Effect of Different Chickpea Genotypes and Its Biochemical Constituents on Biological Attributes of Helicoverpa armigera (Hubner)

Chickpea (Cicer arietinum L.) is the third most important pulse crop grown all over the world. It can provide substantial nutritional and health benefits to the human diet, planted and utilized for an expanding world population for diminishing the poverty and hunger with climate change. Chickpea is grown in Sub-Saharan Africa and South Asia, which accounts for more than 75% of the world chickpea growing area. In India, chickpea a cool-season crop is the most important food legume. India is the largest producer of chickpea accounting for about 66% in area and 65% in production in the world (Dixit et al., 2017). Other major chickpea producing countries are Australia, Myanmar, Ethiopia, Turkey and Pakistan (FAOSTAT, 2016). In India, the area under chickpea was 10.56 million ha with a production of 11.37 million tons and productivity of 1077 kg/ha during Rabi 2017-18 (Anonymous, 2019).
               
Chickpea is infested by an average of about 60 insect-pests and among them the half a dozen species are considered as of economic importance. The gram pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is known to be the key pest (Reed et al., 1987). The yield losses due to H. armigera damage have been reported to the extent of 80% (Ruttoh et al., 2013). The migratory nature, polyphagous, short life cycle, multivoltine and resistance to insecticide makes H. armigera very difficult to control (Parde et al., 2012). There is a need to search for alternative methods to manage H. armigera. The screening of germplasms for resistance to insect pests is the need of the hour. However, the characterization of physiological and biochemical mechanisms conferring resistance to insects is limited (Sharma et al., 2009).

The interactions between the host plants and insects are influenced by the nutritional composition, morphological and secondary metabolites of host plants (Cates, 1980). Insect’s survival and development are affected by host plants nutritional factors and secondary metabolites, which affects insect digestibility. Insect growth and development is dependent on the suboptimal ratio of proteins and carbohydrates (Bennett and Wallsgrove, 1994; Roeder and Behmer, 2014).
       
Phenolics are distinct secondary metabolites such as tannins, flavonoids, lignin and they acquire antioxidant properties. It plays an important role to chelate transition metal ions inhibiting lipid peroxidation by trapping the lipid alkoxyl radicals and scavenges molecular species of active oxygen directly. Moreover, it reduces the fluidity of the membranes by modification of lipid packing order (Sharma et al., 2012). Phenol inhibits the insect larval growth and development by deterring feeding and metabolism (Ballhorn et al., 2011). The tannins precipitate proteins by covalent bonding of protein-NH₂ groups and minimize digestion in the herbivore mid-gut (Bernards and Bastrup-Spohr, 2008). Tannins affects insect from no visual to lower growth and development and even up to mortality (Panzuto et al., 2002).
       
Therefore, to develop the resistant chickpea genotypes against H. armigera it is very important to understand the interrelation between the chickpea biochemical molecules and insect growth and development.

Experiments on the biology of H. armigera on different chickpea genotypes and biochemical constituents estimation in chickpea genotypes were conducted at Division of Entomology and Division of Biochemistry, ICAR-IARI, New Delhi (28°38'N latitude, 77°09'E longitude, 228.61 m altitude) during 2019-2020.
       
Larvae of H. armigera were initially collected from pigeon pea crop cultivated in the experimental fields of Division of Agronomy, ICAR- IARI, New Delhi. The collected larvae were reared on semi-synthetic diet (Chakravarty et al., 2018) in insect rearing laboratory at Division of Entomology, ICAR- IARI, New Delhi under optimum conditions of 26±2°C, 65-75% relative humidity and photoperiod of 12 : 12 [L:D] h. The neonates obtained from F₅ generation were used to study the biology of H. armigera on different chickpea genotypes. Seeds of six chickpea genotypes including resistant and susceptible checks viz., ICCL -86111 (resistant check), ICC-3137 (susceptible check), GL-13001, GL-13042, RSG-959 and NBeG-786 were sown in the plastic pots filled with the potting mixture under net house conditions and the plants were watered as and when needed and fertilized. Utmost care was taken to prevent the plants from insect infestation by covering them in cages with nylon mesh. The genotypes viz., GL-13001, GL-13042, RSG - 959 and NBeG-786 were found tolerant against H. armigera under field condition under all India co-ordinated research trials, so these genotypes were selected along with resistant and susceptible check to study the relationship between biochemical constituents and insect growth and development.
       
