Wheat and Cereal Rye Inter-Row Living Mulches Interfere with Early Season Weeds in Soybean.

Rapid growth of cool-season weeds in the spring exacerbates weed interference during early soybean (Glycine max (L.) Merr.) establishment in northern climates. This study tested the utility of spring-seeded inter-row living mulches in soybean for early season weed suppression using volunteer canola (Brassica napus L.) as a representative model weed species. The effects of the presence or absence of spring wheat (Triticum aestivum L.) or winter cereal rye (Secale cereale L.) living mulches (mulch type) that had been seeded simultaneously with soybean grown using 38 or 76 cm row spacing (spatial arrangement) and the presence or absence of herbicides used for mid-season mulch termination (herbicide regime) were evaluated in three environments in Manitoba, Canada, in 2013 and 2014. Soybean yield was similar in the presence and absence of the living mulches. In the environment that received the lowest precipitation (Carman 2013), the mulches terminated with post-emergence glyphosate resulted in a 55% greater soybean yield compared to the mulches that remained live throughout the growing season. Inter-row mulches that had been living or terminated mid-season reduced volunteer canola seed production by about one-third (up to 9000 seeds m-2). This study demonstrates the utility of wheat or cereal rye inter-row living mulches for enhanced interference with weeds during early soybean establishment.

qPCR assay targeting Bradyrhizobium japonicum shows that row spacing and soybean density affects Bradyrhizobium population.

The ability for a soybean plant to be efficiently nodulated when grown as a crop is dependent on the number of effective Bradyrhizobium japonicum that can be found in close proximity to the developing seedling shortly after planting. In Manitoba, the growing of soybean as a crop has increased from less than 500 000 acres in 2008 to over 2.3 million acres in 2017. Since the large increase in soybean production is relatively recent, populations of B. japonicum have not yet developed. In response to this, we developed a primer pair that can identify B. japonicum, and be used to determine the titre found in field soil. Their utility was demonstrated by being used to determine whether row spacing of soybean affects B. japonicum populations, as well as to follow B. japonicum populations in a soybean field over the course of a field season. The data show that plant density can affect B. japonicum populations. Moreover, evidence is presented that suggests plant development affects overall B. japonicum populations.

Influence of Temperature on Development and Reproduction of Ditylenchus weischeri and D. dipsaci on Yellow Pea.

The ability of the recently described stem nematode of creeping thistle (Cirsium arvense L.), Ditylenchus weischeri, to develop on and parasitize yellow pea (Pisum sativum L.) is uncertain. The current study examined nematode life-stage progression and generation time on yellow pea as affected by temperature with the related pest, D. dipsaci, used as a positive control. Relationships for body length of the two nematode species and life stage were unaffected by rearing on plant hosts compared with carrot disks. Then plant-reared J4 individuals of both nematode species were used to determine the effect of temperature (17, 22, and 27°C) on life-stage progression and minimum generation time with yellow pea. At 17 and 22°C, D. weischeri J4 individuals progressed to only the adult stage whereas, at 27°C, the minimum generation time from J4 to J4 was 30 days or 720 growing degree-days. The minimum generation time for D. dipsaci was 24, 18, and 22 days or 336, 342, and 528 growing degree-days at 17, 22, and 27°C, respectively. The results indicate that development of D. weischeri is temperature dependent and reproduction is unlikely on yellow pea in the Canadian Prairies, where mean daily air temperatures of 27°C are rare and not sustained.

Host Preference and Seedborne Transmission of Ditylenchus weischeri and D. dipsaci on Select Pulse and Non-Pulse Crops Grown in the Canadian Prairies.

