells. The present study describes a new role for PRKA in regulating meiotic resumption in COC and strongly suggests that cumulus cells play an essential role in the control of porcine oocyte maturation through the PRKA metabolic switch.

Fundamental significance of specific phosphodiesterases in the control of spontaneous meiotic resumption in porcine oocytes.

The meiosis of mammalian oocytes begins during the fetal life and stops at the dictyate stage. This study has assessed the role of specific phosphodiesterase (PDE) inhibitors on the control of meiotic resumption in porcine oocytes investigating the influence of PMSG-hCG and cAMP stimulation. Cumulus-oocytes complexes (COCs) and denuded oocytes (DOs) were collected from gilt ovaries obtained at a local slaughterhouse. Oocytes were cultured in NCSU23 with different PDE inhibitors. The EC(50) for oocytes maintained in germinal vesicle (GV) stage was evaluated using different doses of both cilostamide (CIL), PDE3 inhibitor and 3-isobutyl-1-methylxanthine (IBMX), a nonspecific PDE inhibitor. In presence of PMSG-hCG, meiotic resumption is observed after 24 hr of culture. Both CIL and IBMX reversibly blocked meiotic resumption. In absence of PMSG-hCG, meiotic resumption is reduced after 24 hr of culture. After 48 hr of culture, only CIL significantly blocked meiotic resumption. Still in absence of PMSG-hCG, significant effect of treatment was only observed in COCs using the combination of CIL and rolipram (PDE3 and PDE4 inhibitor, respectively) compared to the use of IBMX. To assess the contribution of cAMP synthesis, a low dose of an adenylyl cyclase (AC) stimulator, forskolin, has been used in combination with CIL showing a significant effect of this combination. In CIL-treated COCs and DOs, significant higher percentages of oocytes were maintained in GV stage when cultured in combination with forskolin instead of PMSG-hCG. In conclusion, these results demonstrate that the control of meiotic resumption in porcine oocytes is highly regulated by cAMP. Both the degradation by specific PDE3 enzyme and the synthesis by an active AC are highly involved.

The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats.

A single radish nuclear gene, Rfo, restores Ogura (ogu) cytoplasmic male sterility (CMS) in Brassica napus. A map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687-amino-acid protein with a predicted mitochondrial targeting pre-sequence was found to confer male fertility upon transformation into ogu CMS B. napus. This gene, like the recently described Petunia Rf gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis, although a smaller and related PPR gene is found about 40 kb from this site. The implications of these findings for the evolution of restorer genes and other PPR encoding genes are discussed.