Smart delivery of herbicide for safe and effective control of Egyptian broomrape parasitizing Indian mustard

Abstract

Oilseed Brassica, an important edible oilseed crop of the world, is facing severe threat from the parasitic weed ‘Egyptian broomrape’ [Phelipanche aegyptiaca (Pers.) Pomel] with yield penalty of 0-100%. The problem is more severe due to its physico-biochemical connection with the hosts, where, general weed control measures are not effective to control this parasitic weed. Since, there is no absolute measure to control Egyptian broomrape. The present study was aimed to identify and develop the most effective herbicide formulation and deliver the herbicide to the targeted underground parasite weed for effective and safe control in Indian mustard. Simple (single) and complex (combinational) smart herbicide formulations (NE, ME, SE, SC & ZC) were developed and evaluated under hot-spot conditions. Among all the smart formulations, metsulfuron methyl 5% SC @ 4 ml a.i./ha applied at pre-emergence stage was found most effective in controlling Egyptian broomrape with no phytotoxicity, and recorded higher seed yield of Indian mustard (48–74%) over weedy check plots. Residue analysis using LC-MS/MS revealed that metsulfuron methyl was not detected in soil as well as plant samples thus, it was environmentally safe also. This research successfully demonstrated that metsulfuron methyl-based smart formulations (5% SC) can safely and effectively control Egyptian broomrape in Indian mustard.

Introduction

The Broomrape family (Orobanchaceae) is a group of about 150 species and 17 genera which are mostly of old world natives spread over the Mediterranean, Central and Eastern Europe, Africa, the Middle East and Asia¹. Egyptian broomrape [Phelipanche aegyptiaca (Pers.) Pomel, formerly known as Orobanche aegyptiaca Pers.], is a serious obligate parasite weed of oilseed brassica. Depending upon its infestation severity, ecological conditions and crop competitiveness, the damage caused by broomrape ranges from 0 to 100%². Under severe infestations, 200–300 plants of broomrape per square meter were observed under sandy and loamy sand soils in Indian mustard (personal communication). It acts as ‘Super Sink’, drain water and nutrient from the host and cause severe damage to the host crop, and become a peril to the farmers’ in mustard growing regions of India. It Further, the prolific seed production ability of high longevity leads them to gregarious survival for a long duration. Broomrapes have strong adaptation mechanisms to co-exist with their hosts in the same environment, resulting in strong parasitism that is difficult to control. Besides the difficulty of selectively controlling broomrape in the form of host-attached parasite, eradication of broomrape seed bank is extremely difficult due to prolific production of parasitic seeds, their easy dispersal, physiological dormancy, seed longevity, and germination synchronized with specialized range of host cultivation. Promising new control strategies have been investigated though the majority of them are under development or remain as prototypes to which farmers have not access. Although some examples of successful control do exist for some crops, the majority of commercially available control methods are either not fully effective or not applicable to many of the affected crops, especially in the case of low-input crops³.

Weed control strategy is mainly based on the concept of ‘many little hammers’⁴ or ‘use of technological advancement’⁵ for the selective and effective control of weeds in field crops. Parasitic weed using herbicides has provided effective and cost-efficient control, however, their selectivity and bio-efficacy varied with the crops and environment. Since, the E. broomrape germinates and attaches to the host root underground hence, it is difficult to identify and reach the parasite without using the host plant as a bridge to transfer pesticides to the parasite^6–8. Thus, the researchers have suggested the use of translocated herbicides to transfer the pesticides to the pest but, selectivity to the host plant remained the main difficulty. Few investigators have reported effective and selective control of E. broomrape by herbicides and even fewer have suggested application of soil residual herbicides^6,8,9. Soil fumigants (dazomet, metham, methyl bromide) were used to control parasitic weeds but, recently most of the soil fumigants were banned by WHO. Rimsulfuron selectively controlled E. broomrape in pots, but in field applications under drip-irrigated tomato controlled poorly¹⁰. Directed spray of glyphosate controlled E. broomrape in Indian mustard but, showed phytotoxicity on host plants¹¹. Soil application of 200 kg/ha neem cake and 2 kg/ha copper sulphate at sowing showed 34% and 41% reduction in E. broomrape density in mustard in Rajasthan¹². Drenching of plant holes with copper sulphate (5%) can reduce broomrape infestation by 37% and increase dry leaf yield by 28% in tobacco¹³. Split application with various imidazolinone herbicides on potato, sunflower and parsley foliage selectively controlled Phelipanche ramosa, Orobanche cumana, and Orobanche crenata, respectively¹⁴. Linuron at 0.5 kg/ha at 30 DAS caused complete control of E. broomrape, but simultaneously posed severe phytotoxicity to Indian mustard¹⁵. Soil applied sulfonylurea and imidazolinone herbicides were reported to control Orobanche, but in most cases, low crop selectivity limited their application^8,16,17. Indian mustard is a dominant and versatile oilseed crop of India, recurrently suffers severe yield losses due to E. broomrape infestation. However, there are no absolute control measures to control this perilous weed in rapeseed mustard not in India but, elsewhere in the world.