The stem nematode Ditylenchus weischeri was recently reported on creeping thistle (Cirsium arvense) in Canada. Two greenhouse studies examined host suitability of crops commonly grown in the Canadian Prairies for D. weischeri and the closely related parasite of many crops, D. dipsaci. In the first study, common pulse crops (yellow pea, chickpea, common bean, and lentil), spring wheat, canola, creeping thistle, and garlic were evaluated. Plant biomass and reproductive factor (Rf = nematode recovered/inoculated) 8 weeks postinoculation were used to determine host suitability. Creeping thistle biomass was reduced by D. weischeri whereas D. dipsaci reduced biomass of four of five pea and two of three bean varieties. Two pea varieties were weak hosts for D. weischeri, with Rf slightly >1. D. weischeri aggressively reproduced on creeping thistle (Rf = 5.4). D. dipsaci reproduced aggressively on garlic (Rf = 6.4; a known host), moderately on pea varieties (Rf > 2), and weakly on chickpea and bean (Rf > 1). In the second study, using creeping thistle and yellow pea, D. weischeri was recovered from aboveground parts of the plants and seed of the former and D. dipsaci from the later. The results show that D. weischeri parasitizes creeping thistle but not other crops and that D. weischeri host preference is different from that of D. dipsaci.

Occurrence of Ditylenchus weischeri and Not D. dipsaci in Field Pea Harvest Samples and Cirsium arvense in the Canadian Prairies.

The stem nematode, a parasite of the herbaceous perennial weed, Cirsium arvense (L.) Scop. and identified as Ditylenchus dipsaci (Kühn) Filipjev, was reported in the Canadian prairies in 1979. Recently, D. weischeri Chizhov parasitizing Cirsium arvense was described in Russia, and it has been shown that this species is not an agricultural pest. In this study, we examined Ditylenchus species found in field pea (Pisum sativum L.) grain harvest samples in 2009 and 2010 and from C. arvense shoots in pea fields in the Saskatchewan, Alberta, and Manitoba provinces. Samples from 538 fields (mainly yellow pea) were provided by 151 growers throughout the main pea-growing area of the Canadian prairies. Of the samples collected, 2% were positive for Ditylenchus. The population density of the nematode ranged between 4 and 1,500 nematodes kg(-1) pea harvest sample and related to presence of C. arvense seeds. Positive samples occurred in 2009 but not in 2010 and were from throughout the pea-growing area of the Canadian prairies and not related to cropping history. C. arvense collected from yellow pea fields in Saskatchewan and Manitoba, but not Alberta, were infested with Ditylenchus. Morphological and molecular (ITS-PCR-RFLP) traits indicated that this species belongs to D. weischeri. The results indicated the stem nematode found in yellow pea grain is D. weischeri which resided with C. arvense seeds and debris to pea samples. Unlike D. dipsaci, D. weischeri is not a nematode pest of economic importance; therefore, its presence in the pea harvest samples was not a concern.

Transcriptional changes of antioxidant responses, hormone signalling and developmental processes evoked by the Brassica napus SHOOTMERISTEMLESS during in vitro embryogenesis.

Previous work showed that alterations in Brassica napus (Bn) SHOOTMERISTEMLESS (BnSTM) expression levels influence microspore-derived embryogenesis in B. napus. While over-expression of BnSTM increased microspore-derived embryo (MDE) yield and quality, down-regulation of BnSTM repressed embryo formation [16]. Transcriptional analyses were conducted to investigate the molecular mechanisms underpinning these responses. The induction of BnSTM resulted in a heavy transcriptional activation of genes involved in antioxidant responses, hormone signalling and developmental processes. Several antioxidant enzymes, including catalases, superoxide dismutases, and components of the Halliwell-Asada cycle were induced in embryos ectopically expressing BnSTM and contributed to the removal of reactive oxygen species (ROS). These changes were accompanied by elevated levels of ascorbate and glutathione, which have been shown to promote embryonic growth and development. Induction or repression of BnSTM altered the early cytokinin response, whereas late responses, modulated by Type-A Arabidopsis response regulators (ARRs), were induced in MDEs over-expressing BnSTM. Major differences between transgenic MDEs were also observed in the expression pattern of several auxin transporters and key developmental factors required for normal embryogenesis. While some of these factors, BABYBOOM1 (BBM1) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK), play a key role during early embryogeny, others, CYP78A5, LEAFY COTYLEDON1 and 2 (LEC1 and LEC2), as well as WOX2 and 9, are required for proper embryo development. Collectively these results demonstrate the involvement of BnSTM in novel developmental processes which can be utilized to enhance in vitro embryogenesis.

