Introduction

Toothed dock (Rumex dentatus L.) is a major winter season broadleaf weed of irrigated wheat in India. Its mature plants are generally 30-50 cm taller than wheat with an average plant height of 160±20 cm and produce abundant seeds (16000 fruits/seeds per plant) (Dhawan, 2005). It is highly competitive weed because of its vigorous growth and can reduce wheat grain yield by 70% with infestation of 30 plants/m² (Waheed et al., 2017). Its occurrence in wheat is mainly confined to rice-wheat system (Chhokar et al., 2007b; Singh et al., 1995) and further favored by adopting no-till (NT) in wheat (Chhokar et al., 2007a). The two favorable conditions for this weed, higher soil moisture and shallow seed burial (0-2 cm) are being provided by combination of NT-wheat and rice-wheat system.

The majority of R. dentatus emerges from seeds lying within shallow soil depth of 0-2 cm and fails to emerge with seeding depth of >4 cm (Dhawan, 2005; Singh and Punia, 2008). Although, the presence of perianth around the seed acts as a barrier to germination, but it also helps in concentrating the seeds near to surface during the puddle flooding conditions of rice due to lower seed density (Chhokar et al., 2007a). Moreover, its seed can tolerate the flooding conditions and a flooding duration of 80 days resulted in lowering the emergence of R. dentatus by 46% only (Singh and Punia, 2008). Thus, the abundant seed production, longer seed viability in soil (Lewis, 1973), ability to concentrate seeds near soil surface and withstand flooding conditions made it to fit well in rice-wheat system. Moreover, seeds are less prone to be destroyed by predators (Kumar et al., 2013) and as a result if not appropriately controlled, its abundance increases over the years.

It was not a major problem before the mid-nineties in India, when isoproturon and 2,4-D either alone or in combinations were mainly used for weed control in wheat. These herbicides kept under check the majority of broad-leaved weeds including R. dentatus (Chhokar et al., 2012). However, weed flora shifted in favor of this weed with shift in herbicide usage pattern after the evolution of isoproturon resistance in Phalaris minor Retz during the mid-nineties (Chhokar et al., 2012). The grass herbicides (fenoxaprop and clodinafop) and broad-spectrum herbicide sulfosulfuron recommended for control of single dominant weed isoproturon resistant P. minor, favored R. dentatus due to their ineffectiveness against this weed as well as removal of competition from other weeds. Simultaneously, the introduction of NT wheat in rice-wheat system of northwestern Indian plains further increased R. dentatus infestations because of favorable conditions in the system (Chhokar et al., 2007a). In NT system, it germinates before or along with the crop and can offer stiff competition as weeds germinating earlier or along with the crop are more competitive than late emerging.

For several decades, herbicides have been the preferred weed management tool. Among herbicides, one of the most important mode of action group of herbicides is ALS/AHAS inhibitors, which includes five classes of herbicides: sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidinyl-thio-benzoates (PTBs), triazolo-pyrimidines (TPs), and sulfonylamino-carbonyl-triazolinones (SCTs) (Powles and Yu, 2010; Mehdizadeh, 2016). However, over-reliance on herbicides of limited modes of action of the risk-prone group such as ALS inhibitor herbicides has led to the quick evolution of herbicide resistance (Tranel and Wright, 2002; Beckie and Tardif, 2012; Delye et al., 2013). Among five ALS inhibitor groups, the first introduction is of SU herbicides group during 1980s and the first reported case of resistance from this group is of chlorsulfuron in a biotype of prickly lettuce (Lactuca serriola L.) with selection from chlorosulfuron + metsulfuron mixture in the USA in 1987 (Mallory-Smith et al., 1990). Currently, resistance to ALS/AHAS inhibitors has been documented in 168 weed species including 65 broad-leaved weeds from 54 countries (Heap, 2021).

Metsulfuron-methyl a highly active sulfonylurea herbicide is used globally at low rates ranging 2.0-5.0 g/ha (Chhokar et al., 2015). In India, metsulfuron at 4 g/ha was recommended during 1998 to control broad-leaved weeds including R. dentatus in wheat. The Indian farmers preferred metsulfuron over 2,4-D due to its better crop safety and compatibility with other grass herbicides (Pinthus and Natowitz, 1967; Balyan and Panwar, 1997; Chhokar et al., 2012). Therefore, metsulfuron was extensively used either in sequence with grass herbicides or as tank or ready-mixture with clodinafop and sulfosulfuron for diverse weed flora control in wheat. Since, R. dentatus was highly sensitive to metsulfuron, a reduced dose of 2.0 g/ha with sulfosulfuron 30 g/ha was also recommended (Chhokar et al., 2007b).