The present study emphasized mainly two aspects, first is to find out the most effective and selective herbicide either alone or in combination against E. broomrape, and second is to develop the effective and safe herbicide formulation for its commercial application to control E. broomrape in oilseed brassica. It was also emphasized to develop the formulations only for pre-emergence application, as post emergence application is not feasible in Indian mustard due to its tall plant height. The simple (single herbicide) and complex (combinational herbicides) herbicide formulations were developed at the laboratory scale, and evaluated in-situ in hot-spot areas of E. broomrape parasitizing Indian mustard. The herbicidal properties of these formulations were compared for their weed control efficiency, weed index and seed yield of Indian mustard under progressive field trails in sandy loam soils, and semi arid climate of Rajasthan province in India. Further, these selective and effective formulations can be explored commercially for parasitic weed management in Indian mustard as well as economically important field crops.

Results

Screening of innovative herbicide formulations

Smart formulations of oxyflorfen, pendimethalin and metsulfuron were developed and applied at pre-emergence stage to screen out the most effective herbicide formulation to control Orobanche in Indian mustard under hot-spot condition (Table 2). Though, all the herbicides recorded less Orobanche population (45–58 plants/m²) compared to weedy check plot (137 plants/m²) but, lowest (nil) was recorded with the metsulfuron methyl 20% SC applied at 4 ml a.i./ha. However, the seed yield of Indian mustard was recorded highest with the pendimethalin 5% NE applied at 375 ml a.i./ha. Metsulfuron methyl 20% SC recorded highest WCE (100%), whereas, other herbicide formulation recorded WCE (57–67%). Though, metsulfuron methyl 20% SC recorded highest WCE, but weed index i.e. loss in yield due to herbicide, was 14% which was undesirable. Whereas, pendimethalin 5% NE @ 375 ml a.i./ha recorded lowest WI but, the WCE was only 60%. Thus, among the different herbicide formulations in the screening trial, metsulfuron methyl 20% SC @ 4 ml/ a.i./ha was found effective to control Orobanche (100%), however, showed some phytotoxicity on the host crop and recorded less seed yield and high weed index.

Table 2.

Effect of smart herbicide formulations on Orobanche control and seed yield of Indian mustard (2020-21).

Treatments	Rate of application (a.i./ha)	Orobanche plants/m2	Seed yield (kg/ha)	WCE (%)	WI (%)
Oxyflorfen 5% ME	75 ml	53	1757	61	13
	150 ml	55	1616	59	20
Pendimethalin 5% ME	500	48	1867	65	8
	1000 ml	45	1710	67	15
Pendimethalin 5% NE	375 ml	54	2024	60	-1
	750 ml	58	1725	57	14
Metsulfuron methyl 20% SC	4 ml	0	1743	100	14
Weedy check	–	137	1249	0	38
Weed free	–	0	2020	100	0
LSD (0.05)	–	14	212	10	10

WCE: Weed control efficiency, WI: Weed index. ME: Micro encapsulation, NE: Nano emulsion, SC: Suspension concentrate

Based on the findings of the previous year trial (2020-21), second screening trial (2021-22) was conducted at two famers’ field under hot-spot conditions along with metsulfuron methyl 20% SC, and a new complex SE formulation ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’ (Table 3). These herbicide formulations were applied at different rates as pre-emergence spray to minimize the phytotoxicity in the host crop. Results showed that Orobanche population was recorded significantly lower with the SE formulation ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’ @ 3 ml a.i./ha at farmer 1 (72 plants/m²) and farmer 2 (38 plants/m²) compared to weedy check plots (232 and 120 plants/m²), respectively. However, seed yield of Indian mustard was recorded higher with the metsulfuron methyl 20% SC @ 2/3 a.i./ha at both the farmers’ field. SE formulation ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’ applied at 2/3 ml a.i./ha found the next best treatment in terms of seed yield. Weed control efficiency was recorded highest with the SE formulation ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’ @ 3 ml a.i./ha at both the farmers’ field (68%) followed by metsulfuron methy 20% SC @ 3 ml a.i./ha. However, weed index was recorded higher with the SE formulation ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’ @ 3 ml a.i./ha compared to metsulfuron methyl 20% SC @ 2/3 ml a.i./ha at both the farmers’ field.

Table 3.

