Multiple highly expressed phosphoenolpyruvate carboxylase genes have divergent enzyme kinetic properties in two C4 grasses.

BACKGROUND AND AIMS: Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the carbon-concentrating mechanism (CCM) in plants that perform C4 photosynthesis. This CCM is generally driven by a single PEPC gene product that is highly expressed in the cytosol of mesophyll cells. We found two C4 grasses, Panicum miliaceum and Echinochloa colona, that each have two highly expressed PEPC genes. We characterized the kinetic properties of the two most abundant PEPCs in E. colona and P. miliaceum to better understand how the enzyme's amino acid structure influences its function. METHODS: Coding sequences of the two most abundant PEPC proteins in E. colona and P. miliaceum were synthesized by GenScript and were inserted into bacteria expression plasmids. Point mutations resulting in substitutions at conserved amino acid residues (e.g. N-terminal serine and residue 890) were created via site-directed PCR mutagenesis. The kinetic properties of semi-purified plant PEPCs from Escherichia coli were analysed using membrane-inlet mass spectrometry and a spectrophotometric enzyme-coupled reaction. KEY RESULTS: The two most abundant P. miliaceum PEPCs (PmPPC1 and PmPPC2) have similar sequence identities (>95 %), and as a result had similar kinetic properties. The two most abundant E. colona PEPCs (EcPPC1 and EcPPC2) had identities of ~78 % and had significantly different kinetic properties. The PmPPCs and EcPPCs had different responses to allosteric inhibitors and activators, and substitutions at the conserved N-terminal serine and residue 890 resulted in significantly altered responses to allosteric regulators. CONCLUSIONS: The two, significantly expressed C4Ppc genes in P. miliaceum were probably the result of genomes combining from two closely related C4Panicum species. We found natural variation in PEPC's sensitivity to allosteric inhibition that seems to bypass the conserved 890 residue, suggesting alternative evolutionary pathways for increased malate tolerance and other kinetic properties.

An ABCC-type transporter endowing glyphosate resistance in plants.

Glyphosate is the most widely used herbicide in world agriculture and for general vegetation control in a wide range of situations. Global and often intensive glyphosate selection of very large weedy plant populations has resulted in widespread glyphosate resistance evolution in populations of many weed species. Here, working with a glyphosate-resistant (GR) Echinochloa colona population that evolved in a Western Australia agricultural field, we identified an ATP-binding cassette (ABC) transporter (EcABCC8) that is consistently up-regulated in GR plants. When expressed in transgenic rice, this EcABCC8 transporter endowed glyphosate resistance. Equally, rice, maize, and soybean overexpressing the EcABCC8 ortholog genes were made resistant to glyphosate. Conversely, CRISPR/Cas9-mediated knockout of the EcABCC8 ortholog gene OsABCC8 increased rice susceptibility to glyphosate. Subcellular localization analysis and quantification of glyphosate cellular levels in treated ABCC8 transgenic rice plants and isolated leaf protoplasts as well as structural modeling support that EcABCC8 is likely a plasma membrane-localized transporter extruding cytoplasmic glyphosate to the apoplast, lowering the cellular glyphosate level. This is a report of a membrane transporter effluxing glyphosate in a GR plant species, and its function is likely conserved in crop plant species.

Alternative Options to Glyphosate for Control of Large Echinochloa colona and Chloris virgata Plants in Cropping Fallows.

