Growth stage of Phalaris minor Retz . and wheat determines weed control and crop tolerance of four post-emergence herbicides

Phalaris minor Retz. has evolved multiple herbicide resistance in wheat growing areas in northwestern India. An understanding of the effect of growth stage on herbicide tolerance of wheat and control of P. minor will help in selecting the most appropriate herbicide for different situations. The weed control and crop safety of four commonly used wheat herbicides (sulfosulfuron, pinoxaden, fenoxaprop plus metribuzin and mesosulfuron plus iodosulfuron), each applied at four different wheat growth stages was investigated in field studies for two years. P. minor plants were at 1, 2-3, 3-4 and 7-8 leaf stages when the herbicides were applied at Zadok 12Z12, Z13, Z21 and Z23 stages of wheat, respectively. Sulfosulfuron application at Z12 and Z13 wheat stages (before first irrigation), provided >80% control of P. minor and produced wheat grain yield (4.5-4.7 t/ha) similar to the weed-free check (4.9 t/ha) in both years. Pinoxaden, fenoxaprop plus metribuzin and mesosulfuron plus iodosulfuron application at Z12 and Z13 wheat stages recorded significantly lower wheat grain yield (3.62-3.95 t/ha) due to poor weed control, crop toxicity or both. All the four herbicides were equally effective on P. minor when applied at Z21 wheat stage. At Z23 wheat stage, pinoxaden gave >90% control of P. minor and the highest wheat grain yield (4.82 t/ha). The results are expected to allow changes in the current recommendation of the timing of postemergence herbicides for the management of P. minor in wheat. Additional key words: crop toxicity; crop stages; littleseed canarygrass; selectivity; Triticum aestivum. Correspondence should be addressed to Rubia Rasool: rubiarasool@yahoo.com; rasiarasool@gmail.com Abbreviations used: ALS (acetolactate synthase); DAS (days after sowing); DAT (days after treatment); Z (Zadoks). Funding: University Grants Commission, New Delhi, India, provided financial support to RR (Award letter No. F1-17.1/2012-13/ MANF-2012-13-MUS-JAM-10365). Authors’ contributions: Conceived and designed the experiment; data analysis and interpretation; publication drafting: RR, MSB, GSG. Performed the experiments: RR, MSB. Competing interests: The authors have declared that no competing interests exist. Copyright © 2017 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License. Citation: Rasool, R.; Bhullar, M. S.; Gill, G. S. (2017). Growth stage of Phalaris minor Retz. and wheat determines weed control and crop tolerance of four post-emergence herbicides. Spanish Journal of Agricultural Research, Volume 15, Issue 1, e1001. https://doi. org/10.5424/sjar/2017151-9728 Received: 28 Mar 2016. Accepted: 17 Feb 2017. 15(1), e1001, 8 pages (2017) eISSN: 2171-9292 https://doi.org/10.5424/sjar/2017151-9728