Effect of smart herbicide formulations on Orobanche control and seed yield of Indian mustard (2021-22).

Treatment	Rate of application (a.i,/ha)	Orobanche plants/m2		Seed yield (kg/ha)		WCE (%)		WI (%)
		Farmer 1	Farmer 2	Farmer 1	Farmer 2	Farmer 1	Farmer 2	Farmer 1	Farmer 2
Metsulfuron methyl 20% SC	1.0 ml	114	57	1222	1664	49	52	33	17
	2.0 ml	97	56	1473	1683	57	53	18	15
	3.0 ml	81	52	1443	1948	64	57	20	1
SE ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’	1.0 ml	125	69	1234	1349	47	44	31	32
	2.0 ml	115	52	1401	1738	50	55	22	12
	3.0 ml	72	38	1276	1856	68	68	29	6
Weedy check	–	232	120	1165	1250	0	0	36	37
Weed free	–	0	0	1793	1985	100	100	0	0
LSD (0.05)	–	54	11	251	295	22	8	14	16

WCE: Weed control efficiency, WI: Weed index. SC: Suspension concentrate, SE: Suspo emulsion

Thus, metsulfuron methyl 20% SC @ 3 ml a.i./ha was found inferior than SE formulation ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’ @ 3 ml a.i./ha in terms of Orobanche infestation (plants/m²) and WCE, however, recorded higher seed yield and lower weed index than SE ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’ formulation in both the trials.

Since, higher phytotoxicity was reported in the previous trails (2021-22), again efforts were made to modify the formulations to increase the efficiency and decrease the phytotoxicity in the host crop. Metsulfuron methyl concentration reduced from 20% to 5% SC, and SE formulation modified from ‘Metsulfuron methyl 10% SC+Neem oil 10% EW’ to ‘Metsulfuron methyl 1% SC+Neem oil 18% EW’. A new ZC (zumbo combination) of ‘Metsulfuron methyl 2% SC+Pendimethalin 36% CS’ formulation was developed since, pendimethalin is effective against the annual weeds and a recommended herbicide for Indian mustard (Table 4). These formulations were applied at the same rate (4 ml a.i./ha) as pre-emergence spray at five farmers’ field as replication in the same environment under hot-spot condition. The results were encouraging and showed that metsulfuron methyl 5% SC formulation recorded lowest Orobanche infestation (6 plants/m²) compared to other formulations and weedy check plots (64 plants/m²). Metsulfuron methyl 5% SC also recorded significantly higher WCE (91%) compared to other treatments. However, the highest seed yield and lowest weed index were recorded with the ZC ‘Metsulfuron methyl 2% SC+Pendimethalin 36% CS’ formulation.

Table 4.

Effect of smart herbicide formulations on Orobanche control and seed yield of Indian mustard (mean values of five trials, 2022-23).

Treatment	Orobanche plants/m2	Seed yield (kg/ha)	WCE (%)	WI (%)
Metsulfuron methyl 5% SC	6	2047	91	– 4
SE ‘Metsulfuron methyl 1% SC+Neem oil 18% EW’	12	2065	79	– 5
ZC ‘Metsulfuron methyl 2% SC+Pendimethalin 36% CS’	13	2189	79	– 12
Weedy check	64	1394	0	29
Weed free	0	1963	100	0
LSD (0.05)	9	68	6	4

Thus, metsulfuron methyl 5% SC @ 4 ml a.i./ha proved better in terms of controlling Orobanche (91% WCE) and no yield loss (-4% WI) compared to other formulations, and marginally higher seed yield over the weedy check.

Multi-location bioefficacy evaluation of smart herbicide formulations

Results of the previous screening trials (2020-21 to 2022-23) showed that metsulfuron 5% SC formulation was found quite effective in controlling Orobanche under host spot conditions. So, the present study (2023-24) was conducted to evaluate the bioefficacy of metsulfuron methyl 5% SC formulation and standardize its rate of application (3–5 ml a.i./ha) applied as pre-emergence spray under different soil and environmental conditions. The trials were conducted under two different semi-arid environments at Bharatpur and Jhunjhunu provinces of Rajasthan, hotspot locations for Orobanche in Indian mustard (Table 5).

Table 5.

Effect of smart herbicide formulations on Orobanche control and seed yield of Indian mustard (2023-24).

