Efficacy of Weed Management Techniques on Weed Control, Biomass Yield, and Soil Herbicide Residue in Transplanted Wild Marigold (Tagetes minuta L.) under High Rainfall Conditions of Western Himalaya

Abstract

A reduced herbicide rate with hand-weeding (HW) can act as a safer and sustainable approach for weed control. A field study was conducted at CSIR-IHBT, Palampur, India during 2018 and 2019 to analyze the efficacy of herbicides in combination with manual weeding on weed control and the yield of wild marigold (Tagetes minuta L). The experiment was laid with 12 treatments, consisting of two herbicide dosages with prescribed and reduced rates (R) of pendimethalin, imazethapyr, and carfentrazone-ethyl, along with integration of reduced-rate herbicide treatments with HW. The weed population, dry matter, and the crop biomass yield had a significant effect on different weed-control treatments. Imazethapyr (R) with HW recorded a reduced weed number (9.64 m⁻²) and weed dry matter (13.64 g m⁻²) and a greater biomass yield (235.03 q ha⁻¹). All the herbicides with integration with HW decreased the weed infestation and enhanced the biomass yield. The weed control efficacy of imazethapyr was higher than pendimethalin and carfentrazone-ethyl. Weed infestation in reduced doses of herbicides with HW was lower than in recommended doses. Herbicide residues in the soil of all herbicides at both the dosages were below the detectable limit (<0.001 μg g⁻¹). Therefore, a reduced dose of imazethapyr integrated with HW can be prescribed to T. minuta growers as a more sustainable approach.

1. Introduction

Wild marigold (Tagetes minuta L.; family Asteraceae) has been cultivated as an aromatic plant in different agro-ecological regions of world [1]. It originated in South America and has become established in temperate regions of the world [2]. In India, it is found in Himachal Pradesh, Jammu & Kashmir Uttrakhand, and Uttar Pradesh within an altitudinal range of 1000 to 2500 m amsl [3]. It has pinnate compound leaves with multicellular aromatic glands in its lower surface [4]. It has traditionally been used in diverse health problems such as colds, stomach ailments, and breathing problems, and it acts as a sedative, anti-septic, insecticidal, anti-parasitic, and antispasmodic agent [5]. Its essential oil (EO) is used in food and flavoring, perfumery, and pharmaceutical and agrochemical industries [6]. Its EO has (Z)-ocimene, dihydrotagetone, tagetones (E & Z), and ocimenones (E & Z) as the major components [7,8]. EO with a higher percentage of (Z)-ocimene (35–50%) has a high rate in the international market. During 2016, its EO had a world annual production of about 15 tonnes [9], and its EO market is expected to reach USD 11.8 million by 2025 with a CAGR of 7.0% from 2020 to 2025 (as per EO market report 2020).

Weeds act as major threats to crop growth and reduce yields considerably if not managed timely. Uncontrolled weeds can bring about a yield reduction up to 45–75 per cent [10]. Weeds fight for nutrients, light, and water and also raise labour costs and make it difficult to harvest. During the initial cycle of development, farmers in the western Himalayas reported a major weed problem as this crop is sown in the months of May–June and harvested during autumn (October–November). Its initial growth period coincides with the rainy season, and there is an infestation of weeds, which significantly reduce the plant growth; the presence of weeds also makes harvesting more difficult. In India, a major portion of farmers rely on labour for physical weeding as they are not aware of the use of herbicides and their residual effect on the succeeding crop [11]. Physical weeding is painstaking, wasteful, and time-consuming. Added to the acute labour shortage, this fact renders hand-weeding impossible, and herbicides can act as alternate means. Pre- and post-emergence herbicide testing that can offer a wide variety of weed control in T. minuta under the mid hills of western Himalayan conditions is needed.