Metsulfuron remained effective for about 15 years in India and authors observed the first case of metsulfuron resistance evolution in a population of R. dentatus in 2013 (Heap, 2021) due to the continuous use of metsulfuron. Similarly, in Brazil, Argentina, Canada, and Australia, the strong selection pressure by continuous use of ALS inhibiting herbicides in wheat (Triticum aestivum L.) has documented the emergence of ALS resistant wild radish/radish (Raphanus sp.) biotypes (Yu et al., 2012; Pandolfo et al., 2013; Costa and Rizzardi, 2014; Cechin et al., 2016), causing crop yield losses and making the control costly and difficult. Now, in many fields, R. dentatus populations are escaping the control with metsulfuron even at higher doses (2X=8 g/ha) and repeated applications of the recommended rate leading to increased infestation in wheat fields. At few farmers’ fields, its huge pressure is being observed leading to the problem in both manual and mechanical/combine harvesting of wheat crop along with significant yield losses. The similar maturity period of R. dentatus and wheat causes the contamination of the wheat grains with R. dentatus seeds. As most farmers use their own seed or neighbor farmers, which may further increase its infestation and the spread of resistance problem (Chhokar et al., 2012). The early detection and management of resistance is very crucial to avoid the yield losses. For herbicide resistance management alternative herbicides will always remain the central strategy. So, keeping these in view, the present study was undertaken to identify and quantify the herbicide resistance and cross-resistance patterns to various herbicides in R. dentatus along with its control by alternative herbicides.

Materials and Methods

A series of pot studies were conducted at Resource Management Block, ICAR- Indian Institute of Wheat and Barley Research, Karnal, Haryana, India (Latitude 29^o 43’N, Longitude 76^o 58’E at an elevation of 245 m above mean sea level).

Seed collection of Rumex dentatus populations

Mature seeds of seven R. dentatus populations were collected from wheat fields at maturity (month of April) during 2014 from Haryana state of India. Out of seven, five populations [Nagla, Kurukshetra (NAKUH); Saini Majra, Ambala (SMAMH); Ajrawar, Kurukshetra (AJKUH); Ujjha, Panipat (UJPAH) & Ajanthali, Karnal (AJKAH)] having inadequate or no control with metsulfuron methyl at the recommended (4 g/ha) or higher doses were collected from rice-wheat system. Seeds after collections were stored in paper bags at room temperature in the laboratory till November for bioassay studies. For comparison, populations (IIWBR research farm (DWR); Taraori, Karnal (TAKAH)) earlier confirmed sensitive to all herbicide assayed were used as susceptible (S) stock for resistance detection and quantification and these were from area having different herbicide applications and crop rotations history. The bioassay studies were conducted for three years and for seed collection, R. dentatus populations were raised separately in pots during 2014-15 and 2015-16. In addition, metsulfuron at 4 g/ha was sprayed on five resistant populations at around one-month old seedlings stage, whereas the susceptible population stock was maintained without any herbicide treatment. Moreover, after flowering stage, all the populations were kept in isolation, and also provided with a protected covering of a cloth to exclude any possibility of outcrossing till maturity. Once mature, the seeds were collected and kept separately for further studies in the subsequent season.

Quantification of herbicide resistance profile in R. dentatus

To quantify the herbicide resistance profile, pot studies were conducted for three consecutive Rabi seasons (2014-15, 2015-16, and 2016-17). Each year in November, R. dentatus populations were grown in pots of 15 cm diameter. The pots were filled with soil and well rotten farm yard manure (FYM) in 6:1 ratio by volume after passing through 2 mm sieve. The soil was from the field having no previous infestation of R. dentatus and was a sandy clay loam with pH of 8.1 and an organic carbon of 0.45%. The pots were watered to deplete the soil seed bank, if any, before sowing of R. dentatus.