Treatment	Rate of application (a.i./ha)	No. of Orobanche/m2		Seed yield (kg/ha)		WCE (%)		WI (%)
		Bharatpur	Jhunjhunu	Bharatpur	Jhunjhunu	Bharatpur	Jhunjhunu	Bharatpur	Jhunjhunu
Metsulfuron methyl 5% SC	3 ml	30	0	2047	1108	53	100	-4	16
Metsulfuron methyl 5% SC	4 ml	18	0	2065	1196	72	100	-5	9
Metsulfuron methyl 5% SC	5 ml	12	0	1810	855	81	100	8	35
Weedy check	-	64	250	1394	686	0	0	29	47
Weed free	-	0	0	1963	1307	100	100	0	0
LSD (0.05)	-	8	30	102	188	7	0	5	12

Results showed that metsulfuron methyl 5% SC formulation performed better in terms of decreasing Orobanche infestation, increasing weeds control efficiency and resulting higher seed yield of Indian mustard across the different environments and application rates compared to weedy check plots. Increasing rates of application of metsulfuron methyl 5% SC formulation from 3 to 5 ml a.i./ha decreased Orobanche infestation (30 to 12 plants/m²), increased weed control efficiency (53 to 81%) and weed index (-4 to + 8%) at Bharatpur location. Also, recorded seed yield significantly higher at 3 & 4 ml a.i./ha but, lower at 5 ml a.i./ha compared to weedy check plots at Bharatpur. No Orobanche infestation was observed in the metsulfuron methyl 5% SC treated plots thus, achieved 100% WCE at Jhunjhunu location, whereas, severe infestation (250 plants/m²) was observed in the weedy check plots. Seed yield was decreased with increasing rate of application of metsulfuron methyl 5% SC formulation from 3 to 5 ml a.i./ha, and recorded highest at 3 ml a.i./ha. Though, the yield levels were lower compared to weed free plots. The lowest weed index (9%) was recorded with the metsulfuron methyl 5% @ SC 4 ml a.i./ha at Jhunjhunu.

Thus, metsulfuron methyl 5% SC formulation applied @ 4 ml a.i./ha significantly controlled Orobanche infestation, recorded higher seed yield and lowest weed index at both Bharatpur and Jhunjhunu locations compared to other application rates.

Soil residue study

The samples of soil and mustard plant were analyzed using LC-MS/MS to find residue of metsulfuron methyl in these matrixes. It can be seen from the chromatograms that no residue of metsulfuron methyl was found in plant and soil samples (0–15 and 15–30 cm soil depth). The retention time of peak in the chromatogram of standards (Figs. 1 and 2) was different from the soil samples (Fig. 3) and plant samples (Fig. 4), which infers that there was no residue of metsulfuron methyl.

Fig. 1.

Life cycle of Orobanche and its mechanism of parasitism in Indian mustard.

Fig. 2.

MS Chromatogram for metsulfuron methyl sandard samples.

Fig. 3.

MS Chromatogram for metsulfuron methyl soil samples at 0–15 and 15–30 cm depth.

Fig. 4.

MS Chromatogram for metsulfuron methyl mustard plant samples.

Discussion

Biological similarity between host and broomrape interactions is higher than in other plant pathosystems, that complicates the development of selective methods of broomrape control, without affecting host crop from which it is feeding^18–21. Since the parasite germinates and attaches to the host root underground, it is difficult to identify and reach the parasite without using the host plant as a bridge to transfer pesticides to the parasite^6–8. Researchers have suggested the use of translocated herbicides for that purpose, but selectivity to the host plant remains the main obstacle. Few investigators have reported effective and selective control of Orobanche by herbicides and even fewer have suggested application of soil residual herbicides^6,8,9. Recently, soil applied sulfonylurea and imidazolinone herbicides were reported to control Orobanche, but in most cases, low crop selectivity limited their application^8,16. For example, rimsulfuron, a sulfonylurea herbicide selective to tomatoes, selectively controlled O. aegyptiaca in pots, but in field applications, its efficacy was poor¹⁰.

In case of Indian mustard-Broomrape pathosystem, crop selectivity derived from decomposition, binding or limited transportation of the herbicide in the host plant is not effective since, no herbicide is selective enough to the host plant and showed phytotoxicity. Different herbicides at different concentrations viz., pendimethalin (PE) 1000 g/ha, linuron (PE) l000g/ha, trifluralin (PPI) 1000 g/ha, fluchloralin (PPI) 1000 g/ha, metribuzin (PE/PPI) 175–200 g/ha, sulfosulfuron (PE) 5–10 g/ha, oxyfluorfen (PE) 125–175 g/ha, thiazopyr (PE) 240 g/ha, isoproturon (PE/PPI) 500–1000 g/ha, chlorsulfuron (PE/PPI) 2–6 g/ha and triasulfuron (PE/PPI) 5–10 g/ha were tested in field trials at farmers’ fields in India revealed that these herbicides were found inconsistent in their efficacy against the broomrape in Indian mustard and sometimes showed phytotoxicity²². Similarly, seed treatment of mustard with triasulfuron, sulfosulfuron and chlorsulfuron have been found to delay the emergence and Orobanche attachment but the results were inconsistent and over-dosing of the herbicide seed treatment sometimes caused poor germination and suppression in crop growth²³. Among the proposed herbicides for the control of broomrape, acetolactate synthase (ALS) inhibitors sulfonylureas and imidazolinones have been found to be lethal at relatively low doses both on vegetable and herbaceous crops, but crops may exhibit different tolerance thresholds^24,25. Encouraging results have been obtained with sulfonylureas on tomato in several application trials although on a different species of broomrape (P. aegyptiaca, O. cernua)^25–27. Glyphosate dose range is very limited. Over dosing of glyphosate, resulted in 15–35% toxicity to mustard in terms of marginal leaf chlorosis, slow leaf growth and bending of apical stems and stunting with yield penalty²⁸.