The biological performance of H. armigera on different chickpea genotypes was studied using detached leaf method as described by Sharma et al., (2005a) under controlled conditions in the laboratory with thirty replications for each genotype. The food (fresh terminal leaf twig) was changed at two-day intervals during early instars and daily during later instars. After 10 days, larval weight was recorded separately for each larva on an analytical balance. Larval mortality was recorded during food change and the dead larvae were discarded, this was taken into considerations while calculating the per cent pupation and per cent adult emergence.
       
Observations were recorded on per cent larval survival, larval period, larval weight, per cent pupation, pupal period, pupal weight, per cent adult emergence, adult longevity and total life span. The larval period was recorded separately for each larva and the mean larval period was calculated for the surviving larvae. The data on number of larvae surviving was expressed as per cent larval survival. The data on pupae formed was expressed as per cent pupation and pupal period, mean pupal period was also calculated for surviving pupae. The pupal weight was measured separately for each pupa on an analytical balance after pupation. The data on the number of adults emerged was expressed as percent adult emergence based on the number of larvae released. The emerged adults from the larvae fed on different chickpea genotypes were paired and released into mating jar in a ratio of 1:1 (F:M) and observations were recorded on the pre-oviposition period, oviposition and post- oviposition period and the data on fecundity were expressed as the total number of eggs laid per female. Male longevity, female longevity, male and female total life cycle duration was also calculated with 3 days as incubation period. Growth index (GI) of H. armigera fed on different chickpea genotypes was also calculated by using the formula given by Seth and Sharma (2002):
                
 The test genotypes were also evaluated for field level resistance to H. armigera under natural conditions during 2019-2020. Test chickpea genotypes viz., ICCL-86111 (resistant check), ICC-3137 (susceptible check), GL-13001, GL-13042, RSG-959 and NBeG - 786 were sown in the experimental fields of ICAR-IARI, New Delhi in two rows of 2.5 m length with 30 × 15 cm spacing. The crop was raised by following recommended package of practice and crop was kept unprotected without any insecticide application. After harvest of the crop, per cent pod damage was recorded by counting the total number of pods and pods damaged by H. armigera from 10 randomly selected plants.
Per cent pod damage was calculated using the following formula:
 
Per cent pod damage was converted into pest susceptibility or resistance (%) by using the formula derived from Abbott (1925).
               
Where
P. D = Per cent pod damage.
       
The pest susceptibility/resistance percentage was converted into pest resistance/susceptible rating (PRSR) scale (1- 9) given by Kooner and Cheema (2006).
       
At full vegetative stage of the crop (30 days after sowing), the terminal branches with three to four fully expanded leaves from each genotype were excised and used for the estimation of different biochemical constituents viz., reducing sugar, protein, total phenol and tannins content from NBeG-786, GL-13001, ICC-3137, ICCL-86111, GL-13042 and RSG-959. Reducing sugar content was estimated by following methodology described by Nelson (1944) and modified by Somogyi (1952) method using glucose as a standard. The content of reducing sugar was calculated using a glucose standard curve and expressed as mg g^-1 FW. The protein content was estimated following the method described by Bradford (1976) using Bovine serum albumin (BSA) as standard. The amount of protein concentration was expressed as mg /g of fresh weight of plant tissue. Total phenol content (TPC) was estimated by following methodology described by Singleton et al., (1999) using gallic acid as a standard and was expressed as mg/g of gallic acid equivalent. The tannin content was determined by the Folin Ciocalteu method given by Amorim et al., (2008) and tannin content was calculated with the help of a gallic acid standard curve and expressed as mg/g FW.
       
The data on biological parameters including reproductive fitness of H. armigera reared on different chickpea genotypes and the data on biochemical constituents in chickpea genotypes were subjected to analysis of variance (ANOVA) after suitable transformation wherever necessary in the completely randomized design. The significance of differences between the genotypes was measured by F-test and the treatment means were compared using Tukey’s honestly significant difference (HSD) at P=0.05 using Indian NARS statistical computing portal, ICARIASRI, New Delhi. The relationship between biological attributes of H. armigera reared on different chickpea genotypes viz., larval duration, larval weight, pupal  weight, per cent adult emergence, fecundity, male total life cycle, female total life cycle, growth index and biochemical constituents of chickpea genotypes viz., reducing sugars, protein, total phenols and tannins content was computed using simple correlation co-efficient in Microsoft excel.

