Spinetoram, a Selective Novel Insecticide Able to Check Key Lepidopteran Pests in Cabbage Ecosystem

Pratap A. Divekar1,2*, Sampat K Patel2, Guru Pirasanna Pandi G3, Manimurugan C2,4, Vikas Singh2 and Jagdish Singh1 1Division of Crop Protection, ICAR-Indian Institute of Vegetable Research (IIVR), Varanasi-221305, India. 2ICAR-IIVR, Regional Research Station, Sargatia, Kushinagar-274406, India. 3Division of Crop Protection, ICAR-National Rice Research Institute, Cuttack-753006, India. 4Division of Crop Improvement, ICAR-Indian Institute of Oilseeds Research, Hyderabad-500030, India. Article Information Received 24 July 2022 Revised 07 August 2022 Accepted 26 August 2022 Available online 28 December 2022 (early access)


INTRODUCTION
C abbage (Brassica oleracea var. capitata) is one of the nutrient rich cole crops rich having vitamin C and essential minerals. Cabbage is used in salads, boiled vegetables, fried curries, pickles and vegetables which are dehydrated. Cabbage production in India is 8.76 million tonnes under 0.39 million ha area with productivity of 22.56 tonnes per ha. India's share in cabbage and other brassicas production is 12.3% and occupies second position in the world (Anonymous, 2018). The major yield-limiting factor for cabbage production is insect pest infestation, which causes both qualitative and quantitative loss. Diamondback moth (DBM) Plutella xylostella (Lepidoptera: Plutellidae) is the key pest of cabbage (Talekar and Shelton, 1993) which has high migratory nature and has attained cosmopolitan species status. The annual yield loss due to direct feeding injury and spoilage are estimated to the US $ 2.7 billion worldwide (Zalucki et al., 2012). Larva is the damaging stage in the case of DBM where first-instar larvae act as leaf miners. However, older larvae damage the lower leaf surface and usually devour all tissue except the adaxial wax layer, thus creating a window in the leaf (Samthoy et al., 1989) (Supplementary Fig.  1a). The cabbage butterfly (CB), Pieris brassicae (Linn.) (Lepidoptera: Pieridae), is a serious pest of cabbage and other cole crops across the globe (Feltwell, 1978). CB was identified as a major pest of Brassica oilseeds in eastern Uttar Pradesh and other parts of India (Lal and Ram, 2004). Generally, CB survives the winter in the plains and migrates to hilly regions as the temperature goes up during summer in India. Young caterpillars are gregarious feeders,

O n l i n e F i r s t A r t i c l e
which on the severe attack, devour the plant completely (Ali and Rizvi, 2007) (Supplementary Fig. 1b). DBM and CB were reported as the limiting factors in the cabbage production in India causing yield losses to the extent of 52 % (Krishnamoorthy, 2004) and 40 % (Ali and Rizvi, 2007), respectively. Though the pest management options like host plant resistance (HPR) (Divekar et al., 2019), plant secondary metabolites (Divekar et al., 2022) and bio-control agents (Dukare et al., 2020) are available to control the insect pests in an eco-friendly and sustainable manner, synthetic pesticides are the first choice of farmers. Farmers use insecticides inappropriately for managing insect pests, which involves applying pesticides more than the recommended dosage and applying calendarbased sprays. Across the various states of India, excessive use of pesticides was noticed in cabbage (Weinberger and Srinivasan, 2009). Various studies have reported the DBM resistance to commonly used insecticides like quinalphos, fenvalerate, and cypermethrin and cross resistance to insecticides with different modes of action like cartap hydrochloride, diafenthiuron (Joia and Chawla, 1995). Resistance to newer molecules like emamectin benzoate, indoxacarb, spinosad, flubendiamide and chlorantraniliprole has also been reported (Zhao et al., 2006;Troczka et al., 2017). Injudicious application of insecticides, several generations of insects and the availability of host plant throughout the year have created new challenges like development of insecticide resistance and multiple insect pest attacks in cabbage. To mitigate these challenges, there is an urgent need to find an effective and eco-friendly alternative insecticide that can singly or in combination with the other novel insecticides be used safely to manage lepidopteran insect pests in cabbage. The present investigation was aimed to study the potential of spinetoram and several other novel insecticides for their bioefficacy against lepidopteran insects, safety to natural enemies and phytotoxicity in cabbage ecosystem under field conditions.