In the present investigation, we concentrated to control broomrape at the germination or pre-attachment stage through targeted delivery of a soil active herbicide. A pilot-study conducted under hot-spot condition showed that metsulfuron methyl (20% WP) is the most effective herbicide against broomrape in Indian mustard²⁹, however, its selectivity to the host crop was a concern. The smart formulation, suspension concentrate (SC) of metsulfuron methyl, significantly controlled broomrape (> 90%) under multiple screening trials conducted under hot-spot conditions from 2020 to 21 to 2022-23 with Indian mustard. Since, metsulfuron methyl is a soil active herbicide, its mobility improved with the modified formulation (SC) and delivered at the site of broomrape, deep in soil. Once the metsulfuron delivered at target site under moist conditions, chemical released and inhibited broomrape seed germination by arresting cell division. Thus, physical selectivity was achieved with metsulfuron methyl SC formulation and controlled broomrape seed germination and attachment, and resulted higher seed yield of host crop. Other metsulfuron based formulations like; SE ‘Metsulfuron methyl 1% SC+Neem oil 18% EW’ and ZC ‘Metsulfuron methyl 2% SC+Pendimethalin 36% CS’ were also relatively found effective in controlling broomrape in Indian mustard. The concentration of metsulfuron methyl SC formulation is further decreased from 20 to 5% to minimize its phytotoxicity and tested under multiple trails.

Literature reveals that metsulfuron methyl known to control 60 species as a potential inhibitor of plant growth^30,31. It is a residual sulfonylurea pre and post-emergence herbicide that kills broadleaf weeds and some annual grasses. It is a systemic compound with foliar and soil activity that inhibits the ALS enzyme in susceptible plants³². Inhibition of ALS activity leads to the starvation of leucine, isoleucine and valine amino acids, and ultimately plant die³³. Its mode of action involves inhibiting cell division in shoots and roots³⁴. Since, it is a soil active herbicide, significantly affect the seed germination and tuber growth^35,36. Metsulfuron methyl is also a low residual herbicide and degrade rapidly (> 90%) within 15 days of application³⁷. Studies conducted in Israel revealed that sulfosulfuron, chlorsulfuron and triasulfuron are highly efficient and selective for O. aegyptiaca control in tomato³⁸. Sulfonylurea application affected tuber development causing anomalies but these also varied with products and rates. ‘Atlantic’ yield losses were greater than ‘Lady Rosetta’s yield losses when metsulfuron-methyl was applied³⁵.

Recommended application rate of metsulfuron methyl 20% WP (commercial formulation) is 8 g a.i./ha. The results revealed that metsulfuron methyl 5% SC applied at 4.0 ml a.i./ha was found to recorded higher seed yield and weed control efficiency at Jhunjhunu (100%) and Bharatpur (72% WCE) with minimum weed index at both the locations. Since, the smart SC formulation is having particle size of 3–5 μm which may amplified its mobility, reactivity, efficacy and efficiency at lower rate of application. Rimsulfuron at 100, 150 and 200 g a.i./ha, and sulfosulfuron at 50 and 100 g a.i./ha did not cause any damage to tomatoes and effectively controlled the broomrape³⁹. Pre-emergence sulfosulfuron at 75 g a.i./ha effective in controlling Orobanche in tomato grown under irrigated conditions in India⁴⁰. Three repeated doses of rimsulfuron at 12.5 g a.i./ha each followed by irrigation, sprayed on potato foliage two weeks after crop emergence and re-applied at two-week intervals effectively and selectively controlled O. aegyptiaca with no damage to potato yield or tuber quality⁴¹. Metsulfuron methyl affected F1 generations of Sinapis alba, Centaurea cyanus, and Phacelia tanacetifolia at field dosages of 0.0193 g a.i./ha and higher, showing significantly lower seed germination rates³⁶. The sulfonylurea herbicides are active against broomrapes mainly through the soil solution so they must be incorporated into the soil⁴². Herbicides can be delivered to the target area using mechanical incorporation or by rainfall before host sowing or planting. They can also be delivered into the soil through the irrigation water (herbigation) using either sprinklers or drippers. The success of this mode of herbicide application depends on the availability of herbicide in the soil layer where the host roots are parasitized⁴³.

