Literature DB >> 31935213

Growth behavior and glyphosate resistance level in 10 populations of Echinochloa colona in Australia.

Gulshan Mahajan1, Vishavdeep Kaur1, Michael Thompson1, Bhagirath Singh Chauhan1.   

Abstract

Recently, poor control of Echinochloa colona with glyphosate has been reported in no-till agriculture systems of the northern grain region (NGR) of Australia. Two experiments were conducted using 10 populations of E. colona selected from the NGR of Australia to understand differences in their growth behavior and resistance pattern. Growth studies revealed that these populations differed in plant height (53-70 cm plant-1), tiller production (30-52 tillers plant-1), leaf production (124-186 leaves plant-1) and seed head production (37-65 seed heads plant-1). Days taken to seed heads and shoot biomass in these populations ranged between 40-48 d and 21-27 g plant-1, respectively. Seed production in these populations ranged between 5380 and 10244 seeds plant-1; lowest for population B17/25 and highest for population B17/13. Correlation studies revealed that seed number plant-1 had a positive correlation with tiller number plant-1 (r = 0.73) and negative relation with days taken to seed head initiation (r = - 0.65). The glyphosate dose-response study showed a wide range of responses in these populations and the glyphosate dose required to kill 50% plants (LD50 values) was estimated between 161 to 2339 g a.e. glyphosate ha-1. LD50 values of populations B17/16, B 17/34 and B17/35 were 1086, 2339 and 1153 g ha-1, respectively, making them 6.7, 15.1 and 7.2-fold resistant to glyphosate compared with the susceptible population B17/37. Growth behavior and seed production potential in these populations had no correlation with the resistance index. These results suggest that some populations of E. colona are highly problematic; for example, population B17/34 was not only highly glyphosate-resistant, but also produced a high seed number (9300 seeds plant-1). This study demonstrated that there is a possibility of great risk with the increased use of glyphosate for managing E. colona in the NGR of Australia. The results warrant integrated weed management strategies and improved stewardship guidelines are required for managing glyphosate-resistant populations of E. colona and to restrict further movement of resistant populations to other regions of Australia.

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Year:  2020        PMID： 31935213      PMCID： PMC6959982          DOI： 10.1371/journal.pone.0221382

Source DB:  PubMed          Journal:  PLoS One        ISSN： 1932-6203            Impact factor:   3.240


Introduction

Echinochloa colona (L.) Link (C4 plant) has emerged as a major weed in summer crops in Australia and competes highly for water, sunlight and nutrients [1, 2]. E. colona is widely distributed in the northern grain region (NGR) of Australia [3, 4, 5] and it costs Australian agriculture AU$ 14.7 million annually [6]. Therefore, it affects the economy of Australian agriculture enormously. Emergence of multiple cohorts in the summer season, along with high capacity for seed production and seed dispersal have allowed the spread of E. colona throughout the NGR of Australia. The seeds remain viable in the soil for more than one year, causing continuous recruitment [7]. In Australia, intraspecific variations in E. colona have been reported on the basis of genetic diversity [8]. Morphological studies of these populations may increase our knowledge further and identify how these populations adapt to climate change and play a role in invasiveness. A minor change in morphology or physiology of the plant may affect its adaptability in a changing climate and a large number of dispersed seeds in the field, combined with the ability of this weed to flower under a range of photoperiods, may contribute to its invasiveness [9]. In the NGR of Australia, E. colona is a very common weed in no-till fallow land and glyphosate spray is the most common management practice for managing this weed. Glyphosate was mostly used in orchards (high-value crops) when introduced in Australia during the 1970s, as it was relatively expensive [10]. However, in the 1980s, its price declined, and its application became a common practice for weed control in a pre-seeding and fallow situation in Australia, which enabled the growers to adopt the conservation tillage practice. Glyphosate disrupts the shikimate pathways, reducing aromatic acid production via inhibition of the chloroplast enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Presently, control of some populations of E. colona in the NGR has become difficult with glyphosate as it has evolved resistance. The first case of glyphosate-resistant E. colona was reported in the NGR in 2007 [11]. At present, 41 weed species have been reported as glyphosate-resistant worldwide [12]. The evolved resistance may be due to intensive and repeated use of glyphosate [13, 14]. E. colona has also evolved resistance to four other herbicide modes of action, in addition to glyphosate [15]. A better understanding of the differences between populations for control with glyphosate is essential for developing long-term strategies. Variation in growth, morphological and physiological characteristics may alter herbicide efficacy within a species. Efficacy of glyphosate can be affected by plant species, population, plant development stage and environmental conditions [16]. Further, herbicide-resistant populations can spread from one area to another through pollen, seed or other propagules [17, 18]. Therefore, it is important to understand characteristics of resistant populations of a specific area to make better decisions and long-term strategies for weed control [19, 20]. A dose-response experiment is often conducted to assess the level of resistance in different populations. The dose-response experiment identifies a dose of an herbicide that provides a 50% reduction in shoot biomass [21]. In the NGR of Australia, there is variability in control of E. colona with glyphosate. We hypothesized that the dose required to kill 50% plants (LD50 value) may vary between populations due to development of different levels of glyphosate resistance. It was also hypothesized that the reproduction potential of these populations may differ due to variability in the resistance factor. Information on resistant factor, and growth and reproduction behavior of these populations is limited in the NGR of Australia. Keeping these points in view, this study was planned to evaluate the growth, reproduction behavior and level of glyphosate resistance in different populations of E. colona. In this study, one experiment evaluated the growth and reproduction behavior of 10 populations of E. colona from the NGR of Australia and another experiment evaluated the sensitivity of these populations to glyphosate.

Results and discussion

Growth and seed production

Analysis of variance (ANOVA) for various parameters of E. colona populations has been presented in Table 1. Amongst populations, the final plant height ranged from 53 to 70 cm, where the lowest was B17/35 and highest was B17/16. Populations B17/16, B17/17 and B17/25 attained a similar height, however, they were taller than populations B17/34 and B17/35 (Table 2). Tiller number among different populations ranged between 30 to 52 plant-1, where the lowest was B17/25 and highest was B17/49 (Table 2). Populations B17/7, B17/12, B17/13 and B17/49 produced similar tiller numbers plant-1, however, their tiller production was higher than populations B17/25 and B17/35. Leaf numbers in different populations varied from 124 to 192 leaves plant-1, where the lowest was population B17/16 and highest was population B17/34. Leaf production (numbers plant-1) remained similar for populations B17/34, B17/35 and B17/49, however, leaf production in these populations was higher than populations B17/16 and B17/25. All populations produced similar numbers of seed heads except for B17/25, which produced lower numbers than the other populations (Table 2).
Table 1

Analyses of variance for various parameters in different populations of Echinochloa colona.

SourceDegree of freedom(df)Plant height (cm)Tiller (number plant-1)Leaf (number plant-1)Seed head (number plant-1)Seed head weight (g plant-1)Shoot biomass (g plant-1)Root biomass (g plant-1)Days to seed head initiation (d)Seed production (number plant-1)
Replication72485014259852134729.72205.957.537.345900730
Treatment9203323434058212.539.850.143.018826700
Error6335489321812.7916.327.99.54701360
Table 2

Morphological traits and seed production potential of different populations of Echinochloa colona (mean ± standard error of eight replicates).