The over-reliance on the herbicide glyphosate for knockdown weed control in fallows under minimum and zero-till cropping systems has led to an increase in populations of glyphosate-resistant weeds. Echinochloa colona and Chloris virgata are two major grass weeds in the cropping regions of northern New South Wales and southern Queensland, Australia, that have become harder to kill due to a steady rise in the occurrence of glyphosate-resistant weed populations. Therefore, to help growers contain these hard to kill fallow weeds, an alternate approach to glyphosate application is needed. With this purpose in mind, a pot study was carried out during the summer seasons of 2015 and 2016 at the Tamworth Agricultural Institute, Tamworth, NSW, Australia, to evaluate the efficacy of tank mixtures and sequential applications of Group H (4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor), Group C (inhibitors of photosynthesis at photosystem II), Group A (ACCase inhibitors) and Group L (photosystem I inhibitor) herbicides on late tillering E. colona and C. virgata plants. These herbicide groups are a global classification by the Herbicide Resistance Action Committee. Highly effective results were achieved in this study using combinations of Groups H, C, A and L herbicides applied as tank mixtures for controlling large E. colona plants. Additionally, sequential applications of Group H, C and A herbicides followed by (fb) paraquat were shown to be very effective on large E. colona plants. Late tillering C. virgata plants were generally well controlled by tank mixtures, and sequential applications proved to be highly effective on this grass weed as well. Haloxyfop in combination with paraquat as a tank mixture, via sequential application or as a stand-alone treatment, was highly effective for C. virgata control; however, using combinations of herbicide groups is the preferred choice when combating herbicide resistant weed populations. There was a clear synergy shown using Group H, Group C and Group A herbicides in combination with the Group L herbicide paraquat in this study for controlling advanced E. colona and C. virgata plants. These combinations were shown to be successful on plants grown under glasshouse conditions; however; these treatments would need to be tested on plants grown in a field situation to show whether they will be a useful solution for farmers who are trying to control these weeds in fallow.

Multiple target site resistance to glyphosate in junglerice (Echinochloa colona) lines from California orchards.

BACKGROUND: In California specialty cropping systems such as vineyards and orchards, Echinochloa colona is present as a summer annual weed. It is able to germinate throughout the growing season whenever favorable conditions are present, and management relies heavily on glyphosate applications. Glyphosate-resistant (GR) E. colona biotypes are present in the state, but the levels of resistance observed suggest that there may be differences in mechanisms of resistance among populations. RESULTS: Echinochloa colona lines collected from different regions of California's Central Valley presented resistance levels ranging from 1.4 to 4.3-fold compared to susceptible lines. No differences in the absorption and translocation of [14 C]-glyphosate were observed among lines. Resistant lines accumulated eight-fold less shikimic acid after treatment with 435 and 870 g a.e. ha-1 glyphosate compared to the most susceptible line. Sequencing of a region of the EPSPS gene revealed three single nucleotide changes leading to amino acid substitutions at Proline 106, including Pro106Leu, Pro106Thr and Pro106Ser. CONCLUSION: These results indicate that an altered target site in EPSPS is contributing to resistance in these lines and resistance has evolved independently, multiple times in the Central Valley of California. Additional research is needed to further understand the genomic contributions of resistance loci in this polyploid weed species. © 2018 Society of Chemical Industry.

Comparison of the complete genomes of two Echinochloa species: barnyard grass and jungle rice.

We sequenced and compared the complete chloroplast (cp) genomes of the two Echinochloa species; E. crus-galli (KT983256) and E. colona (KT983255). The size of complete chloroplast genomes of the two species are 139,851 and 139,592 base pairs in length, respectively. They include a pair of inverted repeats (22,640 and 22,618 bp) separated by the small (12,518 and 12,519 bp) and large (82,053 and 81,837 bp) single copy regions. Both chloroplast genomes include a total of 136 genes. Phylogenetic analysis revealed that E. colona was diverged between 2.65 and 3.18 million years ago (Mya) from the E. oryzicola and E. crus-galli.

Susceptibility of Echinochloa populations to cyhalofop-butyl in Southern region of Brazil and impact of the weed phenology on its efficacy of control / Suscetibilidade de populações de Echinochloa a cyhalofop-butyl na região Sul do Brasil e impacto da fenologia da infestante sobre a eficácia do seu controle

