Glyphosate-resistant Lolium multiflorum in Chilean orchards

Summary Lolium multiflorum (Italian ryegrass) seeds suspected of being resistant to glyphosate were collected from fruit orchards at two locations, San Bernardo (SB) and Olivar (OL), Chile, that had been treated an average of three times per year with the isopropylamine salt of glyphosate during the previous 8–10 years. Laboratory experiments were conducted for each orchard population and a susceptible population, a commercial cultivar called Tama (TM), using Petri dishes containing filter paper saturated with 5 mL of glyphosate solution (0–160 mg a.e. L⁻¹) . Pot dose–response experiments were also conducted in the greenhouse. The three L. multiflorum populations were treated with glyphosate (0.00–4.32 kg a.e. ha⁻¹). The dose needed to reduce shoot length (Petri dish experiment) and fresh weight (pot dose–response experiment) by 50% was determined for each population. Compared with the TM population, the Petri dish experiment found that the SB and OL populations were five- and sixfold, respectively, more resistant to glyphosate, whereas the pot dose–response experiment found that the SB and OL populations were two- and fourfold, respectively, more resistant to glyphosate. These results confirm a new case of glyphosate resistance in a novel species, L. multiflorum , and correspond to the first case of glyphosate resistance reported from South America.     

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.     

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.     

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.     

Resistance to glyphosate in Lolium rigidum

Annual ryegrass (Lolium rigidum) is a widespread and important weed of Australia and populations of this weed have developed resistance to most major herbicides, including glyphosate. The possible mechanisms of resistance have been examined in one glyphosate-resistant Lolium population. No major differences were observed between resistant and susceptible biotypes in respect of (i) the target enzyme (EPSP synthase), (ii) DAHP synthase, the first enzyme of the target (shikimate) pathway, (iii) absorption of glyphosate, or (iv) translocation. Following treatment with glyphosate, there was greater accumulation of shikimate (derived from shikimate-3-Pi) in susceptible than in resistant plants. In addition, the resistant population exhibited cross-resistance to 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate, a herbicide which, although structurally similar to glyphosate, acts at an unrelated target site. On the basis of these observations we speculate that movement of glyphosate to its site of action in the plastid is involved in the resistance mechanism. © 1999 Society of Chemical Industry     

Seed germination behavior of glyphosate‐resistant and susceptible Italian ryegrass (Lolium multiflorum Lam.)

Ryegrass (Lolium multiflorum Lam.) is one of the most difficult annual weeds to control in cultivation systems worldwide, especially in temperate regions. The widespread use of herbicides in the past two decades has selected resistant biotypes of ryegrass in crops in Southern Brazil. Ryegrass seeds are dormant when disseminated and germination can be staggered over time (crop‐growing season). Knowledge of the germination behavior of seeds from herbicide‐resistant plants has been little studied, but it would be very useful in integrated weed management. Thus, this study aimed to characterize the dynamics of the soil seed bank of two biotypes of L. multiflorum, one glyphosate‐resistant and the other glyphosate‐susceptible, under a no‐tillage system. The treatments were arranged in a bifactorial scheme, using seeds from biotypes (glyphosate‐resistant and glyphosate‐susceptible) with monthly periods of removal from field (one to 12 months). Seeds of each biotype were placed on the soil surface and covered with soil and straw to simulate no‐till conditions. The percentage of germinated, dormant, and dead seeds was evaluated every 30 days. The ryegrass seed bank of glyphosate‐susceptible and glyphosate‐resistant biotypes was reduced to 11 and 15% of dormant seeds, respectively, at the end of 12 months. However, there was no variation in germination, dormancy, and seed mortality between susceptible and glyphosate‐resistant ryegrass. Seeds of glyphosate‐resistant biotype and susceptible showed germination behavior with similar dynamics in the soil over a period of 12 months.     

Glyphosate resistance in four different populations of Lolium rigidum is associated with reduced translocation of glyphosate to meristematic zones

Weed populations with resistance to glyphosate have evolved over the last 7 years, since the discovery of the first glyphosate‐resistant populations of Lolium rigidum in Australia. Four populations of L. rigidum from cropping, horticultural and viticultural areas in New South Wales and South Australia were tested for resistance to glyphosate by dose–response experiments. All populations required considerably more glyphosate to achieve 50% control compared with a known susceptible population, indicating they were resistant to glyphosate. Translocation of glyphosate within these resistant populations was examined by following the movement of radiolabelled glyphosate applied to a mature leaf. All resistant plants translocated significantly more herbicide to the tip of the treated leaf than did susceptible plants. Susceptible plants translocated twice as much herbicide to the stem meristematic portion of the plant compared with resistant plants. These different translocation patterns suggest an association between glyphosate resistance in L. rigidum and the ability of glyphosate to accumulate in the shoot meristem.     