Modelling the dynamics of feral alfalfa populations and its management implications.

BACKGROUND: Feral populations of cultivated crops can pose challenges to novel trait confinement within agricultural landscapes. Simulation models can be helpful in investigating the underlying dynamics of feral populations and determining suitable management options. METHODOLOGY/PRINCIPAL FINDINGS: We developed a stage-structured matrix population model for roadside feral alfalfa populations occurring in southern Manitoba, Canada. The model accounted for the existence of density-dependence and recruitment subsidy in feral populations. We used the model to investigate the long-term dynamics of feral alfalfa populations, and to evaluate the effectiveness of simulated management strategies such as herbicide application and mowing in controlling feral alfalfa. Results suggest that alfalfa populations occurring in roadside habitats can be persistent and less likely to go extinct under current roadverge management scenarios. Management attempts focused on controlling adult plants alone can be counterproductive due to the presence of density-dependent effects. Targeted herbicide application, which can achieve complete control of seedlings, rosettes and established plants, will be an effective strategy, but the seedbank population may contribute to new recruits. In regions where roadside mowing is regularly practiced, devising a timely mowing strategy (early- to mid-August for southern Manitoba), one that can totally prevent seed production, will be a feasible option for managing feral alfalfa populations. CONCLUSIONS/SIGNIFICANCE: Feral alfalfa populations can be persistent in roadside habitats. Timely mowing or regular targeted herbicide application will be effective in managing feral alfalfa populations and limit feral-population-mediated gene flow in alfalfa. However, in the context of novel trait confinement, the extent to which feral alfalfa populations need to be managed will be dictated by the tolerance levels established by specific production systems for specific traits. The modelling framework outlined in this paper could be applied to other perennial herbaceous plants with similar life-history characteristics.

Occurrence of alfalfa (Medicago sativa L.) populations along roadsides in southern Manitoba, Canada and their potential role in intraspecific gene flow.

Alfalfa is a highly outcrossing perennial species that can be noticed in roadsides as feral populations. There remains little information available on the extent of feral alfalfa populations in western Canadian prairies and their role in gene flow. The main objectives of this study were (a) to document the occurrence of feral alfalfa populations, and (b) to estimate the levels of outcrossing facilitated by feral populations. A roadside survey confirmed widespread occurrence of feral alfalfa populations, particularly in alfalfa growing regions. The feral populations were dynamic and their frequency ranged from 0.2 to 1.7 populations km(-1). In many cases, the nearest feral alfalfa population from alfalfa production field was located within a distance sufficient for outcrossing in alfalfa. The gene flow study confirmed that genes can move back and forth between feral and cultivated alfalfa populations. In this study, the estimated outcrossing levels were 62% (seed fields to feral), 78% (feral to seed fields), 82% (hay fields to feral) and 85% (feral to feral). Overall, the results show that feral alfalfa plants are prevalent in alfalfa producing regions in western Canada and they can serve as bridges for gene flow at landscape level. Management of feral populations should be considered, if gene flow is a concern. Emphasis on preventing seed spill/escapes and intentional roadside planting of alfalfa cultivars will be particularly helpful. Further, realistic and pragmatic threshold levels should be established for markets sensitive to the presence of GE traits.

Modulation of embryo-forming capacity in culture through the expression of Brassica genes involved in the regulation of the shoot apical meristem.