ABSTRACT: Cyhalofop-butyl stands out among the herbicides in the control of imidazolinone-resistant Echinochloa species; but, rice farmers are not always satisfied with the control achieved with this herbicide. The objectives were to evaluate in regional scale the susceptibility of Echinochloa populations to cyhalofop-butyl, and quantify the effect of the weed phenology on its efficacy of control. For this, three trials were carried out under greenhouse conditions with a fully random design, using Echinochloa populations collected in rice fields in the southern region of Brazil. In two trials, the susceptibility level of 156 (2012/13 growth season) and 103 (2013/14 growth season) populations were evaluated with the application of cyhalofop-butyl at 360g ha-1. In other trial, in which treatments were arranged in a bi-factorial design (A = 6 x B = 5), it was evaluated six cyhalofop-butyl rates and five phenological stages of E. crus-galli populations. Echinochloa populations had showed differential susceptibility to cyhalofop-butyl, especially in the 2013/14 growth season, where 20 out of the 103 populations had control lower than 90%. The efficacy of this herbicide was inversely proportional to the phenological stage, and the application timing delay contributed directly to the decrease of susceptibility to the herbicide. Cyhalofop-butyl is an effective alternative to control imidazolinone-resistant Echinochloa populations, as long as the application timing occurs in the early phenological stages (2 to 4 leaves).

RESUMO: Cyhalofop-butyl destaca-se dentre os herbicidas usados no manejo de populações de capim-arroz resistente às imidazolinonas, mas nem sempre o orizicultor fica satisfeito com o resultado obtido com este herbicida. Objetivou-se avaliar a suscetibilidade de populações de capim-arroz (Echinochloa spp.) ao cyhalofop-butyl e, quantificar o efeito da fenologia da infestante sobre a eficácia do seu controle. Para isto, três experimentos foram realizados em casa de vegetação com delineamento inteiramente casualizado, utilizando-se populações de capim-arroz coletadas na região Sul do Brasil. Em dois experimentos, a suscetibilidade de 156 (safra 2012/13) e 103 (safra 2013/14) populações foram avaliadas frente à aplicação do herbicida cyhalofop-butyl na dose de 360g ha-1. Em outro experimento, em que se arranjaram os tratamentos em esquema bi-fatorial, foram avaliados seis doses do herbicida e cinco estádios fenológicos de populações de E. crus-galli. Foi verificada variação na resposta das populações de capim-arroz ao cyhalofop-butyl, em especial na safra 2013/14, em que 20, das 103 populações testadas, tiveram controle menor que 90%. A eficácia do controle foi inversamente proporcional ao estádio fenológico e, o atraso no controle da infestante contribui diretamente para a diminuição da sua suscetibilidade ao herbicida. Cyhalofop-butyl é uma alternativa eficaz para controlar capim-arroz resistente às imidazolinonas, desde que a aplicação ocorra nos estádios iniciais de desenvolvimento (2 a 4 folhas).

Glyphosate resistance in Echinochloa colona: phenotypic characterisation and quantification of selection intensity.

BACKGROUND: A population of Echinochloa colona infesting agricultural fields in the northern region of Western Australia evolved glyphosate resistance after 10 years of glyphosate selection. This study identified two phenotypic (susceptible S versus resistant R) lines from within a segregating glyphosate-resistant population. Estimation of survival, growth and reproductive rates of the phenotypes in response to glyphosate selection helped to characterise the level of resistance, fitness and the selection intensity for glyphosate in this species. RESULTS: Estimations of LD(50) (lethal dose) and GR(50) (growth rate) showed an eightfold glyphosate resistance in this population. The resistant index based on the estimation of seed number (SY(n50)) showed a 13-fold resistance. As a result of linear combination of plant survival and fecundity rates, plant fitness values of 0.2 and 0.8 were estimated for the S and R phenotypes when exposed to the low dose of 270 g glyphosate ha(-1). At the recommended dose of 540 g glyphosate ha(-1) , fitness significantly decreased (fivefold) in S plants but remained markedly similar (0.7) in plants of the R phenotype. Thus, the calculated selection intensity (SI) at 540 g glyphosate ha(-1) was much greater (SI = 17) than at 270 g glyphosate ha(-1) (SI = 4). CONCLUSIONS: The assessment of plant survival and fecundity in response to glyphosate selection in the S and R phenotypes allowed a greater accuracy in the estimation of population fitness of both phenotypes and thus of glyphosate selection intensity in E. colona. The estimation of seed number or mass of phenotypes under herbicide selection is a true ecological measure of resistance with implications for herbicide resistance evolution.

Temperature influences the level of glyphosate resistance in barnyardgrass (Echinochloa colona).