Absorption, translocation, and metabolism of propoxycarbazone-sodium in ALS-inhibitor resistant Bromus tectorum biotypes

Experiments were conducted to investigate the absorption, translocation, and metabolism of propoxycarbazone-sodium in acetolactate synthase-inhibitor resistant (AR and MR) and susceptible (AS and MS) Bromus tectorum biotypes. Absorption and translocation of ^l4C-propoxycarbazone-sodium were similar in all biotypes. One major and three minor metabolites were identified using reverse-phase high performance liquid chromatography. In all biotypes, 80% of the propoxycarbazone-sodium was metabolized by 72 h after treatment (HAT). However, propoxycarbazone-sodium was metabolized more rapidly in the MR biotype than in the other biotypes. The half-life of propoxycarbazone-sodium in the MR biotype was 8.9 h, which was 30, 36, and 40% shorter than in the AS, AR, and MS biotypes, respectively. When ¹⁴C-propoxycarbazone-sodium was applied with 1-aminobenzotriazole, a known cytochrome P450 inhibitor, metabolism decreased 20% 12 HAT in the MR biotype. These results indicate that resistance of the MR biotype to propoxycarbazone-sodium is due to a relatively rapid rate of propoxycarbazone-sodium metabolism compared to other B. tectorum biotypes and that cytochrome P450s may be involved in the metabolism. The fact that these populations evolved so quickly and with different resistance mechanisms is a concern as more ALS inhibitors are introduced into the production systems.     

Efficacy of cultural control methods for combating herbicide-resistant Lolium rigidum

Herbicide-resistant populations of annual ryegrass (Lolium rigidum) are estimated to affect crop production on about 5000 farms in southern Australia. In order to manage resistant populations, some farmers have adopted a two-to-three-year pasture phase which allows use of grazing by sheep, and non-selective herbicides to deplete the weed seed-bank. However, in low-to-medium rainfall zones, where financial returns from pastures are relatively low, farmers have generally combined cultural practices for weed management with the use of alternative herbicides, mainly trifluralin. Used singly, none of the currently available cultural techniques provides an adequate level of weed control. However, when used in carefully planned combinations, extremely effective ryegrass control can be achieved. Some of the important cultural practices for ryegrass control include delayed sowing (sometimes in conjunction with a shallow autumn cultivation); stubble burning; cutting the crop for hay or green manure, increased crop density and capture of weed seeds at harvest. Selection of crop species and cultivars with superior weed suppression potential is also receiving considerable attention. ©1997 SCI     

First glyphosate‐resistant Lolium spp. biotypes found in a European annual arable cropping system also affected by ACCase and ALS resistance

In Europe, glyphosate‐resistant weeds have so far only been reported in perennial crops. Following farmers' complaints of poor herbicide efficacy, resistance to glyphosate as well as to ACCase and ALS inhibitors was investigated in 11 populations of Lolium spp. collected from annual arable cropping systems in central Italy. Field histories highlighted that farmers had relied heavily on glyphosate, often at low rates, as well as in a non‐registered crop. The research aimed at elucidating the resistance status, including multiple resistance, of Lolium spp. populations through glasshouse screenings and an outdoor dose–response experiment. Target‐site resistance mechanism was also investigated for the substitutions already reported for EPSPs, ALS and ACCase genes. Three different resistant patterns were identified: glyphosate resistant only, multiple resistant to glyphosate and ACCase inhibitors and multiple resistant to glyphosate and ALS inhibitors. Amino acid substitutions were found at position 106 of the EPSPs gene, at position 1781, 2088 and 2096 of the ACCase gene and at position 197 and 574 of the ALS gene. Not all populations displayed amino acid substitutions, suggesting the presence of non‐target‐site‐mediated resistance mechanisms. After 39 years of commercial availability of glyphosate, this is the first report of multiple resistance involving glyphosate selected in annual arable crops in Europe. Management implications and options are discussed.     