Somatic embryogenesis in Arabidopsis is achieved by culturing bending-cotyledon embryos on a 2,4-D-containing induction medium for 14 d followed by a transfer on to a hormone-free development medium. Several genes orthologous to Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL) were isolated from Brassica oleracea (Bo), B. rapa (Br), and B. napus (Bn), and ectopically expressed in Arabidopsis to assess their effects on somatic embryogenesis. Ectopic expression of BoSTM, BrSTM, and BnSTM increased the number of somatic embryos, whereas a different effect was observed in lines overexpressing BnCLV1 in which somatic embryo formation was severely repressed. The introduction of BnZLL did not have any effects on Arabidopsis somatic embryogenesis. The increased embryo-forming capacity observed in lines overexpressing Brassica STM was associated with a lower requirement for the inductive signal 2,4-D, and a higher expression of WUSCHEL (WUS) which demarcates the formation of embryogenic cells. This was in contrast to the 35S::BnCLV1 lines which showed the highest requirement for exogenous 2,4-D and a reduced WUS expression. Microarray studies were conducted to monitor global changes in transcript levels during Arabidopsis somatic embryogenesis between the wild-type (WT) line and a BoSTM-overexpressing line, which showed the most pronounced enhancement of somatic embryo yield. The introduction of BoSTM affected the expression of many genes involved in hormone perception and signalling, as well as genes encoding DNA methyltransferases and enzymes of glutathione metabolism. Pharmacological experiments performed to confirm some of the microarray results showed that Arabidopsis somatic embryogenesis is encouraged by a global hypomethylation of the DNA during the induction phase and by a switch of the glutathione pool towards an oxidized state during the subsequent development phase. Both events occurred in the 35S::BoSTM line, but not in the WT line. Altered expression of Brassica STM also had profound effects on B. napus microspore-derived embryogenesis. The yield of microspore-derived embryos increased in lines overexpressing BnSTM and significantly decreased in antisense lines down-regulating BnSTM.

The demography of feral alfalfa (Medicago sativa L.) populations occurring in roadside habitats in Southern Manitoba, Canada: implications for novel trait confinement.

PURPOSE: Feral populations of cultivated crops can act as reservoirs for novel genetically engineered (GE) traits and aid in trait movement at the landscape level. However, little information is available on the potential of cultivated crops to become feral. In this study, we investigated the ferality of alfalfa populations (non-GE version) occurring in roadside habitats. Knowledge on the nature of roadside alfalfa populations would be useful for designing efficient trait confinement protocols and coexistence strategies in alfalfa. METHODS: We investigated roadside alfalfa populations from 2006 to 2009 in three rural municipalities (Hanover, MacDonald, and Springfield) in Southern Manitoba, Canada. We studied the demography of these populations including seedbank, seedling recruitment, and fecundity and examined the impact of road verge mowing on key life stages of these populations. We also compared the growth and reproductive attributes of roadside and cultivated alfalfa populations. RESULTS: Alfalfa is reproductively successful in roadside habitats and capable of establishing self-perpetuating populations. A substantial portion of the alfalfa seeds we extracted from seedbank samples were viable but not germinable, suggesting some degree of seedbank persistence in roadside habitats. In the roadside habitat, alfalfa seedlings recruited successfully, however, seedling mortality was high when seedlings were in close proximity to well-established alfalfa plants. Mowing dramatically reduced the reproductive success of roadside alfalfa. Generally, the growth and reproduction of roadside alfalfa was comparable to cultivated alfalfa except for total fecundity. CONCLUSIONS: Considering the long lifespan (>10 years) of alfalfa and the levels of fecundity, seedbank, and seedling survival we observed, long-term persistence of roadside alfalfa populations seems reasonable. In the context of novel trait confinement, our results suggest that feral alfalfa populations required to be managed if there is a desire/need to confine novel traits in alfalfa.

Roundup Ready soybean gene concentrations in field soil aggregate size classes.

Roundup Ready (RR) soybeans containing recombinant Agrobacterium spp. CP4 5-enol-pyruvyl-shikimate-3-phosphate synthase (cp4 epsps) genes tolerant to the herbicide glyphosate are extensively grown worldwide. The concentration of recombinant DNA from RR soybeans in soil aggregates was studied due to the possibility of genetic transformation of soil bacteria. This study used real-time PCR to examine the concentration of cp4 epsps in four field soil aggregate size classes (>2000 microm, 2000-500 microm, 500-250 microm and <250 microm). Aggregates over 2000 microm in diameter had significantly greater gene concentrations than those with diameters under 2000 microm. The >2000 mum fraction contained between 66.62% and 99.18% of total gene copies, although it only accounted for about 30.00% of the sampled soil. Aggregate formation may facilitate persistence of recombinant DNA.