BACKGROUND: Echinochloa colona is an important summer-growing weed species in cropping regions of northern Australia that has evolved resistance to glyphosate owing to intensive use of this herbicide in summer fallow. RESULTS: Pot trials conducted at 20 and 30 °C on six E. colona populations showed a significant increase in the level of glyphosate resistance in resistant populations at 30 °C compared with 20 °C. However, there was no influence of growth temperature on glyphosate susceptibility of the sensitive population. Sequencing of the target-site gene (EPSPS) of the six populations identified a mutation at position 106 leading to a change from proline to serine in the most resistant population A533.1 only. EPSPS gene amplification was not detected in any of the resistant populations examined. Examining (14) C-glyphosate uptake on two resistant and one susceptible population showed a twofold increase at 20 °C; however, few differences in glyphosate translocation occurred from the treated leaf to other plant parts between populations or temperatures. CONCLUSION: There is reduced efficacy of glyphosate at high temperatures on resistant E. colona populations, making these populations harder to control in summer.

Target-site EPSPS Pro-106 mutations: sufficient to endow glyphosate resistance in polyploid Echinochloa colona?

BACKGROUND: This study confirms and characterises glyphosate resistance in two polyploid Echinochloa colona populations from north-eastern Australia. RESULTS: Glyphosate dose response revealed that the two resistant populations were marginally (up to twofold) resistant to glyphosate. Resistant plants did not differ in non-target-site foliar uptake and translocation of (14) C-glyphosate, but contained the known target-site 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutation Pro-106-Thr and/or Pro-106-Leu. Although plants carrying either a single or two EPSPS mutations were glyphosate resistant relative to the susceptible population, they were still controlled at the field rate of glyphosate (450 g a.e. ha(-1) ) when treated under warm conditions (25/20 °C). However, when treated in hot conditions (35/30 °C), most mutant resistant plants (68%) can survive the field rate, and an increase (2.5-fold) in glyphosate LD50 was found for both the R and S populations. CONCLUSIONS: This study shows that one or two EPSPS Pro-106 mutations are insufficient to confer field-rate glyphosate resistance in polyploidy E. colona at mild temperatures. However, control of these mutant plants at the glyphosate field rate is poor at high temperatures, probably owing to reduced glyphosate efficacy. Therefore, glyphosate should be applied during relatively mild (warm) temperature periods in the summer growing season to improve E. colona control.

Differences in responses to flooding by germinating seeds of two contrasting rice cultivars and two species of economically important grass weeds.

Crop productivity is largely affected by abiotic factors such as flooding and by biotic factors such as weeds. Although flooding after direct seeding of rice helps suppress weeds, it also can adversely affects germination and growth of rice, resulting in poor crop establishment. Barnyard grasses (Echinochloa spp.) are among the most widespread weeds affecting rice, especially under direct seeding. The present work aimed to establish effective management options to control these weeds. We assessed the effects of variable depths and time of submergence on germination, seedling growth and carbohydrate metabolism of (i) two cultivars of rice known to differ in their tolerance to flooding during germination and (ii) two barnyard grasses (Echinochloa colona and E. crus-galli) that commonly infest rice fields. Flooding barnyard grasses with 100-mm-deep water immediately after seeding was effective in suppressing germination and growth. Echinochloa colona showed greater reductions in emergence, shoot and root growth than E. crus-galli. Delaying flooding for 2 or 4 days was less injurious to both species. Echinochloa colona was also more susceptible to flooding than the flood-sensitive rice cultivar 'IR42'. The activity of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) in rice seedlings was increased by flooding after sowing but with greater increases in 'Khao Hlan On' compared with 'IR42'. The activity of ADH and PDC was enhanced to a similar extent in both barnyard grasses. Under aerobic conditions, the activity of ADH and PDC in the two barnyard grasses was downregulated, which might contribute to their inherently faster growth compared with rice. Aldehyde dehydrogenase activity was significantly enhanced in flood-tolerant 'Khao Hlan On' and E. crus-galli, but did not increase in flood-sensitive E. colona and 'IR42', implying a greater ability of the flood-tolerant types to detoxify acetaldehyde generated during anaerobic fermentation. Confirmation of this hypothesis is now being sought.