Target-site mutation associated with glufosinate resistance in Italian ryegrass (Lolium perenne L. ssp. multiflorum)

BACKGROUND: Studies were conducted to elucidate the mechanism of glufosinate resistance in an Italian ryegrass population. RESULTS: Glufosinate rates required to reduce growth by 50% (GR₅₀) were 0.15 and 0.18 kg AI ha^?1 for two susceptible populations C1 and C2 respectively, and 0.45 kg AI ha^?1 for the resistant population MG, resulting in a resistance index of 2.8. Ammonia accumulation after glufosinate treatment was on average 1.5 times less for the resistant population than for the susceptible populations. The glufosinate concentrations (µM ) required to reduce the glutamine synthetase (GS) enzyme activity by 50% (I₅₀) were 31 and 137 for C1 and C2 respectively, and 2432 for the resistant population MG. One amino acid substitution in the plastidic GS2 gene, aspartic acid for asparagine at position 171, was identified in the resistant population. CONCLUSIONS: This is the first report of glufosinate resistance in a weed species that involves an altered target site. Copyright © 2012 Society of Chemical Industry     

Glyphosate‐resistant Italian ryegrass (Lolium multiflorum) on rice paddy levees in Japan

The rapid range expansion of naturalized Italian ryegrass (Lolium multiflorum Lam.) in farmland is a serious problem in Fukuroi city in Shizuoka Prefecture, Japan. Glyphosate has been used to control Italian ryegrass in the levees of rice paddy fields and wheat fields for ～20 years, but this weed in Fukuroi city is poorly controlled by glyphosate. In order to elucidate the level of resistance to glyphosate in Italian ryegrass populations, seed bioassays and a foliar application experiment, using seeds collected from 16 wild populations in and around Fukuroi city and from three susceptible cultivars, were conducted. For the susceptible cultivars and one population from a site where glyphosate had not been applied for >10 years, the shoot length in the seed bioassays was greatly suppressed at a glyphosate concentration of 10 mg ai L^?1 and no seedling survived after the foliar application of glyphosate at a rate of 2.3 kg ai ha^?1. Nine wild populations from levees in the southern part of Fukuroi city showed vigorous shoot growth at a glyphosate concentration of 10 mg ai L^?1 and had at least a 78% survival rate after the application of glyphosate at 2.3 kg ai ha^?1. Four wild populations from levees in the northern part of Fukuroi city showed a slight suppression of the shoot growth as a result of the glyphosate treatment and their survival rates ranged from 20 to 64%. The results suggested that resistance to glyphosate has evolved in the wild populations of Italian ryegrass that are growing on the levees. This is the first report of a glyphosate‐resistant weed in Japan.     

Non‐target‐site mechanism of glyphosate resistance in Italian ryegrass (Lolium multiflorum)

In Shizuoka Prefecture, Japan, glyphosate‐resistant Lolium multiflorum is a serious problem on the levees of rice paddies and in wheat fields. The mechanism of resistance of this biotype was analyzed. Based on LD₅₀, the resistant population was 2.8–5.0 times more resistant to glyphosate than the susceptible population. The 5‐enolpyruvyl‐shikimate‐3‐phosphate synthase (EPSPS) gene sequence of the resistant biotype did not show a non‐synonymous substitution at Pro106, and amplification of the gene was not observed in the resistant biotype. The metabolism and translocation of glyphosate were examined 4 days after application through the direct detection of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) using liquid chromatograph‐tandem mass spectrometer (LC‐MS/MS). AMPA was not detected in either biotype in glyphosate‐treated leaves or the other plant parts. The respective absorption rates of the susceptible and resistant biotypes were 37.90 ± 3.63% and 41.09 ± 3.36%, respectively, which were not significantly different. The resistant biotype retained more glyphosate in a glyphosate‐treated leaf (91.36 ± 1.56% of absorbed glyphosate) and less in the untreated parts of shoots (5.90 ± 1.17%) and roots (2.76 ± 0.44%) compared with the susceptible biotype, 79.58 ± 3.73%, 15.77 ± 3.06% and 4.65 ± 0.89%, respectively. The results indicate that the resistance mechanism is neither the acquisition of a metabolic system nor limiting the absorption of glyphosate but limited translocation of the herbicide in the resistant biotype of L. multiflorum in Shizuoka Prefecture.     

Influence of weed management measures on glyphosate resistance and endophyte infection in naturalized Italian ryegrass (Lolium multiflorum)