1. dentatus seedlings were established by seeding about 60 healthy seeds (without perianth) at 0.5-1.5 cm depth. To remove the perianth, seeds were gently rubbed in a plastic tray. Three pots were used for each herbicide treatment (Table 1). Pots were labeled and arranged in a completely randomized design and watered as required. After three weeks of emergence, thinning was done to maintain fifteen plants per pot. Pre-emergence herbicide (Pendimethalin) was applied at 1-2 DAS (days after sowing) and post-emergence herbicides were sprayed 30-35 DAS (3 to 4 leaf stage of R. dentatus) with knapsack sprayer fitted with flat fan nozzles delivering 350 L/ha of water. For herbicide resistance profile studies, the herbicides and their graded doses used are given in Table-1. Cationic surfactant, Leader Mix (polyethylene amine) from Sumitomo India Ltd. was used at a concentration of 0.35% (v/v) with metsulfuron, triasulfuron, pyroxsulam, florasulam, and ready-mixture of halauxifen + florasulam treatments. Four weeks after the herbicide spray, the fresh weight of seedlings was recorded and, based on the biomass reduction, the 50 and 90% growth reduction (GR₅₀ and GR₉₀) values were determined (Finney, 1971). GR₅₀ could not be calculated for some resistant populations because fresh biomass did not decrease by 50% compared to control, even at the highest dose used. Therefore, GR₅₀ and resistance index (RI) were mentioned in excess of the highest dose tested. Contrary, where the response at the lowest rate was higher than the 50 and 90% observed growth reduction, the GR₅₀ and GR₉₀ values were mentioned as less than the lowest tested dose except for metsulfuron where the calculated response was very close. The resistance index /Resistance Factor (RF) was calculated by dividing the GR₅₀ values of different populations with GR₅₀ value of the most S population. The values >2 were considered resistant (R).

Table 1. Herbicide rates for quantification of herbicide resistance in R. dentatus

Screening of R. dentatus populations against major herbicide groups

Additionally, during two consecutive seasons of 2018-19 and 2019-20, a total of 119 populations collected were screened against six herbicides for knowing the extent of prevalence of metsulfuron resistance and any possibility of further extension of resistance to other mechanisms of action. The procedures for preparing pots and spraying herbicides were similar as mentioned in herbicide resistance profile studies section. However, the herbicide treatments evaluated in this study were metsulfuron at 4 g/ha, carfentrazone 20 g/ha, aclonifen + diflufenican 1000 g/ha, isoproturon 1000 g/ha, fluorxypyr 250 g/ha, 2,4-D-E 250, and 500 g/ha. These herbicides belonged to different chemical families namely sulfonylurea (metsulfuron-methyl), Aryl triazolinone (carfentrazone), urea (isoproturon), phenoxy-carboxylic acid (2,4-D), pyridine carboxylic acid (fluroxypyr), diphenylether (aclonifen), and pyridinecarboxamide (diflufenican). For comparison, untreated controls were also kept and compared to control of the respective population, the growth reductions and mortality under different herbicide treatments were visually assessed and populations were categorized into four groups comprising of 0-25, 26-50, 51-75, and 76-100% growth reductions. Finally, the percentage of the population falling under different % control group of the herbicide treatments was worked out.

Statistical analysis

The shoot fresh weight data of various R. dentatus populations were converted to percent biomass reduction in comparison to the untreated control and these data were pooled over the years due to similar response. The mean per cent growth reduction data were analyzed using non-linear regression procedure to estimate the mean herbicide dose causing 50 and 90% growth reduction (GR₅₀ and GR₉₀) in a SAS 9.3 software (SAS Institute, Cary, NC).

Results and Discussion

Quantification of herbicide resistance profile

The resistance profile of seven R. dentatus populations (DWR, NAKUH, SMAMH, AJKUH, UJPAH, AJKAH, and TAKAH) was studied for three consecutive rabi seasons from 2014-15 to 2016-17 against twelve herbicides (Table 2) belonging to nine herbicide groups namely sulfonylurea (metsulfuron, triasulfuron), triazolopyrimidine sulfonanilide (pyroxsulam, florasulam), synthetic auxin/arylpicolinate (halauxifen methyl), pyridine carboxylic acid (fluorxypyr), phenoxy-carboxylic acid (2,4-D), triazolinone (carfentrazone), protoporphyrinogen oxidase/PPO (flumioxazin), phenylurea (isoproturon), and triazinones (metribuzin). Based on the mean responses across years of R. dentatus populations to different herbicides, the calculated GR₅₀ and GR₉₀ values are given in Table 2. The two biotypes (DWR, Taroari) were sensitive to all the evaluated herbicides. The 50% growth reduction (GR₅₀) values for metsulfuron were 0.12 and 0.11 g/ha for DWR and TAKAH, respectively, whereas 90% growth reduction (GR₉₀) value of DWR and TAKAH for metsulfuron was 0.34 and 0.37 g/ha, respectively. Both these sensitive populations recorded considerably lesser GR₅₀ and GR₉₀ values for metsulfuron than remaining five R populations having GR₅₀ and GR₉₀ values in the range of 7.75-10.30 g/ha and 49.84-104.61 g/ha, respectively. In comparison to the most susceptible response of a population (GR₅₀ of TAKAH and GR₉₀ of DWR) to metsulfuron, the resistant biotypes had GR₅₀ and GR₉₀ values of 73.8-98.2 and 146.6-307.7 times higher (Table 3). Thus, R. dentatus populations exhibited differential response to the application of metsulfuron. The most resistant (R) population (SMAMH) exhibited 308 folds greater GR₉₀ value for metsulfuron than that of the most S population (DWR) and was equivalent to 26.1 times higher than the recommended field rate (X=4 g/ha) in wheat. Whereas, the 90% control (GR₉₀) of S populations was achieved even at 8.5-9.3% of the recommended rate. It means that highly metsulfuron sensitive (MS) R. dentatus has evolved a high level of resistance having RF as high as 308. Similarly, Costa and Rizzardi (2014) showed a very high-level RF (267- fold) of metsulfuron resistance in Raphanus raphanistrum.