PopulationPlant height (cm)Tiller (number plant-1)Leaf (number plant-1)Seed head (number plant-1)Seed head weight (g plant-1)Shoot biomass (g plant-1)Root biomass (g plant-1)Days to seed head initiation (d)Seed production (number plant-1)
B17/762.9 ± 5.847 ± 6.2135 ± 1653 ± 107.7 ± 1.024.8 ± 5.912.7 ± 0.842 ± 1.87022 ± 1245
B17/1261.2 ± 4.947 ± 5.9164 ± 1564 ± 77.5 ± 0.8724.2 ± 5.413.0 ± 1.941 ± 0.98837 ± 1245
B17/1363.7 ± 4.150 ± 4.2156 ± 1159 ± 48.5 ± 0.6925.1 ± 5.318.0 ± 2.640 ± 0.810244 ± 1676
B17/1670.3 ± 7.240 ± 4.6124 ± 1153 ± 39.8 ± 0.9227.3 ± 6.510.1 ± 1.545 ± 1.16986 ± 895
B17/1766.9 ± 6.941 ± 5.0134 ± 1760 ± 78.1 ± 0.8022.7 ± 5.59.7 ± 0.842 ± 0.67801 ± 1004
B17/2566.7 ± 5.830 ± 3.8132 ± 1037 ± 46.2 ± 0.8122.1 ± 5.313.7 ± 1.945 ± 1.45380 ± 729
B17/3456.2 ± 5.243 ± 3.9192 ± 1463 ± 57.9 ± 0.6620.9 ± 4.312.5 ± 1.344 ± 1.89295 ± 892
B17/3553.1 ± 6.139 ± 4.3186 ± 1865 ± 66.2 ± 0.6020.9 ± 5.116.1 ± 3.448 ± 1.76130 ± 893
B17/3763.2 ± 6.343 ± 5.1162 ± 759 ± 57.5 ± 0.9121.1 ± 4.512.4 ± 2.343 ± 0.76387 ± 768
B17/4962.9 ± 6.252 ± 4.7166 ± 1864 ± 69.9 ± 0.8825.4 ± 5.811.9 ± 1.544 ± 0.78298 ± 1214
LSD (0.05)5.77.030.514.21.73.8NS2.72136

NS: nonsignificant.

NS: nonsignificant. The weight of seed heads among different populations varied from 6.2 to 9.9 g plant-1. It was similar for populations B17/7, B17/12, B17/25, B17/34, B17/35, and B17/37 (6.2 to 7.9 g plant-1), however, these populations had a lower seed head weight than populations B17/16 (9.8 g plant-1) and B17/49 (9.9 g plant-1). Shoot biomass among different populations ranged between 20.9 to 27.3 g plant-1 (Table 2). Shoot biomass remained similar for populations B17/13, B17/16 and B17/49, however, in these three populations, shoot biomass was significantly higher than populations B17/34, B17/35 and B17/37. Root biomass did not vary among populations (Table 2). Time taken to seed head initiation in different populations varied from 40 to 48 d. Populations B17/7, B17/12, B17/13, and B17/17 took a similar time for seed head initiation (40–42 d) and produced seed heads earlier than populations B17/16, B17/25 and B17/35, of which B17/35 took the longest (48 d). Seed production in different populations varied from 5380 to 10244 seeds plant-1; where the lowest was population B17/25 and highest was population B17/13. Populations B17/12, B17/13, B17/34 and B17/49 produced a similar number of seeds (8298–10244 plant-1), with their seed production being higher than populations B17/25 and B17/35. A linear positive correlation was found for seed number with tiller number plant-1 (r = 0.73) (Table 3). Tiller number plant-1 had a positive correlation with seed heads plant-1 (r = 0.66). Seed head plant-1 had a negative relation with shoot biomass plant-1 (r = - 0.73); however, relation of seed head plant-1 with leaf number plant-1 was positive (r = 0.67). Shoot biomass had a positive relation with seed head weight (r = 0.76). (Table 3). Plant height had a negative relation with leaf number plant-1 (r = - 0.91). Seed number plant-1 had a negative relation with days taken to seed head initiation (r = - 0.65).
Table 3

Correlation of morphological traits with seed number and R/S factor in different populations of Echinochloa colona (n = 10).

ParameterPlant height (cm)Tiller (number plant-1)Leaf (number plant-1)Seed head (number plant-1)Seed head weight (g)Shoot biomass (g plant-1)Root biomass (g plant-1)Days to seed head initiation (d)Seed production (number plant-1)Resistant index
Tiller(number plant-1)-0.14
Leaf(number plant-1)-0.91*0.28
Seed head (number plant-1)-0.560.66*0.67*
Seed head weight(g plant-1)0.460.60-0.210.28
Shoot biomass (g plant-1)0.610.44-0.56-0.73*0.76*
Root biomass(g plant-1)-0.490.120.410.058-0.41-0.18
Days to seed head initiation (d)-0.34-0.570.23-0.100-0.28-0.33-0.037
Seed production (number plant-1)-0.130.73*0.330.560.470.280.22-0.65*
Resistant index-0.50-0.100.560.290.065-0.26-0.0170.440.23

Critical value of r at 5% = 0.63;

* indicates significant relation.

Critical value of r at 5% = 0.63; * indicates significant relation. The results of this study demonstrated that characteristics like high tillering capacity allow E. colona populations to produce a high leaf number that resulted in a large number of seed heads and seeds. Therefore, there is a need to restrict high tiller production in E. colona to reduce seed numbers. A recent study on crop-weed interference suggested that crop competition could reduce tiller numbers in E. colona [22]. In Australia, farmers are following wide and skip row spacing in crops such as cotton (Gossypium hirsutum L.), mungbean [Vigna radiata (L.) R. Wilczek] and sorghum [Sorghum bicolor (L.) Moench]; therefore, wide space between the rows could provide a better opportunity to E. colona populations with a high tillering capacity nature as compared to when crops are sown in narrow rows. In these environments (wide rows and fallows), E. colona could attain its high tillering potential and could produce a high seed number. E. colona in the present study produced tillers in the range of 39 to 52 plant-1; however, in a previous study conducted in Greece, it produced tillers in the range of 115 to 131 plant-1 [23]. This difference could be due to genotype x environment interactions and differential pot size. In the present study, we observed that populations B17/13 and B17/49 had higher tillers than populations B17/25 and B 17/35. This also suggested that genotypes and environmental interactions played a role in influencing tiller numbers per plant in E. colona populations. The regions where populations are of high tillering capacity are expected to suffer a high crop yield loss due to high E. colona competition. The high seed number observed in populations B17/12, B17/13, B17/34 and B17/49 was largely attributed to a high number of tillers. The number of leaves and seed heads were similar between populations B17/13 and B17/35; however, seed production was lower in B17/35, which could be due to the lower tiller production and seed head weight in B17/35. The time taken to seed head initiation in the present study was similar to a study conducted in northern Greece, in which E. colona attained seed heads between 39 to 45 days after transplanting [23]. The population B17/35 (selected from the Moree region) took a longer time for seed head initiation than other populations (Fig 1). In a previous study in South-East Asia, 12 E. colona populations were studied and it was found that time for seed heads in different populations varied with latitude and plants from a high latitude attained seed heads earlier than from a low latitude [24]. This suggested that growth duration in different populations of E. colona may vary with geographical location. In the present study, the negative relationship between seed head initiation and seed number revealed that late-maturing populations produced fewer seeds as was the case for populations B17/16 and B17/35 when compared with population B17/13. These results suggest that diversity in E. colona traits could result in differential responses to herbicides, cultural practices, and resistance evolution. For example, the early vigor trait in E. colona is an important trait that could affect early crop-weed competition [25] and therefore, management of such populations at an early stage is required to increase crop production and reduce the weed seed bank in the soil.
Fig 1

Location of 10 populations of Echinochloa colona selected from the northern grain region (NGR) of Australia.

In this study, E. colona populations differed in their seed production potential, which ranged between 5380 to 10240 seeds plant-1. Differential seed production in E. colona populations could play an effective role in its spread and population establishment [26]. High seed yields in populations B17/12, B17/13, B17/34 and B17/49 were largely based on a greater number of seed heads and leaf numbers plant-1. High production of leaves in these populations probably maintained a better supply rate of carbon assimilates to seeds. In one study on Brassica, it was found that variation in the supply of carbon assimilates to seeds at or immediately after anthesis could cause a variation in seed production in different populations [27]. Some authors also highlighted the role of the supply of carbon assimilates in determining the seed number in pea (Pisum sativum L.) plant [28]. The present study also revealed that tiller number per plant played a large role in seed production along with leaf number per plant. Population B17/35 had high leaf production but could not produce higher amounts of seeds like B17/13 and B17/34 did, because it had lower tiller production than B17/13 and B17/34. Although this study revealed that the supply of carbon assimilates after anthesis could be a major factor in determining seed production, we could not rule out the possibility of hormonal factors for variation in seed production in these populations. These results suggest that there is also a need to study nutritional and hormonal factors for variation in seed production in these populations [27]. Our study (second experiment) also found that the LD50 value of glyphosate for these populations varied. These results suggest that in these populations, seed viability, seed persistence and fitness penalty may differ and therefore systematic studies need to be investigated. Such knowledge of seed production in these populations is required for understanding the evolution and spread of herbicide resistance particularly for herbicide-resistant populations.