Metsulfuron-methyl is a sulfonylurea in herbicide group B. It is used alone or as a mixture to control broad leaf weeds in cereal crops. Much of the cropping area of south-eastern Australia has alkaline soils. The sulfonylurea herbicides breakdown through both microbial action and chemical hydrolysis. Soil pH has an influence on the rate of breakdown by hydrolysis. The greater the soil pH the less hydrolysis occurs, therefore the breakdown in alkaline soil is mostly through microbial activity. Also with increasing pH, the herbicide is more soluble hence able to leach from the relatively microbe rich topsoil to deeper in the profile where microbial breakdown is less likely to occur.

Conclusion

Broomrape is a serious setback to Indian mustard production in semi-arid regions with inherently poor growing conditions. Metsulfuron methyl is a soil active herbicide effective on plant roots and tubers, proved an applicable solution to control broomrape in Indian mustard. Smart herbicide formulation with less particle size, and more mobility, reactivity and efficiency under pre-emergence application is an advanced technological breakthrough to control parasitic weeds. Metsulfuron methyl SC (5%) formulation significantly controlled Egyptian broomrape in Indian mustard with almost ≈ 100% weed control efficiency in the present study. Also, SC formulation is effective at the half of the application rate (4.0 ml a.i./ha) compared to commercially available WP formulation (8.0 g a.i./ha). Thus, development and use of smart herbicide formulation is a new niche area in weed management especially for parasitic weeds. Metsulfuron methyl based smart formulations (SC, SE and ZC) were developed first time and can be explored commercially for parasitic weed management in economically important field crops in future.

Materials and methods

Study site, climate and soil

The experiments were conducted at two locations; Bharatpur and Jhunjhunu. The site was located between 26.997–27.032° N latitude and 77.272–77.383° E longitude at 183 m mean sea level on the bank of the Utangan river at Bharatpur. The other site was located between 28.12 N latitude and 75.39 E longitude at 450 m mean sea level in Jhunjhunu. The climate is semi-arid (moisture index: -55 to -45) with hot summers and a short monsoon season (July–September). The soils of the location were sandy loam, poor in organic carbon (3.0 g/kg) and available N (120 kg/ha), and medium in 0.5 N NaHCO3-extractable P (18 kg/ha) and 1 N NH4OAc-exchangeable K (152 kg/ha).

Development of herbicide formulations

The herbicide formulations were developed from the technical material of the chemical herbicides as simple and complex formulation with neem oil as per procedure given below⁴⁴.

Emulsion in water (EW)

The EW formulation of neem oil was prepared through an oil-in-water emulsification process using high shear mixing at 3000 rpm. The active ingredient (a.i.) of neem oil was taken as the oil phase. The aqueous phase was prepared by mixing a propylene glycol emulsifier and distilled water in a separate vessel. Then emulsification was initiated by slowly adding the oil phase into the aqueous phase with continuous stirring followed by addition of xanthan gum (rehology modifier). This precise methodology ensured the creation of a well-emulsified and stable EW formulation ready for application.

Suspension concentrate (SC)

The SC formulation involves several precise steps in preparing homogeneous suspensions of high-melting solids. Initially, water-soluble emulsifiers and an antifoaming agent were homogenized under high shear mixing at a speed of 3000–4000 rpm for 15 min. Subsequently, the active ingredients, oil-soluble emulsifier, and an antifreezing agent were added to the solution. This mixture was then subjected to wet grinding for 20 min to micronize the particles in the range of 3–4 μm. Finally, the polymeric stabilizer was added for gravitational stability of the mixture while continuing the mixing process.

Suspo emulsion (SE)

The SE formulations are used to develop a single formulation of two active ingredients of very different physical properties. Here, the SE formulation was developed by combining the suspension concentrate (SC) formulation of metsulfuron methyl with a concentrated aqueous emulsion (EW) formulation of neem oil. After combining both the SC and EW formulations, xanthan gum was added at low shear mixing to ensure gravitational stability.

Nano emulsion (NE)

The NE formulation was developed of karanja oil (KO) + cashew nutshell liquid (CNSL), and of CNSL alone. The water phase containing a predetermined amount of emulsifier, co-surfactant and ethylene glycol was stirred under high shear mixing. The oil phase containing a mixture of karanja oil + CNSL was emulsified into the water phase at 1500 RPM. The solution was then stirred for 15 min to obtain a NE formulation of karanja oil + CNSL. The NE formulation of CNSL was prepared following a similar method, where, CNSL was used alone in the oil phase.