Table 2. Herbicide resistance profile of R. dentatus populations based on response of three years (2014-15, 2015-16 and 2016-17)

Table 3. Herbicide Resistance Index [RI= GR₅₀(R)/GR₅₀(S)] of Rumex dentatus populations for different ALS inhibitor herbicides

The MS populations (DWR and Taraori) were also susceptible to triasulfuron, pyroxsulam, and florasulam (Table 2). However, metsulfuron R (MR) populations exhibited cross-resistance to triasulfuron, pyroxsulam, florasulam, and halauxifen-methyl + florasulam. The GR₅₀ and GR₉₀ requirement for triasulfuron of MR populations ranged from 141.1 to 269.5 g/ha and >480 g/ha, respectively. Five cross-R populations exhibited the GR₅₀ values for triasulfuron >9.4-17.96 times (141.1-269.5 g/ha) of the field application rate (15 g/ha). Similarly, for pyroxsulam and halauxifen + florasulam, the GR₅₀ values of MR populations ranged from 620.5to >2304 g/ha, and 5.81-10.14 g/ha (>551.6-3220 and 37.5-65.4 times of the most S biotype), respectively, while the respective GR₉₀ requirements were >2304 g/ha and 36.21-74.48 g/ha. Thus, compared to S, MR populations needed >1674.4 folds higher pyroxsulam for the same level of 90% growth reduction responses. Furthermore, it is evident that the cross-resistance level was higher for pyroxsulam and florasulam, but lower for ready-mix combination of halauxifen + florasulam. Similarly, the high cross-resistance levels in G. spurium populations of ALS inhibitors (chlorsulfuron, tribenuron, florasulam, triasulfuron, thifensulfuron, and sulfometuron) have been shown by many weed scientists (Hall et al. 1998; Van Eerd, 2004; Beckie and Tardif, 2012). Earlier also, variable and higher SU resistance levels of radish were observed for chlorsulfuron having GR₅₀ values greater than 640 g ha^-1 (Han et al., 2012) and more than 43 g ha^-1 of metsulfuron-methyl (Pandolfo et al., 2013).

In the present study, although the resistance in R. dentatus was observed with metsulfuron but the resistance level was higher with pyroxsulam, which is not yet used. This response might be due to the type of target site alteration selected by metsulfuron (Lamego et al., 2009). Contrarily, Kudsk et al. (1995) pointed the higher resistance to chlorosulfuron, the selecting agent compared to triazolopyrimidine herbicide (flumetsulam), whereas compared to metsulfuron higher RI was reported for other SU herbicides in Kochia (Kochia scoparia [L.] Schrad.) (Saari et al., 1990) and Stellaria media (Kudsk et al., 1995).

The triasulfuron, pyroxsulam, and halauxifen methyl + florasulam are not yet used by the wheat farmers in India because they are either under registration process or not commercially marketed (triasulfuron) for wheat crop. The expected cross-resistance pattern against these herbicides except ready-mixture of halauxifen-methyl + florasulam is because of similar mechanism of action (ALS inhibition). The cross-resistance in pre-mix halauxifen + florasulam is because halauxifen, a synthetic auxin, is poor against R. dentatus (Chhokar unpublished data). Mostly, if resistance evolves to a herbicide, then the other herbicides of that group usually become ineffective. Similarly, in the present study, the MR populations showed cross-resistance to triasulfuron, pyroxsulam, and florasulam.

Similar to the response of R. dentatus against pyroxsulam in the present studies, Sprague et al. (1997) reported the resistant A. rudis biotype >1920 folds more R to chlorimuron at the whole-plant level than the S biotype. The chlorimuron rate required to reach the GR₅₀, in the R biotype was not attained with rates as high as 1000 g/ha (100 times the field use rate). However, the GR₅₀ for the S biotype was 0.52 g ha^-1 much less than the labeled field rate of 10 g ha^-1.