There are no licensed herbicides in India for the cultivation of Tagetes [12]. In order to identify herbicides that offer broad-spectrum weed control in this essential aromatic plant, further study is needed. The mixture of management approaches, such as hand-weeding with herbicides, might increase the effectiveness of decreasing rates of weed control [13]. The economically optimum herbicide dosage is highly affected by the rate of weed infestation and the scale of weeds. Jabran et al. [14] and Kumar et al. [15] observed that total weed density and total weed dry weight were reduced with the use of pendimethalin and imazethapyr, compared with the control. Demer [16] discovered that optimal pendimethalin and imazethapyr doses for the management of T. erecta weeds were still considerably higher than the control doses. Santos et al. [17] reported that weed control efficiency of carfentrazone-ethyl was 90% better in comparison to other herbicides against broadleaf weeds. Indeed, in conjunction with some mechanical weed control, herbicides have proven to be an efficient way of managing weeds [13]. When added to the crop, herbicides experience transformation under environment conditions [18]. Herbicide persistence poses health threats and can impact non-target populations. Therefore, a detailed understanding of dissipation and movement under field conditions is needed for the use of herbicides.

To our knowledge, there is no information on the control of weeds and the utilization of herbicides with hand-weeding for weed suppression in T. minuta. Data on the rate of herbicide degradation are extremely significant because they allow the estimation of levels likely to persist in the soil and enable the risk associated with crop exposure to be evaluated. Using a mixture of physical regulation and herbicides, the effectiveness of treatments will be increased, thus decreasing the cost of weed control as well as the environmental impact. Therefore, this analysis aimed to assess the effects of selected herbicides on the yield and quality of T. minuta at prescribed and reduced rates alone and in conjunction with single-hand-weeding, as well as to understand the dissipation of these herbicides in soil.

2. Material and Methods

2.1. Site Description

This study was conducted at the CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT, Palampur, India; 32°11′ N, 76°56′ E; 1325 m amsl) during 2018 and 2019 with a mean temperature and a mean rainfall of 18 °C and 250 cm, respectively. Rainfall and maximum and minimum temperatures recorded during 2018 and 2019 are reported in Figure 1 [19]. The soil of the experimental field was silty clay with an acidic pH (4.56) and low organic carbon (0.70%).

2.2. Treatments and Field Procedures

During both the years, an experiment with a randomized complete block design (RBD) was carried out with 12 treatments and 3 replications. The treatments consist of recommended doses (on the basis of bioefficacy data obtained from the experiments conducted by SAUs and ICAR institutes) of herbicide pendimethalin (Stomp, EC 30%, BASF), imazethapyr (Pursuit, EC 10%, BASF), carfentrazone-ethyl (Affinity, EC 40%, FMC), reduced doses (50% of the recommended dose or on the basis of its effectiveness) of herbicides only, and in combination with one hand-weeding (HW). During the growing season, weeds in the weed-free plots were removed every week by hand-weeding. No weeding was done in the weedy check treatment.

Table 1. Details of weed management treatments.

S.No.	Weed Management Treatments	Abbreviation
1	Recommended dose of Pendimethalin 1.50 kg a.i./ha (pre-emergence)	p
2	Reduced (R) dose of pendimethalin 1.00 kg a.i./ha (pre-emergence)	PR
3	Reduced dose of pendimethalin 1.00 kg a.i./ha (pre-emergence) followed by (fb) 1 hand-weeding (HW) at 25 days after transplanting (DAT)	PR+HW
4	Recommended dose of imazethapyr 0.10 kg a.i./ha (pre-emergence)	I
5	Reduced dose of imazethapyr 0.05 kg a.i./ha (pre-emergence)	IR
6	Reduced dose of imazethapyr 0.05 kg a.i./ha (pre-emergence) fb 1 HW at 25 DAT	IR+HW
7	Recommended dose carfentrazone-ethyl 0.02 kg a.i./ha (post-emergence) at 30 DAT	C
8	Reduced dose of carfentrazone-ethyl 0.01 kg a.i./ha (post-emergence) at 30 DAT	CR
9	Reduced dose of carfentrazone-ethyl 0.01 kg a.i./ha (post-emergence) at 30 DAT fb 1 HW at 50 DAT	CR+HW
10	Hand-weeding (25 and 50 DAT)	HW
11	Weed-free during the whole season	Weed-free
12	Weedy infested during the whole season	Weedy-check

HW: hand-weeding; fb: followed by; DAT: days after transplanting.