Field efficacy of insecticides against DBM and CB
The field experiment was conducted by growing cabbage var. Golden Acre during the spring season of 2019 (season 1) and 2020 (season 2) at ICAR-IIVR, Regional Research Station, Sargatia, Kushinagar (Latitude NS 26˚ 43' 56.61 and Longitude EW 84˚11' 12.95). Experiments were performed in a randomised block design (plot size: 4m×3m) with twelve treatments including untreated control and the same set of experiment was replicated thrice. The crop was raised as per the recommended package of practices, except for the plant protection measures. The treatment details were given in Table I.
Cabbage seedlings of 35 days old were transplanted in both the seasons. Insecticide spray treatments were applied on 35, 50 and 65 days after transplanting (DAT) on crossing the ETL of lepidopteran pest population. Pretreatment count of the insect pests was noted one day before insecticide application. Larval numbers of DBM and CB were assessed by visual counting from 5 randomly selected plants on 1, 3, 7, 10 and 14 days after each spray from lower and upper surface of the entire plant. The percentage reduction of the pest population over control was calculated by using the formula given by Henderson and Tilton (1955).

Phytotoxicity of insecticides on cabbage
The phytotoxic effects produced by spinetoram 11.7% SC and other insecticides on cabbage leaves and head were also studied. Five plants were selected at random in each plot and the plants were examined for phytotoxic symptoms viz., leaf tip burning, epinasty, hyponasty, necrosis, chlorosis, vein clearing, wilting and resetting on 1, 3, 7, 10 and 14 days after insecticide application. The phytotoxicity symptoms, if any, were graded based on the percent-injured leaves on the scale of 0-10 as per CIBRC, India and percent leaf injury was calculated by using a formula as described by Karthik et al. (2015).

Safety of insecticides to natural enemies
Natural enemies viz., coccinellid complex and spiders were observed and recorded before and after each insecticide application. Immature and adult stages were estimated by visual counting of individuals on lower and upper surface of the cabbage plant. Observations on the number of immature and adults of natural enemies were recorded from five plants per replication on 1, 3, 7, 10 and 14 days after each insecticidal spray application.

Crop damage rating and marketable yield
When plants were ready to harvest, ten plants per plot were scored for larval damage to the head of cabbage using a standard 1-6 scale (Greene et al., 1969). Harvesting was done at 80 DAT. Marketable yield was assessed from ten cabbage heads per plot.

Statistical analysis
The data on the population of lepidopteran insect pests and natural enemies were subjected to square root transformations (Snedecor and Cochran, 1967). The efficacy of different treatments in terms of larval population of DBM and CB, immature and adult of natural enemies, crop damage ratings and marketable yield were studied using one-way analysis of variance (ANOVA) through SPSS 22.0 software. When the F test was significant at 0.05 level, the treatment means were compared using the Tukey's honest significant difference (HSD) test. Additionally, principal component analysis (PCA) was done through SAS University Edition software by using pooled data to find out the affinities between the plant marketable yield, crop damage rating, percent protection over control and insects as well as natural enemy populations (Gowda et al., 2019).

Efficacy of insecticides against DBM, P. xylostella
The lepidopteran insect pests encountered in cabbage throughout the study period in open field conditions were DBM, P. xylostella and CB, P. brassicae. The incidence of DBM before and after three rounds of insecticide treatment during two seasons were summarized in Tables II and III.

O n l i n e F i r s t A r t i c l e
Spinetoram, a Selective Novel Insecticide 5 Infestation of DBM was higher in the second season than the first. Pre-treatment count for DBM larval population was non-significant in both the seasons and was recorded in the range of 5.87 to 6.53 (F (11, 24) = 1.24, p= 0.32) and 8. 73-9.47 ((F (11, 24) =1.64, p=0.15) during season 1 and season 2, respectively. Significant differences were observed for the larval DBM population among the treatments and it was ranged in between 1.73-10.83 (F (11, 24) = 33.24, p˂0.001) and 2. 73-14.40 (F (11, 24) = 48.62, p˂0.001) for the season 1 and 2, respectively. Application of spinetoram @ 60 g a.i./ha, resulted into the highest reduction of DBM in season 1 and 2. Spinetoram and spinosad caused the higher reduction of DBM larval population after three sprays in both season 1 and 2 than the untreated control. Chlorpyriphos produced the least reduction due to spray I (46.33%) and spray II (58.81%). Similarly, chlorpyriphos caused the least reduction after spray I (44.65%) and spray II (58.20%) during season 2. After spray III, least reduction of larval DBM population was recorded due to the application of deltamethrin in season 1 (62.31%) and season 2 (65.20%). DBM can be managed effectively by the application of chemical insecticides at proper time and against appropriate stage of insect.

