Short running title: herbicides for weed control in egyptian clover Essay

Short running title: Herbicides for weed control in Egyptian clover

Core Ideas

· Chicory and scarlet pimpernel were the dominant weed species of Egyptian clover during both the years of the study.

· Imazethapyr (0.1 kg ai ha–1; PRE/POST) and alachlor (0.7 kg ai ha-1; PRE) fb quizalofop-ethyl (0.4 kg ai ha-1; POST) are the potential herbicides for controlling weeds and safe on Egyptian clover

· Imazethapyr (0.1 kg ai ha–1; POST) treated Egyptian clover recorded highest nodule number.

Impact of herbicides on weed control and fodder quality of Egyptian clover [Trifolium alexandrinum L.]

G.Prabhua*, D.R. Palsaniyaa, V. Singhb, R. Srinivasana, M. Chaudharya, S.R. Kantwaa

aIndian Grassland and Fodder Research Institute, Jhansi, UP, India

bTexas A&M University, College Station, TX, USA

*Corresponding author email: [email protected]


Weeds are the serious problem of forage production in India. An efficient herbicide program is necessary for the effective control of weeds in the Egyptian clover (Trifolium alexandrinum L.). This study aimed to evaluate the response of Egyptian clover and weeds to different herbicides that were being used in various cropping systems in Uttar Pradesh, India. The experiments were conducted in a randomized complete block design (RCBD) with three replications in a clay soil during 2013 and 2014 (2 years). A total of 8 different weed species were observed in this study. Scarlet pimpernel (Anagalis arvensis L.), chicory (Cichorium intybus L.), bermudagrass [Cynodon dactylon (L.) Pers], crowfootgrass [Dactylotenium aegyptium (L.) Wild] and littleseed canarygrass (Phalaris minor Retz.) were the dominant weed species during the crop season. Pendimethalin (PRE) resulted in a lowest grass weed density (2 m-2) at 25 DAS, however, severely injured the Egyptian clover. Imazethapyr PRE decreased grass weeds by 75% compared to weedy check but could not control scarlet pimpernel. Both alachlor PRE and pendimethalin PRE controlled 93% of the broadleaf weeds compared with weedy check. At 50 DAS, imazethapyr POST, alachlor PRE fb quizalofop-ethyl POST provided excellent control of weeds, and yielded equivalent to non-treated weed free check. Imazethapyr was safest on Egyptian clover and POST treated crop recorded highest nodule number. Alachlor (0.7 kg ai ha-1; PRE) fb quizalofop-ethyl (0.4 kg ai ha-1; POST) and imazethapyr (0.1 kg ai ha–1; POST) were also safe on soil microbial community compared to other herbicide treatments.


Egyptian clover (Trifolium alexandrinum L.), commonly called as berseem, is a succulent and highly nutritious annual clover, grown in winter and spring season (Gaafar et al., 2011) of Indo-Gangetic Plains. It is a multicut leguminous fodder crop and fixes atmospheric “N” in the soil (33-66 kg N ha–1) for succeeding crops (Knight, 1985; Williams et al., 1990). Egyptian clover provides a high quality fodder with 17–22% crude protein, 42–49% neutral detergent fibre, 35–38% acid detergent fiber, 24–25% cellulose, 7–10% hemi cellulose and 65% digestibility (Kumar et al., 2012). It can be utilized as a potential cover crop in various cropping systems (e.g. Ross et al., 2004). However, it is a slow growing winter crop especially during the seedling stage and poor in competing with weeds (Pathan et al., 2013). The major weeds of Egyptian clover in the region are chicory (Chicorium intybus L.), toothed dock (Rumex dentatus L.), spiny sawthistle [Sonchus asper (L.) Hill], celosia (Celosia argentea L.), greater swinecress [Coronopus didymus (L.) Sm.], bermudagrass [Cynodon dactylon (L.) Pers.], purple nutsedge (Cyperus rotundus L.) and crowfootgrass [Dactylotenium aegyptium (L.) Wild] (Pathan et al., 2013; Singh et al., 2012).