displays the details of weed control treatments. The nursery was sown in the month of April and 60-day-old seedlings were transplanted in a plot with dimensions of 4.2 m × 3.6 m. Pre-sowing fertilization included 30 kg ha⁻¹ N (urea 46% N), 60 kg ha⁻¹ P₂O₅ (single superphosphate 16% P₂O₅), and 40 kg ha⁻¹ K₂O (muriate of potash 60% K₂O), while top dressing consisted of 60 kg ha⁻¹ N (urea) 1/2 each at stem elongation and flowering times. Each plot consisted of 56 plants with 60 cm × 45 cm spacing (with seven rows and eight plants per row). Pendimethalin and imazethapyr were applied within 24 h after transplanting as pre-emergence herbicides, while carfentrazone-ethyl was applied as a post-emergence herbicide at 30 days after transplanting (DAT). A knapsack sprayer (Matabi, Goizper Group, Spain) equipped with a flat fan nozzle was used for herbicide application, which adjusted to deliver 240 L ha⁻¹ at 210 kPa.

2.3. Data Collection

At the initiation of the reproductive stage of T. minuta (at 75 DAT), the weeds from 1 m⁻² area (using two 0.5 m × 0.5 m quadrates per plot) were harvested in order to obtain the weed composition, the weed population, and the weed dry biomass. Weeds were identified, and the weed density (the number of weeds per m⁻² area) was recorded. For recording the weed dry biomass, collected weeds were dried in an oven at 80 ºC for 48 h. From the formula suggested by Bangi et al. [20], the weed control efficiency (WCE) and the weed index (WI) were determined. In order to analyse the outcome of the weed control techniques on the yield, plants were manually harvested in the month of October, and their fresh biomass was recorded.

2.4. Herbicide Soil Residue

Soil from a depth of 0–15 cm was collected at harvest and sampled using hand auger. From each plot, six samples were randomly taken, mixed, and evaluated as one composite sample. Soil samples were thoroughly mixed and sub-samples extracted. Samples were shade dried for 24–48 h and sieved for examination, while residual soil was preserved for use at 4 °C.

2.4.1. Sample Preparation Procedure

In a 50 mL oak ridge centrifuge container, ten grams of dried and sieved soil was taken for further processing. Samples were prepared through the QuEChERS technique according to the procedure stated by Banerjee et al. [21], steps of which are shown in Figure 2. A syringe filter (0.22 μm) was used to pass the final supernatant and was inserted into the LC-MS/MS.

2.4.2. LC-MS/MS Analysis and Validation

LC-MS/MS analysis was done with the help of Shimadzu LC-MS/MS-8030 (UPLC model-Nexera, LC-30AD liquid chromatography, SIL-30AC auto-injector) from the Shimadzu Company, Kyoto, Japan with specifications and a methodology similar to that mentioned in Banerjee et al. [21].

• Column: Zorbax Eclipse Plus C-18 (Agilent Technologies, Santa Clara, CA, USA).
• Column dimensions: 3 mm i.d., 10 cm length, and 3.5 μm column thickness.
• Detector: triple quadrupole mass detector.
• Mobile phase: Solvent A (80:20, 5 mM ammonium format solution in water: methanol) and Solvent B (10:90, 5 mM ammonium format solution in water: methanol). Solvent A and B were mixed and maintained in a 1:1 ratio during the total run.
• Run time: 6 min.
• Flow rate: 0.2 mL min⁻¹.
• Injection volume: 1 μL.
• Heat block and desolvation line temperatures: 400 °C and 250 °C, respectively.
• Drying and nebulizing gas: nitrogen (flow rates: 15 L min⁻¹ and 3 L min⁻¹, respectively).
• CID gas: ultrapure argon.

All three analytes were scanned in positive and negative electrospray ionization (ESI) mode, using Solvent A (80:20, 5 mM ammonium formate solution in water: methanol) & Solvent B (10:90, 5mM ammonium formate solution in water: methanol) through stainless steel union for selection of the proper ionization mode and the precursor ions (

Table 2. LC-MS/MS parameters and regression equation of the herbicides.