Recently, glyphosate‐resistant Italian ryegrass (Lolium multiflorum Lam.) was found on rice paddy levees in a western region of Shizuoka Prefecture, Japan. Naturalized populations of Italian ryegrass are frequently infected with fungal Epichloë endophytes. Endophytes often confer benefits to their host grasses. This study investigated the influence of five weed management treatments on glyphosate resistance and endophyte infection in Italian ryegrass that was growing on paddy levees where glyphosate‐resistant individuals were dominant. The weed management treatments were: (i) mowing once before the grass flowered; (ii) mowing once during flowering; (iii) mowing twice during flowering; (iv) glyphosate application before flowering; and (v) no treatment. The seeds were collected from the treatment plots in 2013 and 2014. The seeds were examined for endophyte infection and the seedlings that had been grown from the seeds were tested for the frequency of glyphosate resistance. The seedlings that had been derived from the glyphosate treatment showed higher frequencies of glyphosate resistance than those seedlings that had been derived from all the other treatments. Endophytes were found in all populations of the seeds from the paddy levees, with higher infection rates in the seeds that had been derived from the glyphosate treatment and the twice‐mowed treatment. There was a significant relationship between the endophyte infection frequency in the seeds and glyphosate resistance in the seedlings that had been grown from the same populations. The results indicate that where glyphosate herbicides are frequently used, selection for glyphosate‐resistant Italian ryegrass occurs, and along with this, the frequency of endophyte infection also increases.     

Improved physical control of glyphosate‐resistant Italian ryegrass (Lolium multiflorum) on rice paddy levees in Japan

Italian ryegrass ( Lolium multiflorum Lam.) is a non‐native annual winter grass that has seriously infested rice paddy levees and wheat fields in Japan. Recently, glyphosate‐resistant Italian ryegrass was found on paddy levees in central Japan, thereby making control of the grass by using glyphosate less effective. In this study, physical control methods were tested that combined the timing and frequency of mowing in order to more effectively control glyphosate‐resistant Italian ryegrass on rice paddy levees. A 3 year field experiment was conducted from 2012 to 2014 in a western region of Shizuoka Prefecture, where glyphosate‐resistant Italian ryegrass has become dominant. Five treatments were tested: (i) mowing once before the flowering of the grass (i.e. conventional mowing measure); (ii) mowing once during flowering; (iii) mowing twice during flowering; (iv) glyphosate application before flowering (i.e. one of the conventional mowing measures); and (v) no treatment. The above‐ground biomass, seed production, soil seed bank and seedling occurrence of Italian ryegrass were measured to determine the effectiveness of these treatments. Mowing during the flowering period resulted in reduced above‐ground biomass, seed production and soil seed bank when compared with the other treatments. Additionally, mowing twice during the flowering period resulted in a lower seedling density than mowing once. The results suggest that, in this region, physical control by mowing during the flowering period would be more effective than conventional measures for controlling glyphosate‐resistant Italian ryegrass.     

Introgression of crown rust (Puccinia coronata) resistance from meadow fescue (Festuca pratensis) into Italian ryegrass (Lolium multiflorum): genetic mapping and identification of associated molecular markers

Crown rust ( Puccinia coronata ) resistance (CRres), which had been introgressed from meadow fescue ( Festuca pratensis ) into the Italian ryegrass ( Lolium multiflorum ) background, was genetically mapped with amplified fragment length polymorphism (AFLP) and sequence tagged site (STS) markers to a terminal segment of chromosome 5. Comparative mapping had previously shown that this region of the Lolium / Festuca genome has a degree of conserved genetic synteny with chromosomes 11 and 12 of rice. Sequences from rice chromosome 12 were used as templates for identifying further STS markers that cosegregated with CRres. The relative genomic positions of molecular markers associated with CRres in L. multiflorum , L. perenne , F. pratensis and oats is discussed, along with their relationships to physical positions on rice chromosomes C11 and C12.     

SSR-based genetic linkage analysis of resistance to crown rust (Puccinia coronata f. sp. lolii) in perennial ryegrass (Lolium perenne)

Crown rust (caused by Puccinia coronata f. sp. lolii) is a serious foliar disease of the pasture and turfgrass perennial ryegrass (Lolium perenne). Previous genetic studies have detected both qualitative and quantitative resistance mechanisms, and interpretation of the genetic system is complicated by variation within the sexually reproducing pathogen. Resistant and susceptible parental genotypes of ryegrass were identified using a composite urediniospore population collected from three geographically distinct locations. A two-way pseudo-testcross mapping population was obtained as the F₁ progeny of the pair-cross between ryegrass parental genotypes Vedette₆ and Victorian₉. Both parents showed intermediate resistance against a pathogen population collected in a single geographical zone (Hamilton, Victoria), but in the F₁ population, significant variation for a range of resistance-associated characters was detected. Statistical analysis of phenotypic data suggested a major gene effect, hence bulked segregant analysis with map-assigned simple sequence repeat (SSR) markers was used to scan the genome. A marker showing strong association with resistance was assigned to linkage group (LG) 2 of perennial ryegrass. Analysis of 11 LG2 SSR markers defined an interval between loci xlpssrh03f03 and xlpssrk02e02 as containing the gene or genes (LpPc1) conferring crown rust resistance. Resistance gene determinants were inherited from both parents, with up to 80% of the total phenotypic variation explained by markers segregating from Vedette₆ and up to 26% of the variation explained by markers segregating from Victorian₉. The two contributions together resulted in an additive increase in effect, with fully resistant individuals requiring determinants from both parents. A conserved syntenic relationship was observed with linkage group B of Avena strigosa, which is the location of a cluster of resistance genes to the oat form of crown rust. The implications of this study for marker-assisted selection of disease resistance in perennial ryegrass are discussed.