Details of agro-techniques

The experiment was conducted in the rabi season (October-March) in hot-spot areas of broomrape parasitizing Indian mustard in a completely randomized block design. Different formulations were randomized and applied as per treatment details in plots of 30 m² (5.0 m x 6.0 m) each. The field preparation was initiated with 2–3 harrowing followed by soil surface leveling. Indian mustard variety DRMRIJ-31 was used as a test variety and planted between 10-15th October at Bharatpur and on 5th November at Jhunjhunu with a seed rate of 4 kg/ha at 45 cm row to row and 15 cm plant to plant distance. The crop was nourished with the recommended dose of N, P₂O₅, K₂O, S, Zn and B at 80.0, 40.0, 40.0, 40.0, 5.0 and 1.0 kg/ha⁴⁴. The N was applied in two equal split doses, first at sowing and second at the time of first irrigation, 30 days after sowing (DAS). Except broomrape, other weeds were uprooted manually at 30 DAS. The broomrape was uprooted from the weed free plots ‘as and when’ it emerged out of soil surface. The crop was harvested when 75–80% silique turned yellow and matured. The year-wise details of the treatments applied are given as below.

Details of field trials

Looking the feasibility of herbicides application in Indian mustard, we have developed formulations for pre-emergence application only. Besides this, our major emphasis was to develop and evaluate the smart formulations effective against Orobanche with added advantage of controlling other annual weeds, if any.

1. Screening of herbicide and formulations

The screening experiments were conducted for three years during 2020-21 to 2022-23 to find out the most potent herbicides and their formulations effective against Orobanche under hot-spot conditions. In the first experiment (2020-21), oxyflorfen, pendimethalin and metsulfuron methyl were selected for evaluation. Since, oxyflorfen and pendimethalin are recommended herbicides in Indian mustard for pre-emergence application to control annual weeds. Based on our previous study and literature also, metsulfuron methyl 20% WP (wettable powder) was found effective against Orobanche. The commercial formulations of oxyflorfen, pendimethalin and metsulfuron were modified to ME (micro emulsion), NE (nano emulsion) and SC (suspension concentrate) formulations, respectively to make them more effective and efficient in controlling weeds including Orobanche. There were total seven smart formulations were developed and applied as pre-emergence spray (Table 1). The ME formulation of oxyflorfen and pendimethalin were applied at recommended rates (150 and 1000 ml a.i./ha) and half of the recommended rates (75 and 500 ml a.i./ha), respectively. The NE formulation of pendimethalin was applied at 375 and 750 ml a.i./ha. Whereas, SC formulations of metsulfuron was applied at the recommended rate (4.0 ml a.i./ha). These formulations were evaluated against weedy check and weed free plots.

Table 1.

Year of study, number of location, smart formulations and the rate of application of herbicides to control Orobanche in Indian mustard.

Year of study	Number of locations	Herbicide formulations	Rate of application (a.i./ha)	Time of application
2020-21	01	1. 1. Oxyflorfen 5% ME	75 ml 150 ml	Pre-emergence
		1. 2. Pendimethalin 5% ME	500 ml 1000 ml
		2. 3. Pendimethalin 5% NE	375 ml 750 ml
		4. Metsulfuron methyl 20% SC	4.00 ml
2021-22	02	1. Metsulfuron methyl 20% SC	1.0 ml 2.0 ml 3.0 ml
		2. SE ‘Metsulfuron 10% SC+neem oil 10% EW’	1.0 ml 2.0 ml 3.0 ml
2022-23	05	1. Metsulfuron methyl 5% SC	4.0 ml
		2. SE ‘metsulfyron methyl 1% SC+neem oil 18% EW’
		3. ZC ‘metsulfuron methyl 2% SC+pendimethalin 36% CS’
2023-24	02	1. Metsulfuron methyl 5% SC	1.0 ml 2.0 ml 3.0 ml

ME: Micro encapsulation, NE: Nano emulsion, SC: Suspension concentrate, SE: Suspo emulsion, EW: Emulsion in water, ZC: Zumbo combination, CS: Capsule suspension.

Taking leads from the previous experiment, the second experiment was conducted at two farmer’s field during 2021-22 to evaluate the metsulfuron methyl based formulations. A new complex formulation of SE (suspo emulsion) was developed by combining metsulfuron 10% SC with neem oil 10% EW formulations. These formulations were applied at different rates as pre-emergence spray and compared with the weedy check and weed free plots (Table 1).