Introduction
Littleseed canarygrass (Phalaris minor Retz.) is one of the most troublesome annual weeds of wheat (Triticum aestivum L.) in northwestern India since many years ago (Bhullar et al., 2016).Globally, it has been reported in more than 60 countries, covering all the continents (Singh et al., 1999).The weed is highly competitive with wheat and can reduce yield by up to 95% (Chhokar et al., 2006).It germinates in different flushes from sowing, to well after first and second irrigations.According to the current recommendation, P. minor in wheat in northwestern India, can be controlled with preemergence herbicides applied within 2 days after sowing (DAS) or with post-emergence herbicides at 30 DAS; post-emergence herbicides are currently most popular (>95%) with the farmers in the region.Rice-wheat is the dominant cropping system in northwestern India, and P. minor often germinates in moist seedbed at the same time as wheat.However in some seasons, rainfall during wheat sowing can stimulate early germination of weeds.Under these situations, P. minor can reach an ideal stage for herbicide application (2-3 leaf) even though wheat crop is not at the labelled stage .At present, the consequences of spraying wheat earlier than the currently recommended stage (Z21) are unclear.
Correct timing of herbicide application plays an important role in achieving effective weed control without causing crop injury.Crop tolerance to herbicides varies with herbicide choice, application dose, application timing, and environmental conditions (Lemerle et al., 1986).Growth stage of wheat and environmental conditions at the time of herbicide application also affect herbicide uptake, translocation and plant's ability to metabolize herbicides thus affecting the extent of herbicide injury and weed control (Tottman, 1980).Sulfosulfuron, pinoxaden, pre-mixes of fenoxaprop plus metribuzin and mesosulfuron plus iodosulfuron are commonly used for P. minor control in wheat in the region.Sulfosulfuron is a sulfonylurea herbicide that inhibits the activity of acetolactate synthase (ALS), an important enzyme necessary for the biosynthesis of branched chain amino acids.It has been widely used locally for the control of P. minor.There have already been some reports of resistance to sulfosulfuron in P. minor in some areas of Punjab (Bhullar et al., 2014) andHaryana (Dhawan et al., 2010) states in northwestern India.The pre-mix of mesosulfuron-methyl and iodosulfuron-methyl-sodium (ALS inhibitor) is also widely used in wheat.GR 50 (dose required for 50% growth reduction) values of mesosulfuron plus iodosulfuron have been reported to increase with time (AICRPWM, 2013).More recently pinoxaden, a selective grass herbicide belonging to the phenlypyrazolin chemistry, has been recommended for post-emergence control of P. minor.Dhawan et al. (2010) and Kaur et al. (2016) reported development of cross resistance in P. minor to pinoxaden in Punjab and Haryana states.Metribuzin is primarily a soil-residual herbicide, which can be applied post-emergence to wheat for the control of annual grasses and dicot weeds (Schroeder et al., 1986;Shaw & Wesley, 1991).Although metribuzin can control P. minor effectively, careful management, including timely application is required to achieve good crop tolerance and weed control (Runyan et al., 1982;Schroeder et al., 1986).The pre-mix of fenoxaprop-p-ethyl plus metribuzin can provide effective weed control but it can also cause some phytotoxic effects on the tillering and grain yield of wheat (Singh et al., 2005).Even though there have been some reports of resistance to sulfosulfuron, pinoxaden and pre-mix of mesosulfuron plus iodosulfuron in P. minor in northwestern India, these herbicides are still extensively used in the region.An understanding of the effect of growth stage on herbicide tolerance of wheat and control of P. minor will help in developing recommendations for selecting the most appropriate herbicide for different situations.The objective of this study was to evaluate the effect of timing of different post-emergence herbicides on crop tolerance and their efficacy on P. minor control.

Description of study site
Field experiments were conducted under irrigated conditions at the research farm of Punjab Agricultural University, Ludhiana, India during winter season of 2013-14 and 2014-15.The experimental soil was sandy loam with organic carbon (0.39%), pH (8), EC (0.13 mmhos/cm) and was low in available nitrogen (214 kg N/ha), available phosphorous (9 kg P/ha) and high in available potassium (337 kg K/ha).The experimental field had been under irrigated rice-wheat cropping system and was planted with dry-seeded rice in the summer seasons.The weekly weather data (maximum and minimum temperature and rainfall) during the two growing seasons are represented in Fig. 1.The wheat crop received 172 mm rain in 2013-14 and 221 mm in 2104-15.