S.No.	Herbicide	Molecular Ion	Retention Time (min)	CE1	Quantifier (Q1)	CE2	Quantifier (Q2)	CE3	Quantifier (Q3)	Regression Equation	Correlation Coefficient (r)
1	Pendimethalin	281.90 (M + H)+	4.992	−10	281.90 > 211.80	−32	281.90 > 42.95	−19	281.90 > 193.85	Y = 2.42428e + 006 × −22898.8	0.9998
2	Imazethapyr	289.70 (M + H)+	2.261	−33	289.70 > 68.95	−22	289.70 > 244.95	−28	289.70 > 176.95	Y = 414367 × − 33238.8	0.9963
3	Carfentrazone-ethyl	430.60 (M + NH4)+	3.074	−13	430.60 > 413.90	−29	430.60 > 347.85	−24	430.60 > 367.85	Y = 3.19132e + 006 × −109164	0.9996

). After identification of the precursor ion, multiple reaction monitoring (MRM) optimization was done for selection of the most sensitive and stable transition with the best product ions for identification and quantification. In each event, the collision energy (CE), the Q1 pre-bias and the Q3 pre-bias, the dwell time, and the pause time were optimized according to the sensitivity of its most stable fragment. Quantification was done on the basis of the calibration curve obtained from LabSolution Browser software in LC-MS/MS. The most intense MRM ion transition was used for quantification (Qantifier), and successive intense transitions were used as the qualifier for identification confirmation (Table 2).

Method validation was carried out by a single laboratory validation method using the following criteria:

Specificity: To achieve the specificities of the pendimethalin, the imazethapyr, and the carfentrazone-ethyl to be extracted from the soil sample, the detector should be able (reinforced by extraction selectivity, clean-up, or separation) to deliver signals that detect the desired peak in a matrix that is unique. In order to ensure the specificity of identification, reagent blank and blank control samples were compared with the fortified sample. Table 2 indicates that the analytes were identified among the matrix without any significant interfering peaks.

Linearity: the linearity of a systematic method is its capacity (inside a given range) to obtain test results, which are proportional to the analyte concentration (i.e., quantity). In order to determine the linear response zone of herbicides, different concentrations of mixed standard solutions ranging from 0.01 to 1 µg mL⁻¹ were injected in LC-MSMS using the optimized method. The response of the detector was recoded against increasing concentration and plotted for the concentration–response curve. The linear response zone was defined from that curve, and the linear regression equation and the coefficient of correlation (Table 2) were calculated by Lab Solution Browser software in LC-MS/MS.

Sensitivity (MDL): the method’s sensitivity was measured in terms of the method detection limit (MDL) for pendimethalin, imazethapyr, and carfentrazone-ethyl in the soil matrix. The MDL is the concentration at which the signal to noise ratio (S/N) for the quantifier ion of each herbicide was ≥3. The MDLs of Pendimethalin, Imazethapyr, and Carfentrazone-ethyl were 0.01, 0.5, and 0.01, respectively (Table 2).

Accuracy (recovery study in soil matrix): recovery tests were conducted to determine the reliability of the analytical method and to evaluate the efficiency of extraction and clean-up measures for this analysis by fortifying the pesticide-free blank soil homogenate with a mixed analytical standard of pendimethalin, imazethapyr, and carfentrazone-ethyl at a fortification level of 1 µg g⁻¹. All samples were analysed in triplicate, and recovery was calculated using the following formula. All the herbicides were recovered from the soil matrix using the method mentioned in Figure 2 and were within the acceptance range of 70–120%.

$$\% Recovery=\frac{Peaked adea of the spiked sample}{Peaked area of the solvent sample}\times 100$$

Precision (repeatability): precision of the method was calculated in terms of the % relative standard deviation (% RSD) within the replicated experiments, and in all the recoveries it was found to be within the acceptable range of ≤20%.

2.5. Statistical Method

The analysis of variance (ANOVA) was done in a randomized complete block design. Weed data was subjected to square root transformation (√(x + 1)) to improve the homogeneity of variance. The significance of treatment differences was compared by the least significant difference (LSD) at the 5% level of significance, and statistical interpretation of the treatments was done. PAST 3 (Paleontological Statistics Software Package for Education and Data Analysis version 3) was used for principal component analysis [22].