The studies on biological performance of H. armigera on six chickpea genotypes revealed that there were significant differences in larval duration (P=0.0003), larval weight (P= <0.0001) and pupal weight (P<0.0001). The larval duration was significantly longer in resistant check, ICCL-86111 (16.84 days) followed by GL-13042 (16.24 days), GL -13001 (15.84 days) and the shortest larval duration was recorded on NBeG-786 (14.44 days) and susceptible check, ICC-3137 (14.12 days). The larval weight after 10 days of releasing into the chickpea genotypes was taken and it was significantly lower in all the test chickpea genotypes as compared to susceptible check, ICC-3137. The lowest larval weight was recorded on the larvae reared on resistant check, ICCL-86111 (191.5 mg) and GL-13042 (230.26 mg). The larval food did not have significant effect on pupal duration. However, the longest pupal duration was observed in the insects reared on GL-13042 (7.7 days), followed by ICC- 3137 (7.6 days), ICCL-86111 (7.57 days), NBeG-786 (7.54 days) and RSG-959 (7.5 days) while the shortest duration was observed on the larvae reared on GL-13001(7.3 days). The pupal weight was significantly lower in all the test chickpea genotypes as compared to susceptible check, ICC -3137. The lowest pupal weight was recorded on the insects reared on resistant check, ICCL-86111 (150.88 mg) and NBeG-786 (159.21 mg). There were no significant differences between the per cent larval survival and per cent pupation, however lowest larval survival and pupation was observed in resistant check, ICCL-86111. The lowest adult emergence was observed in the H. armigera larvae reared on NBeG- 786 (46.43%) but not significantly different from the larvae reared on RSG-959, GL-13001, GL-13042 and resistant check, ICCL-86111 (Table 1).
 

       
The larval food did not had a significant effect on pre-oviposition, oviposition, post-oviposition period, adult longevity andtotal life cycle of male andfemale and growth index, but it had significant effect on fecundity. The fecundity of emerged adults from the larvae reared on chickpea genotypes showed significant difference with highest fecundity of the adults emerged from NBeG-786 (215.8 eggs/female), followed by GL-13042 (122.25 eggs/female), susceptible check, ICC-3137 (98.17 eggs/female), resistant check, ICCL-86111 (89.67 eggs/female), RSG-959 (85.75 eggs/female) while lowest was in GL-13001(74.5 eggs/female). The growth index was highest in the insect reared on susceptible check, ICC-3137 (3.05) but no significant difference was observed as compared to GL-13042 (2.99) and it was followed by GL-13001(2.66), resistant check, ICCL-86111(2.52), RSG-959 (2.34) and NBeG-786 (1.54) (Table 2).
 

       
The per cent pod damage, pest susceptibility/ resistance% and PRSR on different chickpea genotypes in the field condition were varied from 25.9 to 47.84%, 12.91 to 45.86 and from 4 to 5. The lowest pod damage was observed in resistant check, ICCL-86111(25.9%) followed by GL- 13001(35.13%), RSG-959 (35.28%), GL-13042 (40.43%) and NBeG-786 (41.6%) as compared to susceptible check, ICC-3137 (47.84%). Among the test genotypes, the highest pest susceptibility/ resistance % was observed in GL-13001(26.58%) followed by RSG-959 (26.25%), GL-13042 (15.49%) and NBeG-786 (12.91%) than resistant check, ICCL-86111(45.86%). The positive susceptibility / resistance percentage values indicate the increasing resistance while negative values indicate increasing susceptibility. The PRSR rating varied from 4-5 and it was 5 in NBeG-786 and GL- 13042 and 4 in the rest three genotypes, rating of 1-4 shows the resistance and 6-9 susceptibility of genotypes against insect pest (Table 3).
 

       
Similar studies have also been conducted earlier by Sharma et al., (2004, 2005), War et al., (2013) and Golla et al., (2020) who reported that reduced larval survival, delayed developmental period, lower leaf damage, lower larval weight of H. armigera was observed in wild relatives of chickpea as compared to cultivated genotypes suggesting that antibiosis mechanism of resistance or presence of antifeedants in wild relatives of chickpea. War et al., (2012) and Narayanamma et al., (2013) reported that lower leaf damage, larval survival and larval weight were observed in resistant genotypes as compared to susceptible genotypes of groundnut and chickpea against H. armigera suggesting antibiosis mechanism of resistance. Kaur et al., (2017) and Bheemaraya et al., (2019) reported that chickpea genotypes ICC-3137 and L-550 showed higher level of pod damage and was found to be susceptible to H. armigera.
       
Significant differences were observed in reducing sugar content among the test genotypes. Reducing sugar content was significantly higher in GL-13001(0.96 mg/g) followed by NBeG – 786 (0.88 mg/g) RSG-959 (0.60 mg/g), whereas significantly lower reducing sugar content was observed in GL-13042 (0.31 mg/g). The reducing sugar content of all the test genotypes was significantly lower as compared to the resistant check, ICCL-86111 (Table 3). The observations found in this study were in conformity with the report of Singh et al., (1972) who noticed low amount of reducing sugars in the leaves of jassids resistant varieties of cotton in comparison with susceptible check. Haralu et al., (2018) reported that the lowest reducing sugar was noticed in BGD 111-01 chickpea genotype and the percent pod borer infestation showed positive correlation with reducing sugar content.
       