Experimental design and crop management
A heavy pre-sowing irrigation (10 cm) was applied to ensure adequate moisture in the soil at the time of planting.Seedbed preparation consisted of ploughing with a disc harrow followed by two passes with a field cultivator followed by planking.Seeds of P. minor were broadcast uniformly in the field before the last cultivation.Wheat (cv.HD-2967) was seeded on November 19, 2013 and November 12, 2014 using seed rate of 100 kg/ha, in 22.5 cm spaced rows.The crop was fertilized by using Urea as the source of nitrogen at 125 kg N/ha in two splits; half as basal and half with the first irrigation.Phosphorous was applied at 62.5 kg P 2 O 5 /ha through Single Super Phosphate and potassium at 30 kg K 2 O/ha through Muriate of Potash as basal dose.Each year, the experiment was conducted in a randomized complete block design with three replications.Sixteen treatments consisted of four herbicides viz.sulfosulfuron at 25 g/ha, pinoxaden at 50 g/ha, pre-mixes of fenoxaprop plus metribuzin at 275 g/ha and mesosulfuron plus iodosulfuron at 14.4 g/ha, each applied at Z12 (14 DAS), Z13 (21 DAS), Z21 (30 DAS) and Z23 (45 DAS) (Zadoks et al., 1974) (Table 1).A weed-free check and a weedy check was also included for comparison with the herbicide treatments.The gross plot area was 3.25 m × 7 m and net plot was 2.02 m × 5.2 m.The herbicide doses used in the study are currently recommended for the control of P. minor in wheat in the region at 30 DAS.At Z12, Z13, Z21 and Z23 stages of wheat, P. minor plants were at 1, 2-3, 3-4 and 7-8 leaf stages, respectively.All herbicides were applied using knapsack sprayer fitted with a flat fan nozzle which was calibrated to deliver 375 L/ha of spray solution.The crop was supplied with four post-sowing irrigations (each 7.5 cm) at 23, 58, 90 and121 DAS during 2013-14 andat 23, 63, 88 and128 DAS during 2014-15.The irrigation was applied when the soil moisture had declined to onethird of field capacity.All management practices other than the weed control treatments were consistent with the recommended package of practices for the region.In the weed free treatment, weeds were removed by hand weeding throughout the season.P. minor was the major weed in the field; a few broad leaf weed plants present in the field were removed by hand pulling.

Data collection
Phalaris minor plant density was assessed, by using a 0.45 m 2 quadrant placed at two representative locations, in each plot at 30, 45 and 135 (tiller density) DAS.P. minor plants were cut and dried in an oven at 60 o C for 72 h and the above ground biomass recorded at 45, 90 and 135 DAS.The visible crop injury was assessed at 7, 14 and 21 days after herbicide treatment (DAT) and rated on 0-100% scale (0=no toxicity; 100=complete killing).To further quantify wheat response to herbicides, plant height was measured from the ground to the tip of the uppermost leaf from six randomly selected plants in each plot at 45 DAS, and spikes were counted in 1-m 2 at two locations in each plot at 90 DAS and at crop harvest (productive spikes).Grain weight/spike was determined from six representative spikes from each plot; these spikes were then used to obtain number of grains/spike.The crop was manually harvested at maturity on 20 April, 2014 and 22 April, 2015.  [1Wheat stage at time of spray.Z: Refers to the Zadok's stage of wheat.DAS: days after sowing.Data were square-root transformed before analysis; however, back-transformed actual mean values are presented based on the interpretation from the transformed data.

Statistical analyses
Data were analyzed using the GLM procedure in SAS 9.3 to evaluate the differences between treatments (SAS, 2001).Normality, homogeneity of variance, and interactions of treatments and years were tested.In this study, year by treatment interactions were significant; therefore, data were presented separately for each year.Weed density and biomass data were square root transformed and herbicide injury data were arc-sine transformed before performing ANOVA because of high variance.However, non-transformed means are presented with mean separation based on transformed values.Weed free check was not included in the analysis of crop injury, however, all values were compared independently to zero to evaluate treatment differences with the weed free check.Where the ANOVA indicated that treatment effects were significant, means were separated at p<0.05 and adjusted with Fisher's Protected Least Significant Difference (LSD) test.