Weeds compete with Egyptian clover, reduce the forage yield by 20-55% and also contaminate the seed yield (Singh et al., 2012; Wasnik et al., 2017). The economic and quality loss of Egyptian clover due to weeds is considerably higher, but at the same time the growers pay meager attention to weed control because of the management cost involved, labour scarcity, and limited herbicide options for selective weed control (Prabakar et al., 2011). Apart from weed control, herbicide applications can drastically impact the non-targeted plant physiological processes such as photosynthesis, chlorophyll content, enzyme activities, and water use parameters. In a study conducted in Sudan, Wasfi and Ali (2016) reported decrease in chlorophyll-a, and b in corn and cotton after foliar application of pedimethalin at 5 ppm. However, Marenco and Lopes (1994) found no effect of trifluralin, and clomazone on chlorophyll concentrations in soybean but these herbicides had an effect on the nitrogen (N) concentrations in the stems, leaves and roots of soybean. Likewise, application of herbicides has also found to influence the root nodulation process in leguminous plants and soil microbial activity. Shankar et al. (2012) reported that cowpea root nodules number and dry weight of crop decreased with the application of 2",4-D, glyphosate and atrazine. Similar results were found by Eberbach and Douglas (1989) and Zaidi et al. (2004) where above mentioned herbicides (glyphosate and 2",4-D) decreased root nodules of sub-clover (Trifolium subterraneum L. Clare) and greengram (Vigna radiata L. wilczek). Apart from herbicide, rates applied also affected nodulation number (Zaidi et al. 2004). However, no information is available on the impact of potential herbicides for Egyptian clover on its fodder quality, yield parameters and soil microbial activity.

Therefore, the current study was designed with the objectives to 1) find a suitable herbicide for effective weed control and higher productivity of Egyptian clover in the region and 2) determine the effect of different herbicides on fodder quality and soil microbial activity.


Field Experiment

A field experiment was established at the central farm of Indian Grassland and Fodder Research Institute (IGFRI), Jhansi, India from 2013 to 2015 (2 years). The soil type of the experimental site was a clay loam, with a pH of 7.5, and organic carbon content of 0.15%. There were eight preemergence (PRE) and postemergence (POST) herbicide treatments and two non-herbicide treatments; weed free and weedy check, arranged in a randomized complete block design (RCBD) with three replications (Table 1). Each herbicide treatment was applied to a 5 × 4 m plot. Fertilizers, 20 kg N, 80 kg P2O5 and 30 kg K2O ha–1 were applied as a basal dose to all the plots through urea (CH4N2O with 46 % N), single super phosphate [Ca (H2PO4)2 with 16% P2O5 and 11% S] and muriate of potash (KCL with 60 % K2O) respectively. The Egyptian clover was planted during the first week of November in both the years (2013 and 2014). The PRE herbicides were applied at three days after sowing (DAS) whereas, POST herbicides were applied at 22-25 DAS.

The phytotoxic effect of herbicides on the crop and weeds were recorded at 21 days after treatment (DAT). Two cuts of Egyptian clover were taken (first at 55 DAS and second at 85 DAS). Plant samples were collected from treated plots (2 × 2 m2) at the time of harvest, air-dried and then kept in an oven at 65oC for dry matter estimation and converted into yield in metric ton (t) per hectare (ha). Four quadrates of 0.25 m2 (0.5×0.5m) were placed at random in each plot for recording different weed species at 25 and 50 DAS.

Analysis of Chlorophyll and Carotenoids Content

Fresh leaves (0.05 g) of Egyptian clover were collected from the field and then washed, blotted dry and dipped in test tubes containing 5 ml of dimethyl sulfoxide (DMSO) for overnight as described by Sawhney and Singh (2002). The extracted chlorophyll in DMSO was estimated by recording its absorbance at 663 and 645 nm using a double beam UV-VIS spectrophotometer (Motras, Registered office, EA-146, Inderpuri, New Delhi, 110012, India) and was estimated using the formula given by Lichtenthaler and Wellburn (1985).

Where V is the volume of DMSO, A is the Path length and W is the weight of tissue taken.

Number of Root Nodules

Root nodules of Egyptian clover were counted at the time of harvest from five random plants per plot per replication. These data were averaged over sampling units (5 plants) for each plot.