Based on the results for previous experiments, third experiment was conducted during 2022-23 at five farmer’s field as replicates. The metsulfuron methyl 20% SC formulation was modified to metsulfuron methyl 5% SC, and SE formulation of ‘Metsulfuron 10% SC+neem oil 10% EW’ was modified to SE ‘metsulfyron methyl 1% SC+neem oil 18% EW’ formulation to reduce their phytotoxicity on the host crop. A new ZC (zumbo combination) formulations by combining metsulfuron methyl 2% SC with pendimethalin 36% CS were also developed and evaluated. A total three smart formulations (SC, SE and ZC) were applied at the rate of 4.0 ml a.i./ha as pre-emergence spray and compared against weedy check and weed free plots (Table 1).

• 2. Multi-location bioefficacy evaluation of smart herbicide formulations

Based on the results of screening trials (2020-21 to 2022-23), randomized multi-location trials were conducted during 2023-24 at Bharatpur and Jhunjhunu to standardize the rate of application under different environments. Metsulfuron methyl 5% SC formulation were applied at three rates (3.0, 4.0 and 5.0 ml a.i./ha) as pre-emergence spray and compared with weed free and weedy check plots (Table 1).

Observations and weed indices

Equal numbers of rows of Indian mustard were harvested manually from the net plot area (4.5 × 5.5 m) leaving border rows. The harvested produce was Sun-dried and threshed, and the seeds separated from the stover. The seed yield per hectare was calculated by multiplying the factor of net harvested area (24.75 m²) to the total area of one hectare (10000 m²). The number of broomrape plants were counted manually from the net plot area (4.5 × 5.5 m) just after the harvest of Indian mustard, and Sun-dried. The weed indices were calculated as per formulae given below.

Weed index (WI)

It refers to the reduction in crop yield due to the presence of weeds in comparison to weed-free crop.

This is used to assess the efficacy of herbicides. Lesser the weed index, better is the efficiency of herbicides and vice-versa.

Weed control efficiency (WCE)

It refers to the percentage reduction in weed dry matter by any weed control treatment in comparison to weedy check plot. This index is used to compare the different weed control treatments.

This is used to assess the efficiency of applied herbicide or other practices to control weeds. Higher the WCE, better is the efficiency of herbicides in controlling the weeds and vice versa.

Soil and plant residue analysis

Soil and plant samples (10 ± 1 g) were weighed into a 250 mL round-bottom flask, and 50 mL of methanol was added. The mixture was shaken on an orbital shaker for 1 h to ensure efficient extraction. The extract (20 mL) was then passed through anhydrous sodium sulfate into a clean round-bottom flask, and the remaining residue was rinsed with an additional 10 mL of methanol. The combined extract was concentrated to dryness using a rotary evaporator, reconstituted in 4 mL of methanol, vortexed thoroughly, and filtered through a syringe filter prior to instrumental analysis. Quantification was performed using a Shimadzu LC-MS/MS 8050 system (LabSolutions v5.99 SP2) equipped with a C18 column (2 μm, 2.1 × 150 mm). Chromatographic separation was achieved at 35 °C using a flow rate of 0.3 mL min⁻¹ under a gradient elution program with mobile phase A consisting of 5 mM ammonium formate with 0.1% formic acid in water, and mobile phase B consisting of 5 mM ammonium formate with 0.1% formic acid in methanol. The gradient was programmed as follows: 0–3 min, 95% A; 3.5 min, 80% A; 8.5 min, 30% A; 18–20 min, 5% A; and re-equilibration to initial conditions (95% A) by 21 min. Detection was carried out using electrospray ionization (ESI) in LC-MS/MS mode.

Statistical analysis

The data were recorded for different parameters, such as seed yield, WCE and WI, under different botanical simple and complex formulations, and statistically analyzed using the online platform OPSTAT⁴⁵ for randomized block design. The F-test was used to determine the least significant difference (LSD) at p ≤ 0.05 among the treatment effects. Tukey’s HSD Test at p ≤ 0.05 level of significance was also worked out to differentiate the treatment means.

Abbreviations

NE

Nano emulsion

ME

Micro emulsion

SE

Suspo emulsion

SC

Suspension concentrate

ZC

Zumbo combination

Author contributions

R.S.Jat, A. Agarwal, J. Kumar: Conceptualization, methodology, validation, investigation, data curation; A. Agarwal, S. Kala, Nusrat: Formulation development; R.S.Jat: Writing original draft; H.V.Singh, R.L.Choudhary: Writing review and editing; J. Kumar, V.V. Singh: Project administration.

Data availability

The data will be made available by R.S. Jat ( [rs.jat@icar.gov.in]([mailto: rs.jat@icar.gov.in]) ) upon request.

Declarations

Competing interests

The authors declare no competing interests.