Wheat growth and development
Application of fenoxaprop plus metribuzin at Z12 and Z13 wheat stages caused toxicity to wheat plants in both years (Fig. 2).The toxicity symptoms, wilting of foliage followed by whitening/bleaching of the leaf tissue, appeared after the first irrigation and were more severe when the herbicide was applied at Z13 than at Z12 stage.The treatment at Z13 caused 36-38% toxicity at 7 DAT, which reduced to 5-6% at 14 DAT and to 0-2% at 21 DAT.Application of fenoxaprop plus metribuzin at Z12 stage showed 0% toxicity at 7 DAT, 26-28% at 14 DAT which reduced to 2% at 21 DAT.The growth of the surviving crop plants in fenoxaprop plus metribuzin treatment was stunted, and herbicide toxicity reduced wheat plant density by 23-25% at 90 DAS when the spray was done at Z12 and Z13 stages.Application of mesosulfuron plus iodosulfuron at Z12 and Z13 stages suppressed crop growth, caused interveinal chlorosis of leaves and minor necrosis of leaf tips of wheat plants.The toxicity symptoms were more severe when this herbicide treatment was applied at (19-22%) compared to at Z12 (12%).Pre-mixes of fenoxaprop plus metribuzin and mesosulfuron plus iodosulfuron were safe to wheat  when applied at Z21 and Z23 stages.Sulfosulfuron and pinoxaden did not cause any phytotoxicity to wheat plants when applied at any of the stages of wheat.

Wheat grain yield and yield attributes
The application of herbicides at different growth stages of wheat had a significant effect on wheat grain yield and yield attributes (Table 2).When herbicide applications were made at Z12 and Z13 stages, only sulfosulfuron provided yield levels (4.5-4.7 t/ha) similar to the weed-free check (4.9 t/ha) (p>0.05).Mesosulfuron plus iodosulfuron at Z12 and Z13 stages reduced wheat grain yield by 21 and 25%, respectively, as compared to the weed-free check.In case of fenoxaprop plus metribuzin, the yield reduction was 22-26% when application was made at Z12 and 25-27% at Z13.Early application of pinoxaden caused the highest grain yield reductions in wheat (42-44% at Z12 and 22-34% at Z13).All herbicides in this study recorded wheat grain yield at par to weed-free check when applied at Z21 stage.Pinoxaden produced the highest grain yield when herbicides were applied at Z23 stage.
A negative relationship was observed between wheat grain yield and P. minor biomass in the linear regression model (Fig. 3; Table 3).The regression model explained about 98% of the variation in grain yield due to weed biomass and provided a good fit between the grain yield and the weed biomass.Pre-mix of fenoxaprop plus metribuzin applied at Z12 and Z13 wheat stages was excluded from the regression model, as the grain yields under these treatments were influenced by crop toxicity

Treatment
Wheat stage [1]   Plant 0.5 0.5 0.69 0.64 [1] Wheat stage at time of spray.Z: Refers to the Zadok's stage of wheat.DAS: days after sowing.and not just weed control.In all the other weed control treatments, grain yield reduction was mainly influenced by P. minor biomass.