Soil Microbial Count

Enumeration of soil microbial population was carried out by using the standard dilution plate technique. About 10 g of air-dried soil was used for serial dilution and the specific media used for the enumeration of microorganisms is indicated in Table 2. Before the counting of colonies, the agar plates (three replications) were incubated at 28°C for two days for bacteria, seven days for fungi, five days for phosphorus solubilizing bacteria (PSM), and three days for rhizobium.

Soil Dehydrogenase Activity

Dehydrogenase activity was determined by Triphenyl Tetrazolium Chloride (TTC) method as outlined by Page et al. (1982). Air dried soil samples (1 g) were incubated with 0.2 mL of 3% TTC and 0.5 mL of water at 28±0.5 0C for 24 hours. The amount of Triphenyl Formazan (TPF) formed after the incubation period was extracted with methanol (10 ml) and quantified using the spectrophotometer (spectrophotometrically) at 485 nm wavelength using standard TPF. The dehydrogenase activity was expressed as μg Triphenyl formazan (TPF) formed hr–1 day–1.


The amount of rainfall and maximum and minimum temperature for the cropping season 2013-2014 and 2014-2015 is shown in the Fig. 1. The amount and distribution of rainfall varied during two cropping seasons (259.1 mm and 108.6 mm in 2013-14 and 2014-15, respectively). The rainfall received in these seasons were higher than that of long-term average rainfall (56.3 mm) in that season. However, monthly maximum and minimum temperature did not vary much during the period of the study.

Statistical Analysis

Data were subjected to Shapiro-Wilk (normality check) test. The data on weed density and dry matter were subjected to square root transformation before analysis. The analysis of variance (ANOVA) were performed using the Proc-Mixed procedure in SAS (SAS Institute Inc, Cary, NC) and means were separated using Fisher’s Least Significant Difference (LSD) at α = 0.05. Treatments and year were considered fixed effects in the model, whereas blocks (nested within year) were considered as the random effects. The interaction effects of year by treatments were not significant (P ≥ 0.05) for the weed density, biomass and yield; therefore, data from both the years were pooled.


Weed Flora

A total of eight winter weed species were recorded in the treatment plots. These included bermudagrass [Cynodon dactylon (L.) Pers], chicory (Cichorium intybus L.), crowfootgrass [Dactylotenium aegyptium (L.) Wild], greater swinecress [Coronopus didymus (L.) Sm.], Indian sweetclover [Melilotus indicus (L.) All], littleseed canarygrass (Phalaris minor Retz.), scarlet pimpernel (Anagalis arvensis L.), and spiny sawthistle [Sonchus asper (L.) Hill]. At 25 DAS, chicory (Cichorium intybus L.) and scarlet pimpernel (Anagalis arvensis L.) were the prominent weeds in this study. The distribution of weed species in Egyptian clover crop in the region is similar over the past several years. Previously, Kumar and Dhar (2008), reported that chicory, bermudagrass and purple nutsedge were the dominant weeds of Egyptian clover. However, in the current study, at 50 DAS, density of scarlet pimpernel decreased to 42%, whereas, chicory density increased to 133% compared to previous evaluation at 25 DAS in the weedy plot. The regenerative potential of chicory is high, which makes it to grow very well after the harvest of Egyptian clover.

Effect of Herbicide Treatments on Weed Density and Biomass

In general, herbicide treatments decreased the grass and broadleaf weed density in Egyptian clover. (Table 3). At 25 DAS, only PRE applied herbicides were evaluated, such as, pendimethalin, imazethapyr and alachlor. Pendimethalin treated plots recorded a lowest grass weed density (2 m-2) compared with other herbicide treatments, however, it was not safe on Egyptian clover (described later). Imazethapyr and alachlor treated plots recorded a grass weed density of 8 and 9 m–2, which was significantly lower than the non-treated weedy plot (32 m–2). In terms of broadleaf control, efficacy of both alachlor and pendimethalin herbicides were similar and controlled 93% of the broadleaf weeds compared with weedy check. Cantwell et al. (1989) suggested that imazethapyr can be a potential herbicide for the selective control of both grass and broadleaved weeds. However, imazethapyr is not labelled to control the broadleaved weeds, such as, Indian sweet-clover, greater swinecress, scarlet pimpernel and spiny sawthistle. This led to the higher density of broadleaf weeds in the imazethapyr treated plots compared with pendimethalin and alachlor. The grass weed biomass followed the similar trend as the grass weed density. For broadleaf weeds, all PRE herbicides recorded similar biomass, which was 91% lower compared to the weedy plot (Table 3).