3. Results

3.1. Weed Flora

Galinsoga parviflora Cav., Oxalis maritiana L., Bidens pilosa Linn., Commelina benghalensis L., Alternanthera sessilis (L.) DC., Amaranthus viridis Hook. F., Ipomoea purpurea L., Chenopodium album L., Cirsium arvense (L.) Scop., Cynodon dactylon (L.) Pers., Dactyloctenium aegyptium (L.) Willd., Cyperus compressus L., C. rotundus L., and C. haspan L. were the weed flora observed in the experimental field. Weeds such as G. parviflora, B. pilosa, C. bengalensis, C. dactylon, and C. rotundus were found in greater number as compared to other weed species.

3.2. Weed Density (Number of Weeds)

ANOVA presented in

Table 3. Analysis of variance for the effect of weed management treatments on weed parameters at 75 DAT and the yield at harvest of T. minuta.

Source of Variation	df	Weed Density	Weed Dry Biomass	WCE (%)	WI (%)	Biomass Yield (q ha−1)
Year (Y)	1	*	**	ns	ns	**
Weed management (M)	11	**	**	**	**	**
Y × M	11	ns	ns	ns	*	*
Error	46
CV	-	9.79	9.80	8.52	10.96	8.23

* and ** represent significant relationships at p ≤ 0.05 and p ≤ 0.01, respectively. WCE: Weed control efficiency; WI: weed index; ns: not significant; DAT: days after transplanting.

indicated a significant effect of the growing season and weed control treatment on weed population. The first year (2018) recorded a lower weed density (12.84 m⁻²), which was 6.27% lower than the year 2019 (13.70 m⁻²). Among herbicides, the IR+HW treatment observed the lowest weed density (9.64 m⁻²) with a 78.38% reduction in contrast with weedy-check (

Table 4. The effect of year and weed management treatments on total weed density, total dry weed biomass, weed control efficiency, and weed index.

Treatment	Weed Density (per m−2)	Weed Dry Biomass (g m−2)	Weed Control Efficiency (%)	Weed Index (%)
Year
2018	12.84 b	18.11 b	79.73 ns	27.24 ns
2019	13.70 a	19.41 a	78.49 ns	28.35 ns
Weed management *
p	14.91 ghi	21.08 i	79.01 hi	23.99 fg
PR	17.29 k	24.45 k	72.16 k	26.33 gh
PR+HW	15.40 ij	21.79 ij	78.21 hij	18.09 cde
I	9.64 c	13.64 c	91.32 bc	15.86 cd
IR	10.47 cd	14.80 cd	89.85 cd	23.12 f
IR+HW	7.14 b	10.10 b	95.31 b	8.02 b
C	11.21 cdef	15.85 cde	88.38 cdef	49.16 j
CR	12.76 efg	18.05 g	84.95 efg	51.36 jk
CR+HW	10.97 cde	15.52 def	88.98 cde	38.18 i
HW	13.91 gh	19.67 gh	81.94 gh	14.55 c
Weed-free	1.00 a	1.00 a	100.00 a	0.00 a
Weedy-Check	33.03 l	46.71 l	0.00 l	64.86 l

Data with transformed (√(x + 1)) values were used for statistical analysis. Means within each column with similar letters were not significantly different at the 5% probability level. ns: not significant; HW: hand-weeding; fb: followed by; DAT: days after transplanting. * The abbreviations of the weed management treatments were presented in Table 1.

). Recommended doses of herbicides recorded lower weed density as compared to reduced doses. However, comparing reduced dose of herbicides with that in combination with hand-weeding (R doses + HW) showed positive results. Treatments PR+HW, IR+HW, and CR+HW recorded 10.93, 31.80, and 14.02% lower weed density as compared to PR, IR, and CR, respectively. In the case of herbicides, reduced doses of imazethapyr and carfentrazone-ethyl in combination with hand-weeding (i.e., IR+HW & CR+HW) recorded a 25.93 and a 2.14% decrease in weed number than the recommended doses of I and C, respectively.