Efficacy of insecticides against CB, P. brassicae
Results of insecticide evaluation against CB were presented in Tables IV and V. CB, P. brassicae infestation was higher in the first season as compared to the second. CB larval population was non-significant in both the seasons during pre-treatment count and it was observed in the range of 8.00-8.80 (F (11, 24) =1.85, p=0.10) and 5.00-6.07 ((F (11, 24) =1.91, p=0.09) during season 1 and season 2, respectively. Significant differences were noted among the treatments for larval population counts of CB and it was ranged in between 1.96-14.62 (F (11, 24) = 33.24, p˂0.001) and 1.51-9.06 (F (11, 24) = 24.37, p˂0.001) during season 1 and 2, respectively. Highest reduction of CB was observed after application of spinetoram @ 60 g a.i./ha after spray I (78.09%), spray II (86.55%) and Spray III (93.69%) in season 1. Similarly, in season 2 spinetoram 60 g a.i. /ha was found responsible for highest reduction after spray I (72.29%), spray II (84.03%) and Spray III (90.98%). Spinetoram and spinosad were found responsible for higher reduction of CB larval population after three sprays in both the seasons. Chlorpyriphos caused the least reduction due to spray I (46.28%) and spray II (61.33%) and spray III (68.56%) during season 1. Similarly, in season 2 chlorpyriphos resulted into the least reduction after spray I (40.62%) and spray II (55.21%) spray III (64.23%). All the insecticide treatments were found effective against the larvae of CB, P. brassicae over untreated control.   Crop damage and marketable yield assessment Crop damage done by larval feeding of DBM as well as CB and marketable yield obtained after the application of chemical insecticides are shown in Table VI. Insecticide treatments were significantly different with respect to crop damage rating during season 1 (F (11, 24) = 21.85, p<0.001) and season 2 (F (11, 24) = 24.16, p<0.001). The untreated control plots suffered the greatest damage from the larvae of DBM and CB. Crop damage rating was found least in spinetoram 60 g a.i. per ha in season 1 (1.30) and season 2 (1.50). However, amongst the insecticides evaluated, a higher crop damage rating was noted in deltamethrin treated plots during season 1(4.30) and season 2 (4.5).
Significant differences were found among the few insecticides in terms of marketable yield (season 1: F (11, 24) = 29.74, p<0.001 and season 2: F (11, 24) = 34.43, p<0.001). Application of spinetoram 60 g a.i. /ha resulted into greater marketable yield in both the seasons. Among the insecticides, least marketable yield was recorded in deltamethrin treated plots during season 1 (6.43 Kg per ten plants) and season 2 (6.13 Kg per ten plants). Untreated control plots suffered the most and recorded the least marketable yield in both the seasons.

Principal component analysis for mean larval population of DBM and CB, crop damage rating and marketable weight
Three principal components (PCs) were extracted for the different insecticidal treatments with relation to the pooled mean larval population of P. xylostella and P. brassicae, crop damage rating and marketable yield from scree plot with eigen value > 1.0. The relations of the observed parameters on the different insecticidal treatments are presented in Figure 2. PC 1 depicted variation of 88.39% and PC 2 depicted variation of 11.14%, whereas PC 3 displayed only 0.47% variation. Component loadings of different factors governing natural enemy populations among tested chemicals are represented in Supplementary Table I. Among the tested chemicals in PC1, spinetoram 45 and 60 g a.i per ha treatment has a strong positive relation with all the observed parameters followed by spinosad, indoxacarb and chlorantraniliprole. Likewise, deltamethrin and chlorpyriphos have strong relation in components two and three, respectively. Three main groups, comprising different insecticides were identified based on positional proximity in the 2-D biplot. Insecticides such as chlorpyriphos, deltamethrin, flubendiamide and lambda-cyhalothrin, were forming one group as they located close to one another, whereas, spinetoram, indoxacarb, chlorantaniliprole and emamectin benzoate occupied a different location on the 2-D plot. Apart from these groups, control was only located in component two indicating a different trend compared to other parameters.
All the dosages of spinetoram were found safer as maximum number of adults as well as immature coccinellids and spiders were noted. Chorpyriphos and deltamethrin were found harmful as the minimum population of adults as well as immature of coccinellids and spiders were observed.