The variable pattern of resistance and cross-resistance level within and between the various ALS inhibitor herbicides families is generally observed (Poston et al., 2000; Merotto et al., 2009; Pandolfo et al., 2013; Schaedler et al., 2013; Sada et al., 2013; Kuk et al., 2003) in different weed species/populations. This differential response occurs because ALS herbicide families bind to various domains in the enzyme. Thus, if resistance is due to alteration(s) in the binding site, then the affinity of ALS herbicides will be affected differently (Devine et al., 1991; Tranel and Wright, 2002). Resistant biotypes of Lindernia spp. showed very diverse RI values ranging from 60 to 14,100 (Uchino and Watanabe, 2002). Saari et al. (1992) studied four SU resistant weed species and found a higher resistance level to the selecting agent with only one species, i.e. perennial ryegrass. The greater RF with the SUs (thifensulfuron and chlorimuron) than with the imidazolinone (imazethapyr) at the whole plant level was observed in common waterhemp (Lovell et al., 1996), Russian thistle (Saari et al., 1992), and kochia (Saari et al., 1990) in spite of the selecting herbicide family was either SU (Saari et al., 1990, 1992) or imidazolinone (Lovell et al., 1996). In contrast, a smooth pigweed biotype selected with imazaquin demonstrated high resistance levels to imazaquin and imazethapyr, but little or negative cross-resistance to eight SU herbicides at the enzyme level (Manley et al., 1995). Likewise, chlorosulfuron R sugar beet was not cross-resistant to the metsulfuron, imidazolinones, i.e. imazaquin and imazethapyr (Hart et al., 1992).

Our studies indicate SU resistant R. dentatus as cross-resistant to TP. Earlier studies showed a general trend of cross-resistance between sulfonylurea (SU) and triazolopyrimidine (TP) herbicides, and between imidazolinone (IMI) and pyrimidinylthiobenzoate (PTB) herbicides (Devine and Eberlein, 1997). Whereas Mendes et al. (2019) found at least three distinct resistance patterns: resistance to imazethapyr/IMI, chlorimuron/SU (pattern R1), resistance to imazethapyr, chlorimuron, and diclosulam/TS (pattern R2) (the most frequent), and the exclusive resistance to imazethapyr (pattern R3) in Bidens spp. against ALS-inhibitors.

Previous studies showed the higher vulnerability of ALS inhibitor herbicides to resistance evolution and it can occur quickly with four to seven consecutive applications (Mallory-Smith et al., 1990; Primiani et al., 1990; Saari et al., 1994; Hashem et al., 2001; Beckie, 2006). However, even exposures to two applications of either SU herbicide (Sprague et al., 1997) or the imazethapyr (Hora and Peterson, 1995) have led to the appearance of resistance in populations of A. rudis. In our studies, about 10-12 years of metsulfuron usage resulted in resistance evolution. Tranel and Wright (2002) suggested that factors likely to enhance the selection of R- biotypes include “the repeated use of that herbicide over large areas, little or no use of the alternative modes of action herbicides, high efficacy of the herbicide on sensitive biotypes at the rate used, and soil residual activity of the herbicide. All these criteria match with the present case of MR- R. dentatus.

Besides, depending on the nature of herbicides, the resistance evolution also depends on the weed species. Amaranthus species are among the annual broadleaf weeds most prone to develop herbicide-resistant biotypes because of their high genetic variability, high production of rapidly germinating seed, and efficient pollen and seed distribution (Lovell et al., 1996). Similarly, the genus Rumex also shows high genetic variation, abundant seed production capacity and seeds can disperse through wind, water and contamination with crop seeds resulting in an increased selection pressure.

The other auxin herbicide 2,4-D-E had similar effectiveness against both the MR- and MS-R. dentatus. The GR₅₀ values ranged from 69.9 to 91.0 g/ha. Although, auxinic herbicides have been in the market for almost 75 years, resistance to this mode of action has been reported in only 41 weed species to date (Heap, 2021). Also, various R. dentatus biotypes responded similarly for isoproturon, metribuzin, fluorxypyr, carfentrazone, and pendimethalin, and also exhibited the respective GR₅₀ values for these herbicides across populations as <125, <25, <31.25, <2.5, and <125g/ha. Whereas GR₉₀ values observed across populations were 140.2-179.9, 16.0-29.3, 29.8-59.9, 5.0-6.9, and 123.9-157.4 g/ha, for isoproturon, metribuzin, fluorxypyr, carfentrazone, and pendimethalin, respectively. Hamouzova et al. (2019) also observed no differential response of R- and S- populations, to isoproturon.