Response to glyphosate

Out of 10 E. colona populations collected from the NGR of Australia, three populations (B17/16, B 17/34 and B17/35) had greater than 80% survival following treatments with 325 to 2600 g a.e. ha-1 glyphosate. The probit analysis details for each population along with their level of significance is presented in Table 4. The dose-response study of glyphosate for these populations showed a wide range of responses (Fig 2A). The LD50 value of the tested populations ranged from 161 to 2339 g ha-1 (Fig 2A). The susceptible population B17/37 was easily controlled with glyphosate and had a LD50 of 161 g ha-1, below the normal use rate of this herbicide (650 g ha-1). The LD50 values of populations B17/16, B17/34 and B17/35 were 1086, 2229 and 1153 g ha-1, respectively, making them 6.7, 15.1 and 7.2-fold resistant to glyphosate compared with the susceptible population B17/37 (Fig 2B). Correlation studies revealed that growth behavior and seed production potential in these populations had no correlation with the resistance index (Table 4). The most resistant population B17/34 was from the Goondiwindi region, whereas the next most resistant populations, B17/35 and B17/16, were from the Moree and Narrabri regions, respectively. This study has revealed that E. colona populations in the NGR of Australia have different levels of resistance to glyphosate. No-till farming is quite popular in the NGR of Australia for moisture conservation. Therefore, growers use glyphosate in summer fallows to kill weeds and conserve moisture. Repeated and intensive use of glyphosate in this region has evolved glyphosate resistance in these populations [11]. Risk of glyphosate resistance evolution for weeds is higher in those areas where glyphosate has been used for a long time and with minimal soil disturbance [29]. In Australia, glyphosate-tolerant cotton varieties are very popular among cotton growers. Glyphosate-resistant E. colona populations may create serious situations in that production environment. The resistant factor for glyphosate in this study was similar to the first reported case of glyphosate resistance in Australia (7 to 11-fold resistance compared with a susceptible population); but that study was reported for rigid ryegrass (Lolium rigidum Gaud.) [30].
Table 4

Probit transformed response [Intercept + bx (covariate x are transformed using the base 10.0 logarithm)] for different Echinochloa colona populations, *represents significant (P<0.05).

PopulationResponseR2Pearson Goodness- of-fitChi squareSignificance level(Chi square)95% confidence interval
Lower boundUpper bound
B17/7y = - 6.92 + 2.99 x0.980.6380.73*151.0275.3
B17/12y = - 4.78 + 2.09 x0.942.4210.30*142.9282.2
B17/13y = - 6.27 + 2.45 x0.973.5600.17*302.1436.9
B17/16y = - 4.52 + 1.49 x0.990.6630.72*884.41356.5
B17/17y = - 5.79 + 2.52 x1.001.8640.39*157.5283.3
B17/25y = - 3.61 + 1.49 x0.933.0190.22*129.3342.1
B17/34y = - 4.96 + 1.47 x0.981.2490.54*1801.23500.3
B17/35y = - 4.43 + 1.45 x0.981.1850.55*937.71461.3
B17/37y = - 2.83 + 1.46 x1.003.6590.16*76.8227.4
B17/49y = - 4.74 + 1.77 x0.990.5620.58*375.2583.0
Fig 2

a) LD50 dose of glyphosate and b) glyphosate-resistant/susceptible factor in different populations of Echinochloa colona (bars followed by same letter are not significantly different on the basis of 95% confidence interval).

a) LD50 dose of glyphosate and b) glyphosate-resistant/susceptible factor in different populations of Echinochloa colona (bars followed by same letter are not significantly different on the basis of 95% confidence interval). Earlier, glyphosate resistance in E. colona populations was also reported in Australia [11, 31]. There are a number of mechanisms responsible for glyphosate resistance [32], and different mechanisms may result in a different level of resistance [33]. Therefore, these studies suggests that these resistant populations may not carry the same resistance allele, which needs to be investigated. Many reports of glyphosate resistance in different weeds highlight that the reliance on glyphosate for weed control, in the long run, exerts a substantial selection pressure on weeds [34,35,36,37,38,39,40]. Therefore, integrated weed control should be strengthened to reduce selection pressure on these resistant populations, particularly in cotton paddocks. It is quite possible that the mechanism of glyphosate resistance in Australian E. colona populations might be different from resistant E. colona populations reported from California [41] as Australian populations of E. colona have adapted to a dry environment. Therefore, a systematic study is required to understand the evolution of glyphosate resistance in these populations. The evolution of glyphosate resistance in tropical E. colona in Australia suggests that there is a large risk with increased use of glyphosate in fallows and improved stewardship guidelines for glyphosate use are required in the NGR of Australia.

Conclusions

The present study on E. colona populations has increased our understanding of the physiological basis of differences in seed production due to variations in morphological characteristics and resistance behavior. It highlighted that growth parameters such as high tiller production in E. colona populations leads to more seed heads and in turn high seed production. The study further demonstrated that growth behavior and seed production potential in these populations had no correlation with the resistance index. However, this research has posed more questions than it has answered. This study suggested that populations such as B17/34 that are highly glyphosate-resistant, and also produced a high seed number (9300 seeds plant-1) are very problematic. Therefore, systematic research on weed biology, physiology and resistance mechanism is required to answer these questions for better understanding. Efforts need to be made to reduce the invasion of herbicide-resistant populations, such as B17/34. Movement of resistant populations (seeds) from one field to another field via different modes of transportation should be restricted. For the management of resistant populations, crop competition tactics (use of narrow row spacing, competitive cultivars, optimum seeding rates, cover crops, etc.) could be explored in an integrated weed management program to reduce the seed number/seedling recruitment of resistant populations. This study also suggested that there is a need to understand the likelihood of resistance transfer from resistant to susceptible populations through pollen-mediated gene flow and introgression. Such knowledge could be useful in restricting the further spread of glyphosate-resistant populations of E. colona.

Materials and methods

The study was conducted at the QAAFI weed science laboratory and screen house of the University of Queensland, Gatton, Australia. Seeds of 10 different populations of E. colona were collected from the NGR of Australia in March 2017. The coordinates and details of these populations are depicted in Fig 1 and Table 5 respectively. For collection of seeds, permission was obtained from the consultants through phone calls and personal meetings. Seeds were collected from 40–50 plants per site and over an area of >1ha. Seeds were shaken loose from the plants by hand for collection. Only matured plants were chosen for collection of seeds. For each population, about 10, 000 seeds were collected. Germination rate was tested to confirm seed viability and was found to be >80% for each population in both runs. The collected populations were designated on the basis of year and serial number in which these were collected, for example susceptible population B 17/37 indicates sample was collected in 2017 and 37 was the serial number of that population in the collection, while B stands for population. The seeds of each population were cleaned and stored in shade.
Table 5

Detail of 10 populations of E. colona collected from the northern grain region of Australia.