At 50 DAS, the performance of both PRE and POST applied herbicides were evaluated (Table 3). Imazethapyr POST, alachlor PRE fb quizalofo-ethyl POST, propaquizafop POST and tankmix of quizalofop-ethyl + oxyfluorfen applied POST recorded lowest grass weed density compared to the other herbicide treatments. Among these herbicides, imazethapyr and tankmix application of quizalofop-ethyl + oxyfluorfen also provided excellent control of broadleaf weeds and recorded lowest dry biomass of both grasses and broadleaf weeds, whereas propaquizafop was not effective on broadleaf weeds. Imazethapyr was also the safest herbicide on Egyptian clover whereas tankmix application of quizalofop-ethyl + oxyfluorfen and propaquizafop caused severe crop injury. Alachlor PRE fb quizalofop-ethyl POST provided excellent control of grasses but was little weak on broadleaf weeds at 50 DAS. Therefore, imazethapyr was the potential herbicide for Egyptian clover because of its broad-spectrum activity on majority of grass and broadleaf weeds in the current study. In a study conducted on a silty clay loam soil in Mississippi, Shaw and Wixson (1991) reported that 87 to 98% control of pitted morningglory, entireleaf morningglory, johnsongrass, and tall waterhemp was observed with imazethapyr applied at 70 g ai ha–1 as a POST herbicide in soybean. The grass and broadleaf weed biomass (Table 3) followed similar trend as in the case of grass and broadleaf weed density.

Phytotoxicity Effect of Herbicides

Pendimethalin (PRE) and tank-mix application of quizalofop ethyl and oxyfluorfen (POST) caused mortality and severe injury on Egyptian clover (Table 4). The pendimethalin application caused the crop stand loss and alteration of clover morphology (club shaped seedlings). This could be attributed to the inhibition of spindle apparatus and prevention of chromosome alignments during the mitosis by pendimethalin (Senseman, 2007). Similar results have been reported by Mishra (2012), wherein pendimethalin reduced 50% of clover population when applied on a clay loam soil. Furthermore, Jha et al. (2014) reported a decrease in the Egyptian clover yield (17%) with the application of pendimethalin (1.0 kg ai ha–1) compared to weed free plot. It has been indicated that higher dose (> 0.5 kg ai ha–1) of pendimethalin is toxic to Egyptian clover (Barevadia et al., 1998). Similarly, tankmix application of quizalofop + oxyfluorfen also severely injured the crop plants. In a study conducted on a silty loam soil, Dubey et al. (2018) found that straw and grain yield of chickpea in pendimethalin (PRE) fb quizalofop ethyl + oxyfluorfen (POST) treated plots was lower (0.47 and 0.94 t ha–1, respectively) compared to weed free plot (0.92 and 1.80 t ha–1respectively). Other herbicide treatments in this experiment, such as, alachlor (PRE), alachlor (PRE) fb quizalofop-ethyl (POST), imazethapyr (PRE), imazethapyr (POST), were identified as selective herbicides to Egyptian clover because they did not cause significant crop injuries (Table 4). Wasnik et al. (2017) reported that imazethapyr (POST) treated plots recorded a highest dry matter yield (7 to 10 g m–2) of Egyptian clover compared to weedy check, which clearly indicates the selectivity of imazethapyr to clover. Similarly, Singh et al. (2010) found that application of alachlor (PRE) provided higher clover yield and greater weed control efficiency (83.1%) compared to weedy check. These results indicate that alachlor and imazethapyr are safe to use in Egyptian clover.