3.3. Weed Dry Biomass

Analysis of variance (ANOVA) showed that the weed dry biomass was affected significantly (p = 0.05) by the growing seasons and the weed control treatment (Table 3). In 2018, the weed dry biomass was reduced by 6.69% compared with 2019. Among herbicides, treatment IR+HW observed the lowest weed dry biomass density (10.10 g m⁻²) with a 78.37% reduction in contrast with weedy-check, and treatment I was the next with a 70.79% reduction in weed dry biomass (Table 4). Recommended doses of herbicides recorded lower weed dry biomass as compared to reduced doses; however, comparing reduced dose of herbicides with that in combination with hand-weeding (R doses + HW) showed positive results. In treatments PR+HW, IR+HW, and CR+HW weed dry biomass significantly reduced (10.87, 31.75, and 14.01%) compared to PR, IR, and CR, respectively. A reduced dose of imazethapyr in combination with hand-weeding (i.e., IR+HW) recorded a 25.95% decrease in weed dry matter than the recommended dose of I. HW decreased the weed dry matter by 57.88% than weedy-check.

3.4. Weed Control Efficiency (WCE) and Weed Index (WI)

ANOVA showed a significant effect of different treatments on WCE and WI; however, the year did not show any significant effect on both the parameters (Table 3). Amongst herbicides, a higher weed control efficiency was observed in the IR+HW treatment (95.31%) compared with the control and remained statistically at par with I treatment (Table 4). The lowest weed index (8.02%) was also recorded in the IR+HW compared with weedy-check. The weed index in the IR+HW treatment was 87.63% lower as compared to weedy-check.

3.5. Crop Yield

Weed control treatments had a significant effect on T. minuta biomass yield; however, the year had no significant effect (Table 3). A significantly higher biomass yield was observed in all treatments than in weedy-check (Figure 3). In both years (2018 and 2019), the highest biomass yield was observed in the IR+HW treatment (147.65 and 162.04% higher than that of weedy-check, respectively). Treatments PR+HW, IR+HW, and CR+HW recorded statistically higher biomass yields than those in PR, IR, and CR, respectively, in both the years. Moreover, the biomass yields in recommended doses of all the herbicides decreased significantly in comparison to reduced doses in combination with hand-weeding in both 2018 and 2019. The HW treatment increased the biomass yield by 133.52 and 132.04% compared with weedy-check in 2018 and 2019, respectively (Figure 3).

3.6. Crop–Weed Interaction

The biomass yield and the weed data pattern revealed that with a decrease in the weed population and the weed dry biomass, the biomass yield increased. The herbicide treatment with the maximum WCE and the lowest WI recorded the maximum biomass yield. These patterns revealed that treatment I R+HW was the best as compared to other treatments. I R+HW recorded the lowest weed number, weed dry matter, and WI and the highest WCE and biomass yield. The interaction between crop and weed data can be more clearly explained with the help of correlation and principal component analysis.

3.6.1. Correlation Analysis

The correlation analysis among weed and yield parameters was also performed, and the data revealed that weed density (m⁻¹) was significantly and positively correlated with weed dry biomass (r = 0.999; p=0.01) and the WI (r = 0.701; p = 0.01) (Figure 4). However, a significant and negative correlation was observed with WCE (r = −0.948; p = 0.01) and biomass yield (r = −0.700; p = 0.01). It implies that the biomass yield of T. minuta decreased with a proportional increase in weed interference and vice-versa. A highly significant and a positive correlation (r = 0.702) was found between weed dry biomass (g m⁻¹) and WI at the 1% significance level. However, a significant and negative correlation was observed with WCE (r = −0.945; p = 0.01) and biomass yield (r = −0.701; p = 0.01). The positive correlation (r = 0.650; p = 0.01) was also found between the WCE (%) and the biomass yield. A significant (p = 0.01) and negative correlation (r = −1.000) was found between the WI (%) and the biomass yield (q ha⁻¹).