PopulationCoordinatesLocation/PlaceCrop
B17/727.50000000/151.69666667DalbyWheat-Fallow
B17/1230.268508/149.80481NarrabriWheat- Fallow
B17/1330.306499/149.811438NarrabriWheat-Fallow
B17/1630.09099349/149.64890NarrabriLathyrus-wheat/chickpea
B17/1730.38230/149.59679NarrabriFallow
B17/2528.31500000/148.68916667St GeorgeCanal side
B17/3428.58305556/150.36888889MoreeWheat fallow
B17/3529.95805/149.8339MoreeCotton fallow
B17/3727.5514/152.3428GattonWheat-Fallow
B17/4927.5514/152.3428GattonWheat-Fallow

Growth response experiment

In this experiment, 10 populations of E. colona were grown in pots replicated four times. The pots were kept on benches placed outside the screen house. Pots were filled with potting mix (Crasti & Company Pty Ltd, Sydney, Australia). Initially, 10 seeds were sown per pot at 1 cm depth and after establishment, one plant per pot was maintained. The experiment was conducted twice. The first run was started on 27 September 2018 and harvested on 6 December 2018. The second run was started on 3 December and harvested on 5 February 2019. Pots used in the experiment were 20 cm in height and arranged in a completely randomized design. The pots were regularly irrigated. At maturity, plant height was measured from the base of the plant to the tip of the uppermost leaf of the plant. Days taken to seed head initiation was recorded in each pot. For estimating seed production per head, two intact seed heads were chosen randomly from each plant. For the total number of seeds, each rachilla segment (pedicel base) was counted and then, averaged for seeds per head. At harvesting time, tiller numbers, leaf numbers and seed heads per plant were also counted. Harvesting was done when ~80% seed heads matured. At harvest, seed heads were separated from the plants for measuring shoot biomass. After that, all aboveground shoot biomass from each plant was placed separately in a paper bag and dried in an oven at 70°C for 72 hours before being weighed. For root weight data, pots containing potting mix with the root system were first dried in an oven at 70°C for 72 hours. After that, roots were removed from each pot by shaking loose the potting mix. Root biomass of each plant was then measured. Drying of potting mix in an oven helped in the separation of the root system from the potting mix.

Glyphosate dose-response experiment

Seeds of 10 populations including glyphosate susceptible population B 17/37 were sown in pots (9 cm diameter and 10 cm height) filled with potting mix (Crasti & Company Pty Ltd, Sydney, Australia). Initially, 10 seeds were sown per pot at 1 cm depth and after establishment, five plants per pot were maintained. Pots were kept in a screen house under natural light and temperature conditions. The experimental design was a factorial with four replicates where the first factor was population and the second factor was glyphosate dose [0x (no herbicide; control), 0.5x, 1x, 2x, and 4x]. The 1x dose was the recommended dose (650 g a.e. ha−1) for glyphosate. The commercial product of glyphosate named ‘Glymount’ containing active constituent 450 g/L was used and under fallow condition, its labelled rate for control of E. colona in Australia was 0.8–1.2 L ha-1 (commercial dose). The experiment was conducted twice. The first run was started on 5 December 2018 and harvested on 14 January 2019. The second run was started on 25 January 2019 and harvested on 6 March 2019. Glyphosate application was done on 24 December 2018 in the first run and 13 February 2019 in the second run. Plants were kept well-watered and fertilized. Glyphosate was sprayed using a research track sprayer. Plants were treated at the 4–5 leaf stage using a spray volume of 108 L ha-1 and Teejet XR 110015 flat fan nozzles were used. Plants were allowed to grow for 21 days after treatment (DAT) to determine glyphosate sensitivity. Plant survival was assessed 21 DAT, and plant aboveground biomass was harvested, dried for 72 hours at 70°C, and weighed. As there were five seedlings for each population in four replications and the experiment was repeated twice; therefore, results were based on 40 seedlings per population. The susceptible population used in the study was B 17/37.

Statistical analyses

The first experiment was conducted in a randomized completely block design and the second experiment was conducted in a randomized completely block design with a factorial arrangement. In both experiments, there was no interaction between experimental runs and treatments; therefore, the data of the two runs were pooled for ANOVA. All the data met assumptions of normality of residuals and homogeneity of variance. Data of the first experiment were subjected to analysis of variance (ANOVA) using the software Elementary Designs Application 1.0 Beta (AgriStudy.com: www.agristudy.com) (verified with GENSTAT 16th Edition; VSN International, Hemel Hempstead, UK). Treatment means were separated using Fisher’s protected least significant differences (LSD) at P≤0.05. The relationships between parameters were assessed using linear regression analysis (see S1 Data). For the second experiment, LD50 (the dose required to kill 50%) estimates were generated using Probit analysis [IBM SPSS Statistics 20.0 (SPSS, Inc., Chicago, IL, USA)]. The level of significance was tested with a Chi-Square goodness of fit test (see S1 Text). When the calculated value of Chi-Square goodness of fit test was greater than the table value, the null hypothesis was rejected and it was concluded that there was a significant difference between the observed and the expected value and vice versa with values lower than the table value. LD50 values for each population were compared using confidence interval overlap. The resistance index (resistance/susceptibility ratio) was calculated on the basis of the LD50 value to compare the resistance level among different populations.

Additional data and statistical analysis information.

(XLSX) Click here for additional data file.

Probit analysis information.