Our studies indicate R. dentatus populations as highly sensitive to pre-emergence application of pendimethalin and 90% control was achieved at about 16% of the field recommended rate of 1000 g/ha. The main reasons were shallow seeding and prevailing good moisture in pot studies. Gasper et al. (1994) also reported improved weed control with pendimethalin under better soil moisture conditions. Besides, pendimethalin, the excellent control of all Rumex populations was achieved after being treated with the recommended rates of carfentrazone, fluorxypyr, metribuzin, and 2,4-D. Similarly, Cechin et al. (2016) reported alternative post-emergence chemical control options for the ALS inhibitor resistant and cross-resistant radish as synthetic auxins (2,4-D amine), PSII inhibitor (bentazon), and PPO inhibitor (saflufenacil) in wheat crop. Hashem et al. (2001) also found that wild radish (R. raphanistrum) biotypes R to chlorsulfuron were susceptible to 2,4-D. Nevertheless, the rarity of evolved resistance in weeds to bromoxynil/ pendimethalin/ 2,4-D (Heap, 2021) highlights their future role in proactive and reactive herbicide resistant weed management in wheat.

Since, the rate of resistance development relates to the intensity of the selection for resistance (Martinez-Ghersa et al., 1997), the continuous usage of a herbicide or herbicides of the group having the same mechanism of action leads to faster herbicide resistance evolution associated with higher selection pressure (Burnet et al., 1991; Jasieniuk et al., 1996; Beckie, 2006; Powles and Yu, 2010). Similar situation might have happened in R. dentatus case, because the exclusive metsulfuron pressure was imposed in an unbroken rice-wheat sequence, which might have resulted in resistance development. The exclusive dependence on metsulfuron was because the farmers preferred it for its better efficacy against R. dentatus and safety to wheat crop compared to 2,4-D, as differential tolerance to 2,4-D depends on the wheat cultivars and the stage of application, as well. The repeated application of tribenuron in China has resulted in serious sulfonylurea resistance in main weed species Descurainia sophia (L.) Schur. (Cui et al., 2008). Likewise, NT and rice-wheat cropping sequence provide favorable conditions for heavy infestation of R. dentatus resulting in abundant seed production (Chhokar et al., 2007a). Heavy population pressure increases the chances of selection for R- populations as the number of herbicide R- mutants is proportional to population size (Jasieniuk et al., 1996). Moreover, metsulfuron is from high risk of herbicides (ALS inhibitors) and all these conditions favored resistance evolution. With the evolution of MR populations of R. dentatus, the interference in some wheat fields becoming similar to the herbicide resistant P. minor fields causing severe yield reductions to the extent of complete crop failure (Chhokar et al., 2012). Presently, the spread of metsulfuron resistance needs to be checked immediately; otherwise, it may become epidemic very fast and will threaten the food security by limiting the wheat production. One of the key tactics which can reduce the R- population is the early herbicide resistance detection and the use of alternative herbicides.

Based on the results of the present studies, the wheat yield reduction experienced in thearea having MR population can be resolved using pre-emergence pendimethalin and flumioxazin and post-emergence isoproturon, 2,4-D, carfentrazone, fluorxypyr, and metribuzin. In earlier studies (Chhokar et al., 2007b), 2,4-D, carfentrazone and isoproturon have effectively controlled S- R. dentatus.

Metribuzin was found equally effective in controlling both the herbicide R-and S-populations of R. dentatus (Table 2) and it is also effective against the other co-existing problem of multiple herbicide resistant Phalaris minor (resistant to ALS and ACCase inhibitors) (Chhokar et al., 2012). However, metribuzin adoption at farmers’ field will depend on its selectivity on wheat cultivars, as the differential tolerance to metribuzin has been reported earlier (Runyan et al., 1982; Kleemann and Gill, 2007). Similarly, the effectiveness of triazine herbicides (atrazine) against Eastern black nightshade (Solanum ptycanthum) populations R to ALS inhibitors (imazethapyr, flumetsulam, cloransulam, nicosulfuron, prosulfuron, and rimsulfuron) was reported by Ashigh and Tardif (2006).

To control ALS R- populations of R. dentatus, pendimethalin can be used as pre-emergence in conventional-till system; where as in zero tillage (ZT) can be used in combination with non-selective herbicide like glyphosate/paraquat as a pre-seeding option. This pre-seeding herbicide combination will further improve the weed control as one partner will control the existing weed flush (glyphosate) and the other will provide the residual weed control (pendimethalin). Although, the dinitroaniline resistance has been reported in some weeds (Mudge et al., 1984; Heap and Knight, 1986; Vaughn et al., 1990; Morrison et al., 1991; Moss and Cussans, 1991), the extent of resistance is low mainly due to the paucity of a detoxification mechanism in weeds.