3.6.2. Principal Component Analysis (PCA)

In order to analyse variation among treatments, weed parameters (weed density, weed dry weight, WCE, and WI) and the biomass yield from twelve treatments were submitted to PCA. PC−1 and PC-2 jointly explained 98.63% of the total variance (Figure 5). PC 1 (84.09%) accounted for the positive involvement of the weed density, the weed dry weight, and the WI and the negative involvement of the WCE and the biomass yield. PC 2 (14.54%) differentiated the weed density, the weed dry weight, and the biomass yield with positive involvement, while the WCE and the WI were differentiated with negative involvement. Four distinct clusters were observed in the score plot. Cluster I (weed-free, I, IR, IR+HW) explained the lowest weed density and weed biomass and the highest biomass yield. In Cluster II (p, PR, PR+HW, HW), the weed population and the weed dry matter was moderately lower, and the biomass yield was moderately higher as compared to the control. Cluster III (C, CR, CR+HW) showed a higher WCE but a lower biomass yield. In Cluster IV (weedy-check), the highest weed population and weed dry matter, and the lowest biomass yield, were observed.

3.7. Herbicide Residue

Multiple reaction monitoring (MRM) optimizations were carried out in the present study for quantification and identification for each herbicide. A mixture of three herbicides was injected, and the calibration curves for each herbicide were obtained. Table 2 lists the regression equation and the value of the correlation coefficient (r) for various herbicides. An LC-MS/MS representative with a standard curve and soil sample of three herbicides is shown in Figure 6. The analysis of soil samples indicated that the residues of pendimethalin at 1.00 and 1.50 kg a.i. ha⁻¹ were found to be below the limit of quantification in soil samples collected at the harvest stage (

Table 5. Herbicide residues in soil.

S.No.	Herbicides	Dose	Dose Type	Residue at Harvest
1	Pendimethalin	1.50 kg a.i./ha	Recommended	BDL
		1.00 kg a.i./ha	Reduced	BDL
2	Imazethapyr	0.10 kg a.i./ha	Recommended	BDL
		0.05 kg a.i./ha	Reduced	BDL
3	Carfentrazone-ethyl	0.02 kg a.i./ha	Recommended	BDL
		0.01 kg a.i./ha	Reduced	BDL

BDL = below detectable limit (<0.001 μg g⁻¹).

). Similar results were observed at both the dosages in the case of imazethapyr (0.05 and 0.10 kg a.i. ha⁻¹) and the carfentrazone-ethyl (0.01 and 0.02 kg a.i. ha⁻¹). The analysis suggested that pendimethalin, imazethapyr, and carfentrazone-ethyl applied at recommended doses in the present experiment are safe for use.

4. Discussion

4.1. Weed Density and Biomass

The weed population decreased relative to the weed-infested plot during all weed-control treatments. In comparison, hand-weeding greatly decreased the weed density and weed biomass relative to herbicide alone at all lower doses of herbicide application treatments. Hand-weeding of soybeans (Glycine max (L.) Meril) and maize (Zea mays L.) reduced the emergence of herbicides compared to the control [23,24]. Weeds uprooted by hand-pulling or with a hoe died from desiccation due to cell collapse from lack of water. Using hand-weeding in combination with herbicide mixtures in the maize field decreased the weed number and the weed dry weight in comparison to that of herbicide only [24]. In the management of weeds and the development of higher yields in different crops, the combination of herbicides with one supplementary weeding has been found to be very successful [25,26].

Among herbicides, a decrease in weed dry matter was higher in imazethapyr treatments than in pendimethalin and carfentrazon-ethyl treatments. This may be because imazethapyr is effective against most of the grasses and narrow and broad leaf weeds, by inhibiting root and shoot growth of susceptible weed species through disruption of protein and DNA synthesis, in comparison to other herbicides, which are effective either on grasses or broad-leaf weeds [27]. Our results are in line with Kumar et al. [15] in fenugreek (Trigonella foenum-graecum L.) with 69% weed control efficiency over other herbicidal treatments. Our results showed that the prescribed and decreased rate (R) of imazethapyr and carfentrazone-ethyl, using hand-weeding, increased the reduction percentage of weed density and biomass. Imoloame [13] also stated that the application of herbicides at reduced rates integrated with hand-weeding resulted in reduced weed biomass and density. There are several ways to control the weeds including hand or manual weeding and cultural means like hoeing, mulching, and intercropping, which are widely practiced in many aromatic crops. However, studies are still lacking in wild marigold [28]. Organic mulch and herbicides were tested in some Cymbopogon species, and organic mulch was found more effective and economic than herbicides [29].