(DOCX) Click here for additional data file. 9 Oct 2019 PONE-D-19-21773 Growth behavior and glyphosate resistance level in 10 biotypes of Echinochloa colona in Australia PLOS ONE Dear Dr Mahajan, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR: The comments of three reviewers have been received as presented below. Two of them recommended major revision while another one recommended rejection. Please revise the manuscript according to the comments of the three reviewers. Particularly, Rev.# 1 felt that the definition of herbicide resistance should be demonstrated according to reliable literature. Besides, two reviewers reviewed that statistical results have not been sufficiently presented and discussed. Please address all comments one by one and attach a response letter of a list or explanation of changes made to the manuscript in your revised version. ============================== We would appreciate receiving your revised manuscript by Nov 23 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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We look forward to receiving your revised manuscript. Kind regards, Xiao Guo, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 1. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the collection sites access and, if no permits were required, a brief statement explaining why. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Yes Reviewer #3: Yes ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No Reviewer #3: No ********** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: No Reviewer #3: Yes ********** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Overall, the manuscript is written well and clearly. The subject should be of interest of readers of weed science community. My biggest question is one of semantics, I believe. I read the criteria for listing a species as resistant on the website for International Survey of Herbicide Resistant Weeds (I. Heap; http://weedscience.com/Documents/ResistanceCriterion.pdf). There are problems with the research methods used that do not support the results presented. The definition of herbicide resistance is based on plant/population survival not plant growth (biomass). Check the WSSA website for the accepted definition of herbicide resistance. In the case of these studies where there was only few plants per pot, population survival cannot be effectively determined and therefore, it is not possible to definitively characterize the resistance status of the collected populations. Once characterized, plant biomass data can be used to indicate the robustness of the resistance mechanism. However, this is only once resistance has been confirmed. The recommendation is that there is a need for further studies aimed at characterizing the resistance status of the collected biotypes based on survival of a reasonable population size (e.g. 40-50 seedlings). A known susceptible biotype needs to be included and replication is not needed if there is a sufficiently large population size. See methods in Owen et al 2016. The whole plant dose response experiments appear to provide more definitive evidence of resistance. However, based on the author clarification of experimental design the dose response experiment may not be confirmatory if the study was not set up to statistically compare the putative resistant population with the susceptible population. Does the work also need to demonstrate heritability in order to confirm resistance in this case? I suspect literature evidence may be important since experimental evidence is lacking. Is there any plan to relate these phenotypic response back to management, cropping systems, location, and/or soils? SPECIFIC COMMENTS L 97: Please describe results and discussion in two separate sections. L 251-254: How many plants per site were collected? What was the area occupied by those plants per location? Was there a protocol for collecting seed that defined the area of collection? What was the cropping and herbicide use history of each sampled field? How large a seed sample per population was obtained? Were sample sizes similar among populations? What was the germination percent for the sampled populations and did seed viability/germination rate stay constant for the two runs? The authors might consider including the designated names of collected biotypes. L 285-287: How did you come up with 650 g/ha rate of glyphosate as 1X recommended rate? This is a low rate of glyphosate to go with. Who recommended this rate and for what situation? If this was based on chemical fallow, don’t you think 1260 g/ha rate will be much appropriate to go with. No adjuvant was included in those glyphosate doses? Dec 5, 2018 to Jan 14, 2018? L 307-314: The section on statistical analyses is not complete with regard to the glyphosate dose-response analysis. How did you run the probit analysis? Provide the equation used and explained it here. How did you calculate the resistance index when there was no known susceptible biotype included? Figure 2: Are those GR50 values or Resistant/susceptible factor significantly different from each other? If yes, What statistical test did you perform to find this out? Please explain in the figure legend. I don’t see any depository for the raw data on these experiments. Please provide all your raw data from where you build these tables and figures. This is the most important requirement of the journal. Reviewer #2: The topic is important. The whole paper is clear and well- structured. The experiment is reasonably designed. The result is mostly clear and fully described but I personally think that the result is probably not very well-dug. More analysis about the correlations among variables, the effect comparison of glyphosate usage could be conducted. 1. Please make sure with the journal whether the material and method parts are at the end of the paper or after introduction. 2. I might miss some information, but it is better having more descriptions about the codes of the species, for example, B17/34, does it have some relationship with the sampling (Fig.1)? It is not so clear and very easy to get confused with these codes when reading. 3. The Fig.1 seems not so clear and short of legend and north arrow. The color you chose is too similar with each other which makes the whole figure looks not so good. 4. There are correlations among leaf, tiller, amount of seeds, etc. Tables are good, but if you have several scatter plots to show these relationships, it might be clearer. 5. For the influences of height, leaf, etc. to the final tiller of the species, there might be interactive effects among different x variables. Is it better you use the multi- correlation analysis, i.e., y = ax1+bx2+cx3…? This is just a suggestion and you could think about this. 6. Line 134~135, it is not clear using “target” here. 7. I am not sure, for the unit, you use number plant-1 etc., do you think plant-1 here is proper? Is it better just delete it and make some explanations in the text? Because all unit is measured by one individual. Or you just use stem-1or individual-1? 8. In table 2, delete the last number “1”. 9. For table 3, make clear the meaning of each column at the title. “Probit analysis detail” is not clear. 10. Line 275, “by shaking” is not precise, you might find another word to describe this operation. Measures taken to reduce the root loss is better also addressed. 11. Line 305~306, more explanation might be needed for the “Fisher’s protected LSD”? At least give the full name of LSD when it is firstly mentioned. 12. Is the different doses of the glyphosate used well compared? It is not clear in the result. 13. For reference 6 and 7, there are italic styles, make sure with journal and be consist with the others. 14. You declared that the data is available in the supporting information but I did not see any materials attached, please explain this. Reviewer #3: Two experiments were carried out in this study to compare the growth behavior and glyphosate resistance among 10 biotypes of Echinochloa colona in Australia. The growth experiment showed that characteristics like tall nature and high tillering capacity allow E. colona to produce a high leaf number that result in a large number of seed heads and seeds. The glyphosate dose-response experiment showed a wide range of GR50 value in these biotypes, and the biotype B17/34 showed the strongest resistance to glyphosate. The two experiments were conducted rigorously with appropriate replication and sample sizes, and the conclusions were drawn appropriately based on the data presented. In addition, the language of this manuscript is well written and clear. The analysis of variance (ANOVA) and Fisher’s protected LSD were applied to test the difference of growth behavior of E. colona among 10 biotypes. And the probit anaylsis were applied to estimate the GR50 value. These statistical methods were performed appropriately, but the outcome of these statistical results have not been sufficiently presented. Furthermore, I suggest the multivariate analysis on the growth behavior and glyphosate resistance can be applied in this study to test the differences among 10 biotypes. Moreover, there are several major and specific comments on this manuscript: 1. The introduction can be reduced to 3 or 4 paragraphs to make the background and aim clearer. The outline of the introduction can refer to: 1) The Echinochloa colona as a noxious weed emerged in Australia crops. 2) The biological characteristics of growth and reproduction of E. colona. 3) The glyphosate is widely used in Australia to eliminate the E. colona, but induced a variety of glyphosate resistant biotypes. 4）the aim and scientific questions of this study 2. The author combined the “results” and “discussion” in the manuscript. Although this format can be accepted by some journals, but I suggest divided this part into “results” and “discussions” separately. In the present manuscript, the result of two experiment were discussed respectively, and I can not find their relationships. If there is an independent section of discussion, the relationships between the two experiment and their implications can be better integrated. 3. The author proposed “growth behavior and seed production potential in these biotypes had no correlation with the resistance index” (LINE 240-241). However, the statistical methods for analyzing the correlation between growth traits and resistance index were not given in the section of “statistical analysis”. Also, I can not find the relevant result which support this conclusion in the context. 4. The seed of 10 biotypes of E. colona were collected from a large area in northern grain region in Australia. So, do the geographical differences effect on the growth behavior and glyphosate resistance among 10 biotypes of E. colona. I suggest the multivariate analysis such as PCA or RDA can be applied to study whether the biotypes from closer sites have similar characteristics. Moreover, the correlation (or non-correlation) between growth behavior and resistance index may also be revealed by the multivariate analysis. Here are also several specific comments: 5. Figure 1. It’s better to give a small-scale map of Australia, and pointed out the location of northern grain region. 6. Table 1. the standard deviation of these variables should be given. And as the LSD was performed, I suggest the author add lowercase superscript letters after the values to indicate significance at the 0.05 level for the differences among biotypes. 7. LINE 64: “shikmate pathway” might be “shikimate pathway” 8. Line 251-254: it better to give more information on the seed collected location or the farm, like the crop type, soil type, glyphosate dose and so on, which can help readers know the growth environment of these biotypes. 9. Line 252: how to identify different biotypes? Do they just collected from different sites，or have distinct heritable characteristics? 10. Line 301: I did not find the ANOVA result 11. Line 307-308 Which variable did the “GR50” in this study based on? The shoot biomass, tiller number, or individuals? ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Liping Li Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. 4 Nov 2019 ============================== ACADEMIC EDITOR: The comments of three reviewers have been received as presented below. Two of them recommended major revision while another one recommended rejection. Please revise the manuscript according to the comments of the three reviewers. Particularly, Rev.# 1 felt that the definition of herbicide resistance should be demonstrated according to reliable literature. Besides, two reviewers reviewed that statistical results have not been sufficiently presented and discussed. Please address all comments one by one and attach a response letter of a list or explanation of changes made to the manuscript in your revised version. ============================== We have addressed all comments of three reviewers and attempt has been made to incorporate the critical comments of reviewer#1 carefully. 1. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the collection sites access and, if no permits were required, a brief statement explaining why. Yes, permissions were obtained from the consultants through phones and personal meetings. Reviewers' comments: ________________________________________ Reviewer #1: Overall, the manuscript is written well and clearly. The subject should be of interest of readers of weed science community. My biggest question is one of semantics, I believe. I read the criteria for listing a species as resistant on the website for International Survey of Herbicide Resistant Weeds (I. Heap; http://weedscience.com/Documents/ResistanceCriterion.pdf). There are problems with the research methods used that do not support the results presented. The definition of herbicide resistance is based on plant/population survival not plant growth (biomass). Check the WSSA website for the accepted definition of herbicide resistance. In the case of these studies where there was only few plants per pot, population survival cannot be effectively determined and therefore, it is not possible to definitively characterize the resistance status of the collected populations. We have also recorded the data for survival %. We again reanalysed the data and calculated LD50 value on the basis of survival %. We used several doses, so the number of plants used per pots were sufficient. Yes, for only one population, they were not enough. Once characterized, plant biomass data can be used to indicate the robustness of the resistance mechanism. However, this is only once resistance has been confirmed. The recommendation is that there is a need for further studies aimed at characterizing the resistance status of the collected biotypes based on survival of a reasonable population size (e.g. 40-50 seedlings). A known susceptible biotype needs to be included and replication is not needed if there is a sufficiently large population size. See methods in Owen et al 2016. There were five seedlings for each biotype in four replications. So 20 seedlings for each biotype. As the experiment was repeated twice; therefore, results are based on 40 seedlings per biotype. We had a susceptible biotype B 17/37. The data has been reanalysed for survival%. The whole plant dose response experiments appear to provide more definitive evidence of resistance. However, based on the author clarification of experimental design the dose response experiment may not be confirmatory if the study was not set up to statistically compare the putative resistant population with the susceptible population. Does the work also need to demonstrate heritability in order to confirm resistance in this case? I suspect literature evidence may be important since experimental evidence is lacking. We followed the publication of Gaines et al. 2012 for experimental design and planning that was published in Weed Technology and they did not demonstrate heritability in order to confirm resistance. Yes, we had a susceptible biotype named B 17/37 in these 10 biotypes. Gaines T.A., Cripps A., Powles S.B. Evolved resistance to glyphosate in junglerice (Echinochloa colona) from the tropical Ord River region in Australia. Weed Technology, 2012 26: p. 480-484. SPECIFIC COMMENTS L 97: Please describe results and discussion in two separate sections. Results and discussion are combined here according to the format of the journal. Moreover, combined results and discussion is good to avoid repetition. However at the end, we have concluded our results in a general discussion. L 251-254: How many plants per site were collected? What was the area occupied by those plants per location? Was there a protocol for collecting seed that defined the area of collection? How large a seed sample per population was obtained? Were sample sizes similar among populations? What was the germination percent for the sampled populations and did seed viability/germination rate stay constant for the two runs? The authors might consider including the designated names of collected biotypes. There were 40-50 plants per site and the area occupied by those plants per location was >1 ha. Seeds were collected directly from plants by shaking. Only matured plants were chosen for collection of seeds. There were about 10, 000 seeds per population. The sample size was different depending on infestation levels. The germination rate (seed viability) was >80% for each biotype in both the experimental runs. The collected biotypes were designated on the basis of year and serial number in which these were collected for example 17/37 was designated as sample was collected in 2017 and 37 was the serial number of that biotype in collection. L 285-287: How did you come up with 650 g/ha rate of glyphosate as 1X recommended rate? This is a low rate of glyphosate to go with. Who recommended this rate and for what situation? If this was based on chemical fallow, don’t you think 1260 g/ha rate will be much appropriate to go with. No adjuvant was included in those glyphosate doses? Dec 5, 2018 to Jan 14, 2018? The commercial product of glyphosate named Glymount containing active constituent 450 g/L was used and under fallow condition in Australia, its labelled rate is 0.8 -1.2 L/ha https://growchoice.com.au/wp-content/uploads/2015/05/Glymount-DFU.pdf. Therefore, this is not a low rate of glyphosate in Australia. No adjuvant was included in glyphosate doses. We have corrected the date- it is Jan14, 2019. L 307-314: The section on statistical analyses is not complete with regard to the glyphosate dose-response analysis. How did you run the probit analysis? Provide the equation used and explained it here. How did you calculate the resistance index when there was no known susceptible biotype included? Probit analysis was done using software IBM SPSS Statistics 20.0 (SPSS, Inc., Chicago, IL, USA). The equation has been provided and explained. Resistance index was calculated on the basis of susceptible biotype named B 17/37 on a factor basis. Equation (response curve) has been now provided for each biotype. The equation used for estimating response is as: y = [Intercept + bx (covariate x are transformed using the base 10.0 logarithm)] Figure 2: Are those GR50 values or Resistant/susceptible factor significantly different from each other? If yes, What statistical test did you perform to find this out? Please explain in the figure legend. In the revised version, we have now calculated LD50 value and these values were compared using confidence interval overlap as described in table 3. The probit analysis detail of each biotype has been provided in the supplementary file (SPSS software) . I don’t see any depository for the raw data on these experiments. Please provide all your raw data from where you build these tables and figures. This is the most important requirement of the journal. We have now provided all raw data on these experiments as supplementary file . Reviewer #2: The topic is important. The whole paper is clear and well- structured. The experiment is reasonably designed. The result is mostly clear and fully described but I personally think that the result is probably not very well-dug. More analysis about the correlations among variables, the effect comparison of glyphosate usage could be conducted. 1. Please make sure with the journal whether the material and method parts are at the end of the paper or after introduction. Yes, the material and method parts are at the end of the paper as per journal style. 2. I might miss some information, but it is better having more descriptions about the codes of the species, for example, B17/34, does it have some relationship with the sampling (Fig.1)? It is not so clear and very easy to get confused with these codes when reading. For B17/34 indicates B is for biotype; 17 indicates sample was collected in 2017 and 34 is the serial number in collection. We reported the biotypes as per our designation in the weed library. 3. The Fig.1 seems not so clear and short of legend and north arrow. The color you chose is too similar with each other which makes the whole figure looks not so good. The figure 1 has been modified with clear legend and north arrow. 4. There are correlations among leaf, tiller, amount of seeds, etc. Tables are good, but if you have several scatter plots to show these relationships, it might be clearer. Many correlation between different parameters were significant, therefore, to avoid the confusion, we preferred to show this presentation in table form. 5. For the influences of height, leaf, etc. to the final tiller of the species, there might be interactive effects among different x variables. Is it better you use the multi- correlation analysis, i.e., y = ax1+bx2+cx3…? This is just a suggestion and you could think about this. We have correlation matrix in table 2 and critical differences were observed at 5% level of significance. 6. Line 134~135, it is not clear using “target” here. We have replaced this with ‘restrict high’ 7. I am not sure, for the unit, you use number plant-1 etc., and do you think plant-1 here is proper? Is it better just delete it and make some explanations in the text? Because all unit is measured by one individual. Or you just use stem-1or individual-1? It is the standard trend that we have followed and easy to understand. 8. In table 2, delete the last number “1”. Deleted 9. For table 3, make clear the meaning of each column at the title. “Probit analysis detail” is not clear. Added. However, all detail of probit analysis has been provided in supplementary file. 10. Line 275, “by shaking” is not precise, you might find another word to describe this operation. We feel that shaking best describes our method and have used this word in our previous papers to describe this. 11. Line 305~306, more explanation might be needed for the “Fisher’s protected LSD”? At least give the full name of LSD when it is firstly mentioned. Full name of LSD (least significant differences) has been mentioned now. 12. Is the different doses of the glyphosate used well compared? It is not clear in the result. LD50 values have now been calculated for each biotype and the values compared with a 95% confidence interval. 13. For reference 6 and 7, there are italic styles, make sure with journal and be consist with the others. Italics have been removed. 14. You declared that the data is available in the supporting information but I did not see any materials attached, please explain this. We have attached all raw data now. Reviewer #3: Two experiments were carried out in this study to compare the growth behavior and glyphosate resistance among 10 biotypes of Echinochloa colona in Australia. The growth experiment showed that characteristics like tall nature and high tillering capacity allow E. colona to produce a high leaf number that result in a large number of seed heads and seeds. The glyphosate dose-response experiment showed a wide range of GR50 value in these biotypes, and the biotype B17/34 showed the strongest resistance to glyphosate. The two experiments were conducted rigorously with appropriate replication and sample sizes, and the conclusions were drawn appropriately based on the data presented. In addition, the language of this manuscript is well written and clear. Thank you for your comments. The analysis of variance (ANOVA) and Fisher’s protected LSD were applied to test the difference of growth behavior of E. colona among 10 biotypes. And the probit anaylsis were applied to estimate the GR50 value. These statistical methods were performed appropriately, but the outcome of these statistical results have not been sufficiently presented. Moreover, there are several major and specific comments on this manuscript: 1. The introduction can be reduced to 3 or 4 paragraphs to make the background and aim clearer. The outline of the introduction can refer to: 1) The Echinochloa colona as a noxious weed emerged in Australia crops. 2) The biological characteristics of growth and reproduction of E. colona. 3) The glyphosate is widely used in Australia to eliminate the E. colona, but induced a variety of glyphosate resistant biotypes. 4）the aim and scientific questions of this study All your points have been addressed and the introduction has been reduced to 4 paragraphs. Differences among growth behaviour of biotypes were compared with LSD values. 2. The author combined the “results” and “discussion” in the manuscript. Although this format can be accepted by some journals, but I suggest divided this part into “results” and “discussions” separately. In the present manuscript, the result of two experiment were discussed respectively, and I can not find their relationships. If there is an independent section of discussion, the relationships between the two experiment and their implications can be better integrated. Results and discussion are combined here according to the format of the journal and we feel combining the results and discussion avoids repetition. The relationship between the two experiments and the implications has been discussed in general discussion at the end of the article (Conclusion section). 3. The author proposed “growth behavior and seed production potential in these biotypes had no correlation with the resistance index” (LINE 240-241). However, the statistical methods for analyzing the correlation between growth traits and resistance index were not given in the section of “statistical analysis”. Also, I can not find the relevant result which support this conclusion in the context. Statistical methods for analysing the correlation has been provided under heading statistical analysis. The relationships between parameters were assessed using linear regression analysis. Suggested lines have been added in the conclusion section. The analysis detail of correlation has been provided in the supplementary file. 4. The seed of 10 biotypes of E. colona were collected from a large area in northern grain region in Australia. So, do the geographical differences effect on the growth behavior and glyphosate resistance among 10 biotypes of E. colona. I suggest the multivariate analysis such as PCA or RDA can be applied to study whether the biotypes from closer sites have similar characteristics. Moreover, the correlation (or non-correlation) between growth behavior and resistance index may also be revealed by the multivariate analysis. We tested these biotypes under one location. Therefore, it is not justified to run PCA analysis. There was no such trend that close sites have similar characteristics. We are studying the genetic diversity of these biotypes in a different experiment. Here are also several specific comments: 5. Figure 1. It’s better to give a small-scale map of Australia, and pointed out the location of northern grain region. We have modified figure 1 to incorporate these suggestions. 6. Table 1. the standard deviation of these variables should be given. And as the LSD was performed, I suggest the author add lowercase superscript letters after the values to indicate significance at the 0.05 level for the differences among biotypes. Letters are to be used in DMRT (Duncan multiple range test) design. LSD values have been given to identify the differences between biotypes. If the difference between two values is more than LSD value, it means the treatments are significantly different. However, we have included the standard deviation in the table. 7. LINE 64: “shikmate pathway” might be “shikimate pathway” Corrected. 