In another study, a total of 119 populations were tested against six herbicides of different group for knowing the extent of prevalence of metsulfuron resistance and any possibility of further extension of resistance to other mechanisms of action (Figure 1). Of the total population, 52.9, 16.0, 7.6, and 23.5% were classified as having 0-25, 26-50, 51-75, and 76-100% control with metsulfuron, respectively. A significant population (68.9%) exhibited ≤50% control with metsulfuron 4 g/ha indicating large-scale infestation of MR- R. dentatus in wheat fields of north Indian Plains. The continuous monitoring of MR- R. dentatus including its distribution and spread is imperative for resistance management.

Figure 1. Rumex dentatus population percentage having different level of control with various herbicides during 2018-19 and 2019-20 (N= 119 populations)

While the alternative herbicides namely carfentrazone 20 g/ha, aclonifen + diflufenican 1000 g/ha, isoproturon 1000 g/ha, fluorxypyr 250 g/ha, 2,4-D-E 250, and 500 g/ha effectively controlled (76-100%) all the populations of R. dentatus. These herbicides belonged to different chemical families namely sulfonylurea (metsulfuron), triazolinone (carfentrazone), urea (isoproturon), phenoxy-carboxylic acid (2,4-D), pyridine carboxylic acid (fluroxypyr), diphenylether (aclonifen), and pyridinecarboxamide (diflufenican).

Herbicide group rotation and tank-mixing having different mechanism of actions are highly effective in preventing/delaying the resistance evolution and can be key component of an integrated weed management program (Jasieniuk et al., 1996; Cavan et al., 2000; Gressel, 2009; Beckie and Harker, 2017). A mixture of an ALS inhibitor with a photosystem-II inhibitor (Mallory-Smith and Retzinger, 2003) or auxinic herbicide or both is a much more effective tactic than rotation to delay resistance. Therefore, the alternative herbicides in mixture can be explored for managing and delaying the further extension of resistance. Alternative herbicide combinations such as isoproturon + 2,4-D, isoproturon + fluorxypyr, 2,4-D + carfentrazone, isoproturon + carfentrazone can be used for control of diverse broad-leaved weed flora including MR- Rumex in wheat. Brosnan et al. (2012) also reported that carfentrazone accelerates broad-leaved weed control with metsulfuron. However, for herbicide mixtures to be effective in delaying or preventing resistance, the component with the alternate mode of action should: (a) control the same spectrum of weeds; (b) have the same persistence; (c) affect a different target site; and (d) be degraded by different metabolic systems (Wrubel and Gressel, 1994).

Since no herbicide is invulnerable to selecting for resistant biotypes, excessive dependence on chemical weed control system is not a sustainable weed management strategy (Shaner, 2014). Thus, more focus has to be given to non-chemical weed control measures. Among non-chemical methods, the crop rotation is the most critical agronomic strategy to lower the selection pressure (Gressel and Segel, 1990) through implementation of different management options and restoration of diversity in weed flora. Crop rotation consisting of cover crops, competitive crops (barley/mustard) or short duration crops restricting weeds to form seeds due to difference in maturity period (pea and potato or the other vegetable crops between rice and wheat) or repeated cutting (green fodder crops like Egyptian clover, Lucerne, and Oat) will help in depleting the weed seed bank and thereby, the problem in the next season. Thus, crop rotation through its effect on weed seed germination and mortality affects the weed seed bank in soils.

Wheat varieties having early vigor with smothering and allelopathic effects on weeds also need integration. Adjusting the sowing time either early or late can also reduce R. dentatus impact. In Indian conditions, early wheat sowing (last week of October) reduces R. dentatus emergence along with crop due to higher temperature and less humidity leading to faster depletion of soil moisture in upper layers leading to reduced competition, whereas the late sowing deplete the soil seed bank by allowing germination followed by killing with herbicide or tillage operations.

Tillage also influences the weed flora abundance and it has been observed that NT favors the build-up of R. dentatus, but reduces the P. minor population. Similarly, conservation tillage and NT adoption increased the horseweed (Conyza canadensis) population and made it one of the most problematic weeds (Bhowmik and Bekeck, 1993; Brown and Whitwell, 1988). However, NT seeding also provides an opportunity to restrict the yield reductions due to control of herbicide resistant multiple weeds populations by pre-seeding application of tank-mix combination of non-selective herbicides (glyphosate/paraquat/glufosinate) with pendimethalin or metribuzin (Chhokar unpublished data). Moreover, if NT system with surface residue retention (conservation agriculture) is adopted then benefits are more in reducing the weed infestation (Chhokar et al., 2009). Kumar et al. (2013) also observed that residue retention drastically reduces the infestation of R. dentatus in wheat.