4.2. Crop Yield

Our results indicated that a combination of all herbicides with hand-weeding improved the biomass yield, and the maximum yield was recorded in IR+HW treatment. The increase in biomass yield in hand-weeding treatments could be due to the decrease in weed population and weed dry matter, which leads to a higher availability of nutrients due to less weed competition (Table 4, Figure 3). Imoloame [24] reported that, in maize, hand-weeding treatments increased the growth and the biomass yield better than the control. The biomass yield at all imazethapyr treatments was higher than pendimethalin and carfentrazone-ethyl. Imazethapyr provided a 78% weed control with a higher yield than Sericea lespedeza [30]. Kumar et al. [15] in fenugreek and Ram and Singh [31] in soybean also recorded maximum yields with the use of imazethapyr as compared to other herbicides. A higher biomass yield was obtained in reduced dosages of herbicide in combination with hand-weeding as compared to recommended doses; this will help in a safe approach to weed control with a view to reducing the use of herbicides in the cropping method.

4.3. Herbicide Residue

The preservation or dissipation of an herbicide is largely influenced by crop management activities in the field and by environmental factors, including temperature, soil physics, and soil microbial activity. The analysis of soil samples reported that herbicides’ residues (pendimethalin, imazethapyr, and carfentrazone-ethyl) at both the dosages were below the detectable limit (<0.001 μg g⁻¹) in soil samples collected at the harvest stage (Table 5). The tagetes field received approximately 2825 and 1661 mm of rainfall during the experimental period in 2018 and 2019, respectively, which was higher than the average rainfall in this region. This might have increased the leaching because of greater herbicide solubility in water, resulting in a decrease in herbicides and allowing no residues to be contained in the soil when harvested [18,32]. Marsh and Lloyd [33] recorded that imazethapyr persisted in Romanian soil for a longer time and displayed residual effects even after two to three years on succeeding crops. High microbial activity increases the rate of deterioration, which may also induce a low concentration of herbicides in the soil [18,34].

5. Conclusions

Our results indicated that imazethapyr had higher efficacy in controlling weeds of T. minuta as compared with pendimethalin and carfentrazone-ethyl, with a higher weed control efficiency and biomass yield. The use of hand-weeding with herbicides at different application rates lowered the weed population and weed dry matter and improved the crop yield. The decreased rate of imazethapyr incorporation with hand-weeding (IR+HW) showed the highest decrease in weed density and weed dry biomass with increased T. minuta yield and decreased use of herbicides in the cropping system as well. Therefore, imazethapyr at a prescribed rate could be changed with imazethapyr at a reduced rate in combination with hand-weeding (IR+HW) and recommended to aromatic plant growers as a more eco-friendly solution for the environmental management of weeds and for the healthier development of T. minuta. Herbicide residues of pendimethalin, imazethapyr, and carfentrazone-ethyl at both the dosages were below the detectable limit (<0.001 μg g⁻¹) in soil samples collected at the harvest stage, indicating that recommended doses in the present experiment are safe for use. There is also a need to research the feasibility of the application of organic mulches instead of hand-weeding with reduced doses of herbicides for weed control in T. minuta in the future, which will decrease the use of herbicides and increases the sustainable production in the cropping system. Medicinal and aromatic plants perform better with organic manures, biofertilizers, and mycorrhizal association. Weeds could be managed effectively with mulches. Crop rotations and intercropping also add value per unit area and are helpful for the control of disease, pests, and weeds. Organic manures and mulches not only improve the yield and control weeds but also provide organic matter and nutrients to the soil, ultimately improving the soil’s health. The future global market is brighter for organically grown products with premium prices than those grown with conventional farming. Therefore, it is essential to increase the pace to move from chemical or conventional farming to organic farming in the medicinal and aromatic plant sector.