Phenotypic and molecular genetic characterization indicate no major race‐specific interactions between Xanthomonas translucens pv. graminis and Lolium multiflorum

Bacterial wilt of forage grasses, caused by the pathogen Xanthomonas translucens pv. graminis (Xtg), is a major disease of forage grasses such as Italian ryegrass (Lolium multiflorum). The plant genotype‐bacterial isolate interaction was analysed to elucidate the existence of race‐specific responses and to assist the identification of plant disease resistance genes. In a greenhouse experiment, 62 selected plant genotypes were artificially inoculated with six different bacterial isolates. Significant differences in resistance were observed among L. multiflorum genotypes (P < 0·001) and in virulence (intensity of disease symptoms) among Xtg isolates (P < 0·001) using the area under the disease progress curve (AUDPC). No significant genotype‐isolate interaction (P > 0·05) could be observed using linear regression modelling. However, additive main effects and multiplicative interaction effects (ammi ) analysis revealed five genotypes which did not cluster close to the origin of the biplot, indicating specific interactions between these genotypes and some bacterial isolates. Simple sequence repeat (SSR) markers were used to identify marker‐resistance associations using the same plant genotypes and bacterial isolates. The SSR marker NFA027 located on linkage group (LG) 5 was significantly associated with bacterial wilt resistance across all six bacterial isolates and explained up to 37·4% of the total variance of AUDPC values. Neither the inoculation experiment nor the SSR analyses revealed major host genotype‐pathogen isolate interactions, thus suggesting that Xtg resistance, observed so far, is effective across a broad range of different bacterial isolates and plant genotypes.     

Metabolism, uptake and translocation of 14C-paraquat in resistant and susceptible biotypes of Crassocephalum crepidioides (Benth.) S. Moore

The metabolism, uptake and translocation of paraquat in resistant (R) and susceptible (S) biotypes of Crassocephalum crepidioides (Benth.) S. Moore (redflower ragleaf) at the 10-leaf stage was investigated. A study on the properties of leaf surface was carried out to examine the relationship between leaf surface characters and paraquat absorption. The extractable paraquat was not metabolized by the leaf tissue of either the resistant or susceptible biotypes. Differential metabolism, therefore, does not appear to play a role in the mechanism of resistance. Both biotypes did not show any significant difference in the amount of cuticle and trichome densities. Furthermore, both biotypes are identical in the structure of stomata, trichomes and epicuticular wax. The results of the leaf surface studies are in agreement with the findings of the uptake study. Both biotypes demonstrated no significant difference in absorption between the resistant and susceptible biotypes. However, 10% of the absorbed ¹⁴C-paraquat into the S biotype was translocated basipetally, but not in the R biotype. The results of this study suggest that in C. crepidioides , differential translocation may contribute to the mechanism of resistance at the 10-leaf stage.     

The effect of temperature on glutathione S-transferase activity and glutathione content in Alopecurus myosuroides (black grass) biotypes susceptible and resistant to herbicides

Herbicide‐resistant populations of Alopecurus myosuroides (black grass) have become widespread throughout the UK since the early 1980s. Previous observations in this laboratory have demonstrated that natural climatic fluctuations caused increases in endogenous glutathione S‐transferase (GST) enzyme activity in A. myosuroides plants as they mature, which is thought to be linked to herbicide resistance in this species. The present study has investigated the effects of plant growth at 10°C and 25°C, and reports GST specific activity and glutathione (GSH) pool size in resistant and susceptible A. myosuroides biotypes. Findings demonstrate differences in GST activity between resistant and susceptible populations, which are transient at lower growth temperatures. The GSH pool size was elevated at lower growth temperature in both biotypes. We speculate that these endogenous responses are part of a natural mechanism of acclimation to environmental change in this species and suggest that resistant plants are more able to adapt to environmental stress, as indicated in this instance by temperature change. These observations imply that the control of resistant A. myosuroides by graminicides may be more effective when applied at lower temperatures and at earlier growth stages.     

Glyphosate resistance in perennial Sorghum halepense (Johnsongrass), endowed by reduced glyphosate translocation and leaf uptake