Discussion
Sulfosulfuron and fenoxaprop plus metribuzin provided effective control of P. minor when applied at Z12 and Z13 wheat stages, all four herbicides were equally effective when applied at Z21, and only pinoxaden worked effectively at Z23 wheat stage.Sulfosulfuron is absorbed both through root and foliage (Senseman, 2007) and has a broad window of application in wheat and longer residual effects (Shaner, 2014).P. minor plants were at 1-4 leaf stages, when herbicides were applied between Z12-21 stages of wheat, and would have been killed through both root and foliage uptake.The efficacy of sulfosulfuron against 2-3 leaf stages of P. minor has been reported earlier (Chhokar & Malik, 2002;Chhokar et al., 2008).In contrast, Singh et al. (2003) recorded higher control of P. minor with sulfosulfuron used at Z13 compared to at Z21 wheat stage at Pantnagar, India.The results of this study suggest that even in situations where weeds germinate early after first irrigation, the current recommendation for sulfosulfuron at Z21 stage still provides effective weed control and crop yield response.
Fenoxaprop and metribuzin have contrasting modes of entry into plants; metribuzin enters the plant mainly from the soil via roots and fenoxaprop enters entirely through the foliage; their joint action may be the reason for enhanced efficacy against P. minor, particularly when it was applied at Z12-13 wheat stages.In an earlier study, Chhokar et al. (2008) reported effective control of P. minor with metribuzin applied at Z23 stage of wheat, but in our study metribuzin plus fenoxaprop had a lower efficacy than its earlier timing, which may be attributed to the large size of P. minor plants (7-8 leaf stage).The lower efficacy of the mesosulfuron plus iodosulfuron on small plants of P. minor when applied at Z12-13 wheat stages, may be due to low leaf area of weeds as these herbicides are primarily absorbed through foliage (Yuan et al., 2013) and known for more consistent results from later applications (Tickes, 2000;Crooks et al., 2004).In an earlier study, mesosulfuron provided better control of Lolium perenne at 4-5 leaf than at 2-3 leaf stage (Grey et al., 2012).This was also supported by our results where the best control of P. minor (>85%) with meosulfuron plus iodosulfuron was recorded at more than 3-leaf stage.Pinoxaden is also absorbed through the foliage only (Hofer et al., 2006), hence its lower efficacy when applied at Z12 and Z13 wheat stages could be related to lower leaf area of P. minor, and its failure to prevent germination of P. minor seeds after its application.Consequently, pinoxaden worked most effectively once all target grasses had fully emerged.Therefore, the best weed control with this herbicide was observed when it was applied at Z21-23 wheat stages.Kieloch et al. (2006) indicated that pinoxaden can provide high levels of weed control even at late weed growth stage which was also supported by this study.
Wheat plants showed excellent tolerance to sulfosulfuron and pinoxaden at all growth stages.In the case of pinoxaden, the safener (cloquintocetmexyl) present in the formulation provides very good tolerance in wheat (Hofer et al., 2006).The pre-mixes of fenoxaprop plus metribuzin and mesosulfuron plus iodosulfuron were toxic to wheat plants at Z12 and Z13 stages and significantly reduced spike density compared to weed free check (p<0.05).These results are supported by the chlorophyll fluorescence (Fv/Fm) data, which showed reduced chlorophyll fluorescence in wheat plants compared to unsprayed plants indicating that the treated plants were under stress (data not shown).The higher crop toxicity by these herbicide mixtures when used at Z13 than at Z12 stage could be related to the short time interval between herbicide application and irrigation which increased herbicide uptake.The herbicide application at Z13 stage was followed by irrigation after 24 hours, and 10 and 42 mm rainfall was received one week after the spray during 2013 and 2014, respectively (Fig. 1).The close association between metribuzin toxicity in crops and soil moisture Table 3. Correlation (r) among growth parameters associated with crop-weed competition.
Parameters [1]  Weed has been reported earlier (Blackshaw et al., 1994;Kleemann & Gill, 2008).Crooks et al. (2003) reported toxicity to wheat plants 2-4 weeks after the treatment with mesosulfuron plus iodosulfuron and the toxicity reduced significantly when herbicide application was delayed to the tillering stage of wheat (Grey et al., 2012).These studies suggest that tolerance of wheat to this herbicide increases close to the tillering stage.In our case, the crop also showed excellent tolerance to both pre-mixes when applications were made at Z21-23 stages which represent the tillering stage of wheat, and may be attributed to the enhanced capacity of wheat plants to metabolise the herbicides at these stages (Das, 2014).The differential sensitivity of wheat to metribuzin applied at its different growth stages (Shaw & Wesley, 1991;Grey & Bridges, 2003) and different doses (Balyan, 1999) has been demonstrated earlier.