Among the various factors, the seed burial depth and soil moisture dramatically affect R. dentatus germination and emergence. For higher emergence, R. dentatus prefers higher soil moisture and shallow burial depth (Chhokar et al., 2007a; Singh and Punia, 2008). These findings need to be utilized for its management by creating unfavorable conditions through modifications in tillage crop establishment techniques involving zero-tillage, surface crop residue retention, and soil moisture management. Under rice-wheat system, after rice harvest, the major proportion of the R. dentatus seed bank remains near the soil surface. Allowing this seed bank to emerge and kill by either pre-planting herbicides combinations such as glyphosate/paraquat + pendimethalin in NT system or non-inverting shallow tillage in CT system can significantly lower its infestations. Moreover, the higher soil moisture requirement for seed germination of R. dentatus compared to wheat can also be utilized for its management. Chhokar et al. (1999) observed 64.7% germination of wheat even under osmotic potential of-10 bars, whereas Rumex germination is completely inhibited at osmotic stress higher than -2 bars (Dhawan, 2005). Thus, wheat could be seeded (5-6 cm depth) after slight depletion of moisture in the upper 3-4 cm soil layer of which R. dentatus emerges in maximum number, ultimately reducing the R. dentatus emergence. Similarly, burying R. dentatus seeds deeper than 4 cm by initial or primary tillage followed by subsequent shallow tillage operations so that seeds buried deeper do not come in the upper layer integrated with depleted soil moisture conditions in upper layer (3-4 cm) will reduce and delay the emergence leading to reduced competition to crop. To create the depleted soil moisture in upper soil layer, integration of early sown CT system and stale seed bed can be useful. In this scenario, the R. dentatus likely to emerge can also be controlled with application of pendimethalin just before first irrigation. Moreover, in north India, its population is increasing over the years along with increased number of infested fields. One of the reasons is sowing of contaminated wheat seeds with R. dentatus seeds. The combine harvesting is mainly responsible for its contamination with wheat grains due to their similar maturity. Likewise, seeds of new variety move very fast from farmer to farmer and pose risk of resistance spread through movement of contaminated seeds. The other factors which can also spread resistance are the use of un-rotten FYM. Measures should be taken to check the spread of R- populations to new areas by encouraging the use of certified seed and well rotten FYM.

With restricted discovery of new modes of action chemistries, growers have to face the issue of a reduced number of herbicide options in future. So, concerted efforts are required to prolong the effectiveness of the available/possible alternative herbicides by assisting them with other weed control practices. Moreover, developing herbicide resistant wheat to glyphosate and glufosinate will add to chemical control option and simplify the weed management by tackling multiple types of grass and broad-leaved weeds resistant to ALS, ACCase, and photosynthetic inhibitor herbicides in wheat (Harker et al., 2012; Chhokar et al., 2012). Thus, herbicide resistant/tolerant wheat will be an additional tool to the weed management toolbox to control herbicide resistant and tolerant weeds. It will also promote wheat-based NT and CA systems, leading to lower energy requirements. However, the long-term resistance management strategies should include the diversified IWM consisting of herbicide rotation, herbicide mixture, crop rotation, competitive variety, higher seed rate, closer spacing, tillage practices, sanitation practices (weed free crop seeds and manure), herbicide resistant varieties, and stale seedbed for sustaining wheat production.

A detailed study on R. dentatus biology is also required for further strengthening the herbicide resistance management by identifying the weak points in its life cycle and targeting them. Whenever and wherever possible, consideration should also be given to the use of mechanical weed control to remove weeds surviving the herbicide application before seed setting as well as use of weed seed harvesting/mechanical harvester for reducing the weed seed bank. The integration of all these approaches will lower the evolution and spread of herbicide resistance, ultimately improving wheat production and farm income.

Conclusions

The persistent and intensive metsulfuron use for R. dentatus control in wheat led to the evolution of ALS resistant populations in the northern Indian plains. Bioassay studies exhibited high level of metsulfuron resistance (RF of 308) in R. dentatus having survival up to 16-folds the recommended field dose (4g/ha). Furthermore, MR populations showed high but variable level of cross-resistance to multiple ALS-inhibiting herbicides thus eliminating several ALS groups (SU and PTB) options in resistant areas. However, old herbicides, 2,4-D and pendimethalin along with carfentrazone, fluorxypyr, and metribuzin are quite effective in controlling MR populations. These herbicides in combination and rotation can be used to reduce the selection pressure for resistance evolution and thereby wheat yield reductions. However, long term sustainable weed management should include the integration of chemical and non-chemical weed control methods.

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