8. Line 251-254: it better to give more information on the seed collected location or the farm, like the crop type, soil type, glyphosate dose and so on, which can help readers know the growth environment of these biotypes. We have provided the GPS coordinates on map. However, we are including all information here for your information Biotype Coordinates Location/Place Crop B17/7 27.50000000/151.69666667 Dalby Wheat-Fallow B17/12 30.268508/149.80481 Narrabri Wheat- Fallow B17/13 30.306499/149.811438 Narrabri Wheat-Fallow B17/16 30.09099349/149.64890 Narrabri Lathyrus-wheat/chickpea B17/17 30.38230/149.59679 Narrabri Fallow B17/25 28.31500000/148.68916667 St George Canal side B17/34 28.58305556/150.36888889 Moree Wheat fallow B17/35 29.95805/149.8339 Moree Cotton fallow B17/37 27.5514/152.3428 Gatton Wheat-Fallow B17/49 27.5514/152.3428 Gatton Wheat-Fallow 9. Line 252: how to identify different biotypes? Do they just collected from different sites，or have distinct heritable characteristics? They were collected from different sites. Heritable characteristics were not studied in this experiment. 10. Line 301: I did not find the ANOVA result ANOVA results have been provided as annexure. Table 1. ANOVA for different parameters Source Degree of freedom Plant height (cm) Tiller (number plant-1) Leaf (number plant-1) Seed head (number plant-1) Seed head weight (g plant-1 ) Shoot biomass (g plant-1) Root biomass (g plant-1) Days to seed head initiation (d) Seed production (number plant-1) Mean square Replication 3 59.9 52.2 702.9 129.3 0.179 5.78 5.863 8.91 1621914 Experimental run 1 17155.2* 9812.4* 64139.5* 7940.1* 202.1* 15401.2* 315.96* 214.5* 0.433155* Biotype 9 202.8* 323.5* 4340.5* 582.4* 12.5* 39.761* 50.14 43.0* 32654110* Experimental run x Biotype 9 48.8 60.9 962.2 119.9 2.59 24.083 47.84 22.5 1001201 Error 57 32.0 44.1 925.5 200.9 2.75 14.584 24.5 7.34 5510769 * indicates significant at 5% level 11. Line 307-308 Which variable did the “GR50” in this study based on? The shoot biomass, tiller number, or individuals? It was based on biomass. However, reviewer# 1 suggested to do on survival % basis. Therefore, now we have provided on survival % basis. ________________________________________ 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Yes Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Liping Li Reviewer #3: No We are okay with Reviewer#2. Submitted filename: Response letter (6).docx Click here for additional data file. 2 Dec 2019 PONE-D-19-21773R1 Growth behavior and glyphosate resistance level in 10 biotypes of Echinochloa colona in Australia PLOS ONE Dear Dr Mahajan, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR: The manuscript has been greatly improved. However, there is still plenty of questions to answer and comments to address, as has been pointed out by reviewer #3. Besides, reviewer #1 asked that “Is there any plan to relate these phenotypic response back to management, cropping systems, location, and/or soils?” in the comments. But I did not find any response to this comment. Please response to this point in the next version. ============================== We would appreciate receiving your revised manuscript by Jan 16 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Xiao Guo, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed Reviewer #3: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes Reviewer #3: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: No ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes Reviewer #3: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes Reviewer #3: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: Personally, I currently do not work a lot on spv files. So I think the supplemental materials are better in xlsx, or txt format. And it is better to have a list of the supporting information. Reviewer #3: The manuscript PONE-D-19-21773 has been improved since the previous version. However, several problems have not yet been solved in the manuscript, although some of them were replied in the response letter. For example, I can see the ANOVA table and site information in the response letter, but can not find them neither in the manuscript nor in the supporting files. Moreover, the ANOVA statistics for the growth characteristics need to be reanalyzed, because the replication was set as a treatment, which I can not agree with. Another problem is, I think there needs some evidence or reference to prove that, the seeds being collected from one site are belong to the same biotype. 1. Line 248-250: By definition, the “biotypes” is a group of organisms having the same specific genotype. So, plants with same biotype must have common heritable traits, by which the biotype can be distinguished. In this study, the seeds of one biotype were collected from 40-50 plants per site and over an area >1ha. I wonder whether the seeds of same biotype were collected from one clone, or distinguished by any trait during the seed collection. Otherwise they can only be call “population” but not “biotype”. Since the experimental evidence for heritability traits is lacking, it is better to provide references in discussing this point. 2. Line 296, “14 January 2018” should be “14 January 2019” 3. Line 312-314, the author supplied the ANOVA table in the response letter, but I can not find it neither in the manuscript nor in the supporting material in xlsx files. Maybe it was included in the SPV format files which I could not open. The SPV file can only be open by SPSS, which is a commercial software and very expensive. I believe that many readers, just like me, have not installed the SPSS software. So, I suggested the contents in SPV files can be transformed into a common format, such as xlsx or txt, which can be opened by all readers. Moreover, in the ANOVA table, the replicates should not be treated as a factor. 4. Line 319, remove “the growth of” 5. Figure 1, the author gave a table of the site information in the response letter, but I can neither find it in the manuscript nor supporting information. 6. Table 2, please give the sample size involved in the correlation analysis given in the table caption or note, like “n=?” 7. Figure 2 I found the biotypes of B17/7, B17/12, B17/17, B17/25 had no significant difference with the susceptible biotype. Do this mean the five biotypes being susceptible as B17/37? If it is, I suggest separate the all 10 biotypes into two groups, and compare their growth traits between groups. Maybe you can find significant differences of growth characteristics between susceptible and glyphosate resistant biotypes. This is just a suggestion and you could think about this. 8. Line 328: Furthermore, do the difference of LD50 and resistance index among the 10 biotypes were compared using LSD method? Please add the information of statistical methods . 9. Line 334, The “funding acquisition” can be removed because there was “no special funding was obtained for this work” in the funding information. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. 8 Dec 2019 Dr Xiao Guo, Academic Editor PLOS ONE Subject: Ref.: PONE-D-19-21773R1 Growth behavior and glyphosate resistance level in 10 biotypes of Echinochloa colona in Australia PLOS ONE Dear Dr Guo, We thank you again for the critical look on the manuscript and highly appreciate the remarks of the reviewers for further improvement of the manuscript. The manuscript has been revised in the light of useful comments. All the comments have been incorporated in the revised version carefully. For clarity, the comments and suggestions will appear in the black colored text; while our response will appear in the blue text. In the manuscript also, specific changes can be seen in the highlighted text. We hope that the revised manuscript is now acceptable in “PLOS ONE”. We look forward to hearing from you in due course. Sincerely yours Gulshan Mahajan ============================== ACADEMIC EDITOR: The manuscript has been greatly improved. However, there is still plenty of questions to answer and comments to address, as has been pointed out by reviewer #3. Besides, reviewer #1 asked that “Is there any plan to relate these phenotypic response back to management, cropping systems, location, and/or soils?” in the comments. But I did not find any response to this comment. Please response to this point in the next version. ============================== This study suggested that glyphosate resistant populations such as B17/34 that produced a high seed number are very problematic. Attempt should be made to reduce its invasion/further infestation. Movement of seeds from one field to another field via various mode of transportation should be restricted. For management of resistant population, crop competition tactics (narrow row spacing, competitive cultivars, optimum seeding rate, cover crops etc.) could be explored in an integrated weed management program to reduce the seed number of resistant populations. The related information has been added in the manuscript. Reviewer #2: Personally, I currently do not work a lot on spv files. So I think the supplemental materials are better in xlsx, or txt format. And it is better to have a list of the supporting information. Agreed. The supplemental material of probit analysis has been attached in a Word file now. Reviewer #3: The manuscript PONE-D-19-21773 has been improved since the previous version. However, several problems have not yet been solved in the manuscript, although some of them were replied in the response letter. For example, I can see the ANOVA table and site information in the response letter, but can not find them neither in the manuscript nor in the supporting files. Moreover, the ANOVA statistics for the growth characteristics need to be reanalyzed, because the replication was set as a treatment, which I can not agree with. Another problem is, I think there needs some evidence or reference to prove that, the seeds being collected from one site are belong to the same biotype. The data have been again reanalysed with eight replications as per suggestions. An ANOVA table and site information has been added in the manuscript. The term biotype has been replaced with population. 1. Line 248-250: By definition, the “biotypes” is a group of organisms having the same specific genotype. So, plants with same biotype must have common heritable traits, by which the biotype can be distinguished. In this study, the seeds of one biotype were collected from 40-50 plants per site and over an area >1ha. I wonder whether the seeds of same biotype were collected from one clone, or distinguished by any trait during the seed collection. Otherwise they can only be call “population” but not “biotype”. Since the experimental evidence for heritability traits is lacking, it is better to provide references in discussing this point. As seeds were not collected from one clone. Therefore, to avoid confusion, we have replaced the term biotype with population. 2. Line 296, “14 January 2018” should be “14 January 2019” Corrected. 3. Line 312-314, the author supplied the ANOVA table in the response letter, but I can not find it neither in the manuscript nor in the supporting material in xlsx files. Maybe it was included in the SPV format files which I could not open. The SPV file can only be open by SPSS, which is a commercial software and very expensive. I believe that many readers, just like me, have not installed the SPSS software. So, I suggested the contents in SPV files can be transformed into a common format, such as xlsx or txt, which can be opened by all readers. Moreover, in the ANOVA table, the replicates should not be treated as a factor. Now we have transferred the content from SPV file to txt file (probit analysis) and the file has been attached as a supplementary file. We have reanalysed the data again with eight replications. An ANOVA table has been added in the text. 4. Line 319, remove “the growth of” Corrected 5. Figure 1, the author gave a table of the site information in the response letter, but I can neither find it in the manuscript nor supporting information. Site information from Figure 1 has been added in the manuscript. 6. Table 2, please give the sample size involved in the correlation analysis given in the table caption or note, like “n=?” Added. n=10. 7. Figure 2 I found the biotypes of B17/7, B17/12, B17/17, B17/25 had no significant difference with the susceptible biotype. Do this mean the five biotypes being susceptible as B17/37? If it is, I suggest separate the all 10 biotypes into two groups, and compare their growth traits between groups. Maybe you can find significant differences of growth characteristics between susceptible and glyphosate resistant biotypes. This is just a suggestion and you could think about this. Yes, populations B17/7, B17/12, B17/17, B17/25 were susceptible as 17/37. However, they differ in their growth behaviour. We tried to analyse with your suggestion and found these cannot be grouped together on the basis of growth traits. 8. Line 328: Furthermore, do the difference of LD50 and resistance index among the 10 biotypes were compared using LSD method? Please add the information of statistical methods . No, these were not compared using LSD method. LD50 values for each population were compared using confidence interval overlap in probit analysis (Table 4). Resistance index is a factor (resistance/susceptibility ratio); therefore, these were also compared on the basis of LD50 value. 9. Line 334, The “funding acquisition” can be removed because there was “no special funding was obtained for this work” in the funding information. Yes, it has been removed. Submitted filename: Response letter (7).docx Click here for additional data file. 30 Dec 2019 Growth behavior and glyphosate resistance level in 10 biotypes of Echinochloa colona in Australia PONE-D-19-21773R2 Dear Dr. Mahajan, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Xiao Guo, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed Reviewer #3: All comments have been addressed ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes Reviewer #3: (No Response) ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: (No Response) ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes Reviewer #3: (No Response) ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes Reviewer #3: (No Response) ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: The file “Probit analysis detail” looks strange to me. And is there a supplement material list? I did not see. Reviewer #3: (No Response) ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No Reviewer #3: No 6 Jan 2020 PONE-D-19-21773R2 Growth behavior and glyphosate resistance level in 10 populations of Echinochloa colona in Australia Dear Dr. Mahajan: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Xiao Guo Academic Editor PLOS ONE
  6 in total