Effect of Herbicide Treatment on Total Chlorophyll and Carotenoid Content

The chlorophyll and carotenoids content were not affected by the application of any of the herbicides (Table 5). The findings of the current study exemplify that herbicides used in this study did not have any impact on the photosynthetic activity of Egyptian clover. However, a study conducted on a clay soil in Egypt, El-Nady and Belal (2013) reported that application of pendimethalin significantly increased chlorophyll pigments (chlorophyll a, b and total chlorophyll) at cotyledon and first true leaf stage of Cucumis sativa. It was speculated that the increase in chlorophyll pigment content was accompanied with the increase in mesophyll tissue thickness. Similarly, Amaregouda et al. (2013) revealed that total chlorophyll content (1.34 mg g–1 of fresh weight) was higher in soybean when applied with pendimethalin 1350.5 g ai ha–1, imazethapyr 87.5 g ai ha–1 (1.26 mg g–1 of fresh weight) and tepraloxydim 100 g ai ha–1 (1.24 mg g–1 of fresh weight). However, a lack of significant effect of herbicides on the chlorophyll content of the Egyptian clover in the current study is not yet clear and needs further investigations.

Effect of Herbicide Treatment on Root Nodulation of Egyptian Clover

Root nodule counts of Egyptian clover were affected by the application of herbicides (Table 5). In general, the nodulation was lower with PRE herbicides. It was believed that the PRE application of herbicides, probably, had chemical induced stress on the development of root nodules. The highest root nodule number was found with imazethapyr POST (38) followed by premix application of imazethapyr + imazamox POST (31), compared to the other treatments. Curley and Burton (1975) reported that pesticides may affect the rhizobia potential to stimulate nodulation in legumes at the seedling stage. Moreover, Aggarwal et al. (2014) reported that the application of imazethapyr POST at 100 g ha–1 had resulted in a greater nodule number in blackgram (47% higher) compared to control treatment, which supports the findings of this study. Several other studies have also found a higher nodule counts with imazethapyr application (Raman and Krishnamoorthy, 2005; Chattha et al., 2007; Kumar et al., 2017).

Effect of Herbicides Treatments on Soil Dehydrogenase Activity

Soil dehydrogenase activity was influenced by the herbicide treatments (P ≤ 0.05). In general, the herbicide treatments have reduced the dehydrogenase activity by 10 to 60% compared to weed free plot (Table 5). A lower soil dehydrogenase activity in herbicides applied treatments has also been reported by Sebiomo et al. (2011). The greater dehydrogenase activity in the weed free plot might be due to the lack of inhibitory effect of herbicides and a greater soil water content due to absence of weeds. Glinski et al. (2000) and Wollinska and Bennicelli (2010) established that decrease in the soil water content significantly increases the oxygen diffusion rate (ODR) and redox potential of a soil. Furthermore, the authors have reported that optimum level of ODR for the dehydrogenase activity should be at least 35µg O2 m–2 s–1. In this study we did not measure the ODR and diffusion rate of the experimental plot because it was not the focus of this study. However, decreased ODR level in the weed free plot could be a possible reason for the higher dehydrogenase activity.

Among the herbicides, alachlor treated plots recorded a lowest (87.7 µg TPF g–1 24 hr–1) dehydrogenase activity compared to other herbicide treatments. The half-life of alachlor ranged from 20 to 60 days at soil surface (Pothuluri et al. 1990; Walker et al. 1992) but studies have shown that half-life of alachlor can extend under moisture stress and lower temperature conditions. A study conducted in United Kingdom, Walker et al. (1992) indicated that the half -life of alachlor increased to 244.8 days at 5 °C soil temperature when compared with 25 °C (34.8 days). In the current study, the average temperature during the crop season (October to March) was below 25 °C (Fig.1). Furthermore, Felsot et al. (1995) reported that the alachlor at higher concentrations (longer half-life) inhibits the bioactivity of micro-organisms which indeed decrease the dehydrogenase activity. However, alachlor fb quizalofop-ethyl treated plots had higher dehydrogenase activity (214.23 µg TPF g–1 24 hr–1), which was equivalent to weed free plot (Table 5), fb quizalofop-ethyl + oxyfluorfen. These herbicides provided excellent control of grasses and broadleaf weeds leading to lesser density of crop/weed plants and lower ODR, which could result in higher dehydrogenase activity. The high dehydrogenase activity in these herbicide treated plots could also be attributed to the utilization of applied herbicides as a carbon source that might have stimulated the microbial activity (e.g. Panettieri et al. 2013).

Effect of Herbicide Treatments on Soil Microbial community

Herbicide treatments influenced the soil microbial community of the experimental site (P

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