The higher grain yield with sulfosulfuron, when applied at Z12 and Z13 wheat stages than later in the season, could be attributed to its excellent crop safety and lower biomass of P. minor.The selectivity of preand post-emergence sulfosulfuron in wheat was reported by Levy (2008).The results of our study indicated that sulfosulfuron can be adopted for the control of P. minor at these stages (Z12-13).At those early stages of wheat, fenoxaprop plus metribuzin showed toxicity, pinoxaden had poor weed control and mesosulfuron plus iodosulfuron showed both (toxicity and poor weed control) (Table 1; Fig. 2).The crop toxicity and/or poor weed control reduced wheat plant density, spike density and grains per spike, which lowered the grain yield by 21-44% compared to the weed free check (p<0.05)(Table 2).These results are supported by the strong positive correlation among spike density and grains per spike with grain yield (p<0.01) (Table 3).The weed biomass at 90 and 135 DAS are strongly correlated and has strong negative effects on spikes/m 2 and grain number per spike as well as grain yield (p<0.01).Hence treatments that provided poor weed control allowed large weed biomass, which resulted in lower grain yield.Further, grains/spike and grain number/m 2 are strongly related to grain yield, which also explain the differential response among different weed control treatments.The yield reduction with both the pre-mixes was greater when applied at Z13 than at Z12, which could be related to greater herbicide absorption associated with higher leaf area of the crop.
All four herbicides applied at the Z21 crop stage provided effective control of P. minor and were safe to wheat, and produced wheat grain yield similar to the weed-free check (p>0.05).When herbicide treatments were applied at Z21 stage, P. minor and wheat plants were within the recommended 3-4 leaf stage, which could be the reason for high herbicide efficacy and selectivity.
Effective control of P. minor at Z21 wheat stage with these herbicides was also reported earlier (Chhokar & Malik, 2002;Singh et al., 2005;Chhokar et al., 2008).Pinoxaden provided excellent control (>90%), even with large plants of P. minor (7-8 leaf), which was the reason why it provided wheat grain yield similar to weed free check when applied at Z23 wheat stage.Weed biomass for the other three herbicides was greater than with pinoxaden, which could be associated with the differences in wheat grain yield.Singh et al. (2011) found higher P. minor control with pinoxaden compared to the pre-mix of fenoxaprop plus metribuzin when the herbicide application was made at wheat stages beyond Z23 stage.The negative linear relationship between wheat grain yield and P. minor biomass explained about 98% of the variation in grain yield (Fig. 3; Table 3).The application of different herbicides reduced P. minor biomass by 25-98% as compared to the weedy check (192-195 g/m 2 ).This level of competition from P. minor in the weedy check plots reduced crop density, spike density (35-40%) and wheat grain yield by 50% as compared to the weed free check.
As conclusions, the post-emergence application of 25 g/ha sulfosulfuron at Z12 and Z13 stages of wheat provided effective control of P. minor and was safe to wheat.Pre-mixes of fenoxaprop plus metribuzin and mesosulfuron plus iodosulfuron were toxic to wheat plants when applied at these stages.Pinoxaden application at 50 g/ha provided effective control of large P. minor (7-8 leaf).This study provides background information required to make changes to the current recommendation of timing of post-emergence herbicides in wheat in northwestern India.

Figure 3 .
Figure 3. Relationship between the wheat grain yield and Phalaris minor biomass.The line represents a linear model fitted to the average of the data for both years.

Table 1 .
Effect of different weed control treatments on density and biomass of P. minor during 2013-14 (I) and 2014-15 (II) growing seasons at Punjab Agricultural University Research Farm near Ludhiana, India.

Table 2 .
Effect of different weed control treatments on growth, yield attributes and grain yield of wheat, during 2013-14 (I) and 2014-15 (II) growing seasons at Punjab Agricultural University Research Farm near Ludhiana, India.