BACKGROUND: In a large cropping area of northern Argentina, Sorghum halepense (Johnsongrass) has evolved towards glyphosate resistance. This study aimed to determine the molecular and biochemical basis conferring glyphosate resistance in this species. Experiments were conducted to assess target EPSPS gene sequences and ¹⁴C‐glyphosate leaf absorption and translocation to meristematic tissues. RESULTS: Individuals of all resistant (R) accessions exhibited significantly less glyphosate translocation to root (11% versus 29%) and stem (9% versus 26%) meristems when compared with susceptible (S) plants. A notably higher proportion of the applied glyphosate remained in the treated leaves of R plants (63%) than in the treated leaves of S plants (27%). In addition, individuals of S. halepense accession R₂ consistently showed lower glyphosate absorption rates in both adaxial (10–20%) and abaxial (20–25%) leaf surfaces compared with S plants. No glyphosate resistance endowing mutations in the EPSPS gene at Pro‐101–106 residues were found in any of the evaluated R accessions. CONCLUSION: The results of the present investigation indicate that reduced glyphosate translocation to meristems is the primary mechanism endowing glyphosate resistance in S. halepense from cropping fields in Argentina. To a lesser extent, reduced glyphosate leaf uptake has also been shown to be involved in glyphosate‐resistant S. halepense . Copyright © 2011 Society of Chemical Industry     

Evolution of resistance to diclofop-methyl in ryegrass (Lolium multiflorum): investigation of the role of introgression with related species

Populations of Lolium multiflorum (ryegrass) exposed to increasing levels of herbicide selection have variability in the rate of evolution of herbicide resistance. We tested the hypothesis that herbicide dose and gene flow with related species are important factors regulating the rate of evolution of herbicide resistance in L. multiflorum. Seeds of the latter were planted in pure stands and in mixture with Festuca rubra and subjected to four herbicide rates. The level of herbicide resistance attained by the offspring after two years of selection was evaluated. Evolution of resistance observed in the field was compared to that calculated by Gressel and Segel's rotational model. ©1997 SCI     

Differentially expressed genes in dicamba‐resistant and dicamba‐susceptible biotypes

In an ongoing effort to investigate the mechanism of auxinic herbicide resistance in Kochia scoparia (kochia), polymerase chain reaction‐based cDNA suppression subtractive hybridization was used to identify genes that are differentially expressed between dicamba‐resistant (HRd) and dicamba‐susceptible (S1) kochia biotypes in response to herbicide treatment. Both the HRd and S1 adaptor‐ligated cDNAs were used in separate hybridizations in order to generate biotype‐specific clones. A total of 710 cDNAs, representing putative differentially expressed mRNAs, were isolated and subjected to further screening. The false‐positive cDNAs were removed by conducting two colony hybridizations and at least one Northern hybridization. Differential or biotype‐specific expression was confirmed for six clones each from the HRd and S1 plants. The S1‐related genes were constitutively expressed at higher levels than in the HRd plants, but none had significant sequence similarity to known genes. Among the HRd‐related genes, HRd‐88 had 42% amino acid sequence identity to a conserved domain within thiol peptidases, which might be involved in auxin‐regulated gene expression. The constitutively expressed and inducible (by the dicamba treatment) HRd‐39 had 40% identity and 60% similarity to a domain from the Fe(II)/α‐ketoglutarate‐dependent hydroxylase superfamily. The HRd‐39 gene product had the characteristics of an enzyme that is able to detoxify dicamba via oxidative hydroxylation and thus its overexpression might confer the dicamba resistance phenotype.     

A survey of Lolium rigidum populations in citrus orchards: Factors explaining infestation levels

The presence of herbicide‐resistant Lolium rigidum in Mediterranean (Spanish) citrus orchards was reported in 2005 and it poses a serious threat to crop management. The main objective of this research was to investigate which components could be responsible for the persistence of annual ryegrass populations in Mediterranean mandarin and orange orchards. This is the first study regarding L. rigidum populations in Mediterranean citrus orchards. Surveys were conducted in 55 commercial citrus orchards in eastern Spain in 2013 by interviewing technicians who were working in cooperatives about crop management. The level of infestation by L. rigidum and the presence of harvester ants (Messor barbarus) then were estimated in the same orchards. The variables were subjected to a two‐dimensional analysis and both univariate and multivariate logistic regression models were fitted for each of the three L. rigidum density levels that had been established. The multivariate models showed the significant factors that were associated with various L. rigidum densities: (i) at a low density, the herbicides that were applied, the number of applications in 2013 and the type of irrigation (flood or drip); (ii) at a medium density, the presence of harvester ants; and (iii) at a high density, the herbicides that were applied in 2013. The results indicated that drip irrigation and one application of glyphosate mixed with other herbicides (or herbicides other than glyphosate) were associated with a lower L. rigidum density. The alternative management options that are presented here should help farmers to reduce weed problems in Mediterranean citrus orchards. Future research is required to better understand the presence of herbicide‐resistant populations, as well as the possible beneficial presence of granivorous ant species.