There were significant differences in protein content among the test genotypes. Protein content was significantly higher in NBeG-786 (34.75 mg/g) followed by RSG-959 (28.06 mg/g), GL-13001(26.72 mg/g) whereas significantly lower protein content was observed in GL-13042 (22.88 mg/g) as compared to resistant check, ICCL-86111(27.27 mg/g) (Table 3). Protein-based compounds mediate wide-ranging defense responses in plants (War et al., 2013). War et al., (2012) reported that the higher protein content may be attributed to the greater activity of plant defensive enzymes and also other defensive protein production. Blanco-Labra et al., (1995) reported that the plant proteinase inhibitors are reserve proteins that deter insect feeding and digestion. Kansal et al., (2008) reported that the prolonged larval period, lower larval weight, survival and adult emergence of H. armigera was recorded in diet containing chickpea trypsin inhibitor, indicating the antibiosis mechanism.
       
Significant differences were observed in total phenol content among the test genotypes. Phenol content was significantly higher in NBeG-786 (0.90 mg/g) followed by RSG-959 (0.77 mg/g) whereas significantly lower phenol content was observed in GL-13001(0.66 mg/g) as compared to resistant check, ICCL-86111(0.76 mg/g) (Table 3). Kaur et al., (2014) reported that resistant pigeon pea genotypes against H. armigera showed higher phenol content. The higher phenolic compounds in resistant genotypes than in intermediate and susceptible pigeon pea genotypes might be the reason for insect resistance (War et al., 2013). Quinones resulting from phenols oxidation bind covalently to leaf proteins and inhibit insect digestibility. The phenolic constituent’s accumulation such as phenols, o-dihydroxyphenols, flavonols in plants act against herbivores and become toxic to insects (Walling, 2000; Bhonwong et al., 2009).
       
There was significant difference in the tannin content among the test genotypes. The significantly higher tannins content was observed in NBeG-786 (2.49 mg/g) followed by GL-13001(2.22 mg/g), GL-13042 (2.19 mg/g) whereas significantly lower tannin content was observed in RSG- 959 (1.58 mg/g) as compared to resistant check, ICCL- 86111(1.84 mg/g) (Table 3). Many researchers reported that the tannins affect the development and survival of many insect pests (Bernards and Bastrup-Spohr, 2008; Sharma et al., 2009) by nonspecifically reducing nitrogen mineralization and/or digestion in the midgut of herbivore (Bernards and Bastrup-Spohr, 2008). The increase in tannins content helps in reducing the plant tissue damage by feeding deterrence (Kaur et al., 2014).
       
Chickpea biochemical components showed significant influence on the survival and development of H. armigera except tannins. Relationship between biological attributes of H. armigera and biochemical constituents in different chickpea genotypes revealed that reducing sugars, protein, total phenols and tannins content showed negative association with majority of biological attributes of H. armigera reared on chickpea genotypes. Protein and total phenol content showed significant negative relation with per cent adult emergence (r= -0.894*, r= -0.848*) and growth index (r= -0.932*, r= -0.846*) (Table 4). Golla et al., (2020) reported that the protein content was significantly and negatively correlated with larval weight, pupation and adult emergence. Phenol content was negatively correlated with larval weight, pupation, adult emergence, pupal weight and fecundity whereas positively correlated with pupal period. Tannins content was positively correlated with larval weight, pupation and adult emergence. Haralu et al., (2018) reported that the percent pod borer infestation was negatively correlated with phenol content but positively correlated with the contents of total sugar, reducing sugar, protein and total chlorophyll.
 

The test chickpea genotypes viz., GL-13001, GL-13042 and RSG-959 exhibited moderate level of resistance with prolonged larval duration, decreased larval and pupal weight, reduced per cent adult emergence with low fecundity, lower pod damage and higher amounts of anti-nutritional factors viz., phenols and tannins content as compared to susceptible check, ICC-3137 indicating antibiosis mechanism of host plant resistance. These chickpea genotypes can be used a source of resistance to develop varieties with a stable resistance against H. armigera.

Authors thankfully acknowledge Head and Professor of Entomology Division, ICAR-IARI, New Delhi for providing required support during these studies. We are also thankful to post graduate school, IARI, New Delhi as this is a part of M. Sc thesis of the senior author.