Real-time polymerase chain reaction monitoring of recombinant DNA entry into soil from decomposing roundup ready leaf biomass.

Glyphosate-tolerant, Roundup Ready (RR) soybeans account for about 57% of all genetically modified (GM) crops grown worldwide. The entry of recombinant DNA into soil from GM crops has been identified as an environmental concern due to the possibility of their horizontal transfer to soil microorganisms. RR soybeans contain recombinant gene sequences that can be differentiated from wild-type plant and microbial genes in soil by using a sequence-specific molecular beacon and real-time polymerase chain reaction (PCR). A molecular beacon-based real-time PCR system to quantify a wild-type soybean lectin ( le1) gene was designed to compare amounts of endogenous soybean genes to recombinant DNA in soil. Microcosm studies were carried out to develop methodologies for the detection of recombinant DNA from RR soybeans in soil. RR soybean leaf litterbags were imbedded in the soil under controlled environmental conditions (60% water holding capacity, 10/15 degrees C, and 8/16 h day/night) for 30 days. The soybean biomass decomposition was described using a single-phase exponential equation, and the DNA concentration in planta and in soil was quantified using real-time PCR using sequence-specific molecular beacons for the recombinant cp4 epsps and endogenous soybean lectin ( le1) genes. The biomass of RR soybean leaves was 8.6% less than nontransgenic (NT) soybean leaves after 30 days. The pooled half-disappearance time for cp4 epsps and le1 in RR and of le1 in NT soybean leaves was 1.4 days. All genes from leaves were detected in soil after 30 days. This study provides a methodology for monitoring the entry of RR and NT soybean DNA into soil from decomposing plant residues.

Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation.

The presence of the recombinant cp4 epsps gene from Roundup Ready (RR) corn and RR soybean was quantified using real-time PCR in soil samples from a field experiment growing RR and conventional corn and soybean in rotation. RR corn and RR soybean cp4 epsps persisted in soil for up to 1 year after seeding. The concentration of recombinant DNA in soil peaked in July and August in RR corn and RR soybean plots, respectively. A small fraction of soil samples from plots seeded with conventional crops contained recombinant DNA, suggesting transgene dispersal by means of natural process or agricultural practices. This research will aid in the understanding of the persistence of recombinant DNA in agricultural cropping systems.

Mycorrhizal and rhizobial colonization of genetically modified and conventional soybeans.

We grew plants of nine soybean varieties, six of which were genetically modified to express transgenic cp4-epsps, in the presence of Bradyrhizobium japonicum and arbuscular mycorrhizal fungi. Mycorrhizal colonization and nodule abundance and mass differed among soybean varieties; however, in no case was variation significantly associated with the genetic modification.

Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis.

Roundup Ready (RR) genetically modified (GM) corn and soybean comprise a large portion of the annual planted acreage of GM crops. Plant growth and subsequent plant decomposition introduce the recombinant DNA (rDNA) into the soil environment, where its fate has not been completely researched. Little is known of the temporal and spatial distribution of plant-derived rDNA in the soil environment and in situ transport of plant DNA by leachate water has not been studied before. The objectives of this study were to determine whether sufficient quantities of plant rDNA were released by roots during growth and early decomposition to be detected in water collected after percolating through a soil profile and to determine the influence of temperature on DNA persistence in the leachate water. Individual plants of RR corn and RR soybean were grown in modified cylinders in a growth room, and the cylinders were flushed with rain water weekly. Immediately after collection, the leachate was subjected to DNA purification followed by rDNA quantification using real-time Polymerase Chain Reaction (PCR) analysis. To test the effects of temperature on plant DNA persistence in leachate water, water samples were spiked with known quantities of RR soybean or RR corn genomic DNA and DNA persistence was examined at 5, 15, and 25 degrees C. Differences in the amounts and temporal distributions of root-derived rDNA were observed between corn and soybean plants. The results suggest that rainfall events may distribute plant DNA throughout the soil and into leachate water. Half-lives of plant DNA in leachate water ranged from 1.2 to 26.7 h, and persistence was greater at colder temperatures (5 and 15 degrees C).