Principal component analysis for pooled mean population of natural enemies
Three principal components (PCs) were extracted from the different insecticidal treatments with relation to spiders and coccinellids population for scree plot with eigenvalue > 1.0. The coccinellid and spider population on different insecticidal treatments are presented in Figure 1. PC1 depicted a variation of 86.99% and PC2 depicted variation of 11.49%, whereas PC3 displayed 1.52% variation. Component loadings of different factors governing natural enemies populations among tested chemicals are represented in Supplementary Table II. Among the tested chemicals in PC1, chlorantraniliprole has strong positive relation followed by spinetoram. Likewise, lambda-cyhalothrin and spinetoram have strong relation in components two and three, respectively. Four main groups, comprising different insecticide parameters, were identified based on positional proximity in the 2-D biplot. Insecticides such as lambda-cyhalothrin, flubendiamide, deltamethrin were separated from other chemicals, whereas, indoxacarb, chlorantraniliprole occupied a different location from control on the 2-D plot. Apart from these groups, spinetoram and emamectin benzoate were only located close to control indicating a safer trend compared to other parameters.

Phytotoxicity of insecticides on cabbage leaves and head
None of the phytotoxic symptoms appeared on cabbage leaves and heads after the application of each insecticidal treatment.

DISCUSSION
DBM and CB were the lepidopteran insect species observed throughout the study period in cabbage. In the present investigation, applications of spinetoram, spinosad, chlorantraniliprole and indoxacarb were significantly effective in reducing the DBM and CB larval population on cabbage when sprayed thrice at 15 days interval providing significantly greater marketable yield with relatively safer to the natural enemies throughout the cropping season. Studies by Legwaila et al. (2014) wherein they reported a higher efficacy of spinosad against DBM egg and larvae under laboratory conditions. Muthukrishnan et al. (2013) reported that spinetoram at 45 and 54 g a.i./ha significantly reduced Spodoptera litura larval population in tomato. Chlorantraniliprole 52 mg a.i /L under field conditions was reported as effective as indoxacarb and spinosad furthermore significantly effective than emamectin benzoate in managing Pieris rapae giving marketable yield with three spray application (Su et al., 2017). Our results showed that insecticides viz., chlorpyriphos, deltamethrin, flubendiamide and lambda-cyhalothrin were less effective in controlling the larval DBM and CB population in cabbage. Similarly, field trials conducted by Patra et al. (2017) revealed that chlorpyriphos was least effective against DBM in comparison to the novel molecules. Spinetoram showed highest efficacy in managing of DBM and CB with higher safety to natural enemies and without any visual symptoms of phytotoxicity (Fig. 2). This may be due to its unique mode of action i.e. stomach, contact, systemic and translaminar activity of this molecule. Similar findings were reported by Shimokawatoko et al. (2012). In comparison with conventional insecticides, novel molecules conserved natural enemies. However, as compared to untreated plots, populations of natural enemies were lower in insecticide treated plots. Apart from spinetoram, novel molecules namely spinosad, chlorantraniliprole, indoxacarb and emamectin benzoate were also safer to natural enemies. Dhanalakshmi and Mallapur (2008) reported that emamectin benzoate and spinosad were as safe as untreated control to natural enemies. Reduction in coccinellid and spider population following the application of chlorpyriphos, deltamethrin, flubendiamide and lambda-cyhalothrin might be due to the non-targeted impacts on their biology, growth and O n l i n e

F i r s t A r t i c l e
Spinetoram, a Selective Novel Insecticide 9 reproduction. Detrimental effects of chlorpyriphos on coccinellid predators were reported by El-Hawary et al. (2010). Adverse effects of lambda-cyhalothrin on seven spotted lady beetle, Coccinella septumpunctata were reported by Tengfei et al. (2019) under in-vitro conditions. Deltamethrin was reported as the most detrimental compound with 100 % mortality against two-spotted lady beetle, Adalia bipunctata (L.) (Coleoptera: Coccinellidae) (Garzon et al., 2015).

CONCLUSION
Spinetoram can be effectively used to manage DBM and CB in cabbage ecosystem that is safer to the natural enemies like coccinellids and spiders. Apart from spinetoram, other novel molecules viz., spinosad, indoxacarb, chlorantraniliprole and emamectin benzoate were also effective in field conditions. Spinetoram can be incorporated into IPM programs and need to be used alternatively or sequentially in insecticide resistance management strategies.

Funding
This work was supported by ICAR-Indian Institute of Vegetable Research, Varanasi, U.P., India.

Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Ethical approval
The research work does not contain any studies with human participants and animals performed by any of the authors.

Supplementary material
There is supplementary material associated with this article. Access the material online at: https://dx.doi. org/10.17582/journal.pjz/20220724130756

Statement of conflict of interest
The authors have declared no conflict of interest.