Review 1.  Global perspective of herbicide-resistant weeds.

Authors:  Ian Heap
Journal:  Pest Manag Sci       Date:  2014-01-15       Impact factor: 4.845

2.  Geographical variation in resistance to acetyl-coenzyme A carboxylase-inhibiting herbicides across the range of the arable weed Alopecurus myosuroides (black-grass).

Authors:  Christophe Délye; Séverine Michel; Aurélie Bérard; Bruno Chauvel; Dominique Brunel; Jean-Philippe Guillemin; Fabrice Dessaint; Valérie Le Corre
Journal:  New Phytol       Date:  2010-03-22       Impact factor: 10.151

Review 3.  What have the mechanisms of resistance to glyphosate taught us?

Authors:  Dale L Shaner; Richard Bradley Lindenmeyer; Michael H Ostlie
Journal:  Pest Manag Sci       Date:  2011-08-12       Impact factor: 4.845

4.  Concerted action of target-site mutations and high EPSPS activity in glyphosate-resistant junglerice (Echinochloa colona) from California.

Authors:  Rocío Alarcón-Reverte; Alejandro García; Susan B Watson; Ibrahim Abdallah; Sebastián Sabaté; María J Hernández; Franck E Dayan; Albert J Fischer
Journal:  Pest Manag Sci       Date:  2014-09-05       Impact factor: 4.845

Review 5.  Herbicide-resistant weed management: focus on glyphosate.

Authors:  Hugh J Beckie
Journal:  Pest Manag Sci       Date:  2011-05-05       Impact factor: 4.845

Review 6.  Overview of glyphosate-resistant weeds worldwide.

Authors:  Ian Heap; Stephen O Duke
Journal:  Pest Manag Sci       Date:  2017-11-29       Impact factor: 4.845
  6 in total

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