Biochemical Components Conferring Resistance to Pigeonpea Genotypes against Callosobruchus chinensis (L.) (Coleoptera: Bruchidae) under Semitropical Storage Conditions

India is the largest producer, consumer and also the largest importer of pulses in the world. Pulses are grown in around 24-26 million hectares of area with a production of 17-19 million tonnes annually in India and accounts for over 33.33 percent of the total world area and over 20% of total world production (Upadhyay et al., 2017). Pigeonpea, C. cajan is one of the most common tropical and subtropical legume crops belonging to the family Leguminosae, mostly being cultivated for its edible seeds in Africa, Asia and Americas.
       
Generally, plants possess toxic secondary metabolites which act either directly on insect pests through antibiosis or develop non-preference for insects feeding on the seeds (War et al., 2012). The host plants contain both nutritional and anti-nutritional compounds. The nutritional compounds may enhance the growth and development and on the contrary the anti-nutritional compounds, may deter the survival and development of insects (Ding et al., 2000 and Cipollini et al., 2005).
               
However, the unpredictability in pigeonpea production due to poor genetic background and damage by insect pests and diseases limits the seed yield of this crop. Under storage conditions, bruchids, C. chinensis and C. maculatus crusade extensive losses to all the food legumes worldwide (Sharma et al., 2017) and causing 40-50% losses of pulses in storage (Gosh and Durbey, 2003). Keeping this in view, the present research focuses mainly on the possibilities of exploring biochemical sources of resistance to bruchids in cultivated as well as wild relatives of pigeonpea.

Test insects and maintenance
         
Bruchids culture was reared and maintained in Entomology unit of International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India. Bruchids were reared on chickpea variety (L 550) throughout the study period. Sex determination for male and female was done using key characters (Arora, 1977). The culture was maintained in the (Bio-Oxygen Demand, BOD) incubator with 28±2°C temperature and 70±5% relative humidity. Sub culturing of adult bruchids were done with one kg chickpea/pigeonpea seeds in a five-liter capacity plastic jar.
 
Bioassay technique
 
Fifteen genotypes of pigeonpea were evaluated for resistance to C. chinensis in multi-choice (MC) and single-choice (SC) tests under laboratory conditions. In the first test, all pigeonpea test genotypes were subjected to C. chinensis infestation freely (no restriction). Seeds of each genotype were kept equidistantly in circular manner in a wide mouth plastic basin measuring 9 cm in length and 2 cm in depth. Each plastic basin was considered as one replication and there were three replications for each genotype. Ten pairs of 0-24 hours old adults of C. chinensis were released in the center of each plastic basin using an aspirator device and covered with food wrapping film containing tiny perforations. Under single choice test (SC), C. chinensis was allowed incursion only to a single genotype. The seeds of each genotype were placed separately in plastic bioassay cups measuring 4 × 10 cm (L × B) and five pairs of freshly emerged 0 to 24 hours old adults of C. chinensis were released. Each bioassay cup was considered as one replication and thereafter the same procedure was followed for multi-choice test (MC). The whole experiment was conducted in an incubator maintained at 28±2°C and 70±5% relative humidity and 12: 12 hours (L:D) of photoperiod. Seed damage was expressed as the percentage of damaged seeds for each genotype and the percentage damage incidence was determined using the formula described by Khattak et al., (1995) Sathish et al., (2020) :  
Oviposition preference
 
In multi choice test, the ovipositional preference of the female C. chinensis to pigeonpea grains of different genotypes was studied. The seeds of each genotype were randomly placed equidistantly in a plastic cup; ten pairs (male, female) of C. chinensis were introduced into the center of the plastic basin and covered with a perforated food wrapping plastic film. The total number of eggs laid on each genotype were counted after 7 days for the assessment of ovipositional preference. In single choice test, to study the ovipositional preference of the female C. chinensis, pigeonpea grains of each genotype were placed in a bioassay cup separately and five pairs of C. chinensis were released. Thereafter, the same procedure was followed as in the multi-choice test.
 
Adult emergence (%)
 
The freshly emerged beetles were counted at 30 days after release and removed daily to avoid the chances of their recounting and the data was pooled to get the total number of adults emerged. The number of eggs laid on each genotype were counted after 7 days of bruchids release. The per cent adult emergence was recorded using the formula (Howe, 1971):
 
Seed weight loss (%)
 
Seed weight loss was measured with a digital balance (Sartorius, CP124S, Sartorius AG Gottingen, Germany) before and after 45 days of bruchid infestation. The weight loss % was calculated using the formula:
   
Number of exit holes
 
The genotypes were examined on a bi-weekly basis after F₁ progeny emergence to record the number of exit holes per genotype by visual observation.
 
Biochemical basis of resistance to bruchids
 
Biochemical compounds were estimated using standard protocols. The total phenols were estimated using the method described by Bray and Thorpe (1954); tannins by Vanillin-Hydrochloric acid method (Burns, 1971); total of flavonoids by Vanillin reagent method (Swain et al., 1959) and the total soluble sugars by Phenol-Sulphuric acid method (Dubois et al., 1956).
 
Statistical analysis
 
The experiments were designed statistically by completely randomized design (CRD) with three replications. Results from the data were subjected to analysis of variance (ANOVA) and the ‘F’ tests were carried at 5% level of significance using SPSS 16^th version. The Pearson’s correlation coefficient (r) analysis was performed using GenStat 14th edition. The diversity among the genotypes was assessed based on seed damage percentage under single choice and multi choice tests using principal coordinate analysis (Genstat, 2014).

Seed damage by the bruchids on different pigeonpea genotypes
 
The seed damage percent was significantly different among pigeonpea genotypes both under multi-choice and single choice conditions (Fig 1). The results revealed that seeds of wild species C. platycarpus ICPW 68 were highly resistant to damage by C. chinensis. It kept the C. chinensis population under check under storage conditions. The lowest percentage of seed damage was recorded on ICPW 68 (4.33% and 18.9%) while, ICPL 161 exhibited highest seed damage percent (59.78% and 74.40%) under multi-choice and no choice conditions respectively (Fig 1). In the present study, none of the 15 pigeonpea genotypes tested were found to be free from the damage by pulse beetle, but there were appreciable differences in their susceptibility to C. chinensis. The results of the present studies are in corroboration with the findings of Jadhav et al., (2012) who reported that bruchid damage ranged between 14 and 30% in wild relatives of pigeonpea, compared to 78% in the commercially cultivated pigeonpea variety ICPL 85010. The present findings are also in confirmation with the reports of Eker et al., (2018) who evaluated the kabuli accessions of chickpea YAR and ILC 8617 and reported 100% seed damage in multi-choice test, but in no-choice test, the accession CA 2969 exhibited 93% seed damage followed by the accession YAR (87.0%) and in desi chickpea, the accession ICC 4957 and C. reticulatum accession AWC 612, the seed damage was 83.0 per cent.
 

 
Oviposition preference of the bruchids on grains of Pigeonpea genotypes
 
The oviposition response of C. chinensis both under multi-choice and single choice conditions varied significantly (p=<0.005) across different pigeonpea genotypes (Fig 2). The results revealed that minimum number of eggs  were laid on ICPW 68 both under single choice (49.37) and multi–choice (21.33) conditions, followed by ICPL 20186 (28.40) and ENT 11 (33.67). The highest number of eggs were recorded on ICPL 161 (171.38 and 68.00) under single and multi-choice tests, respectively. In other words, none of the test genotypes were found resistant to C. chinensis in terms of oviposition preference due to their physical and morphological traits. However, ICPW 68 was found to be statistically superior genotype among all the genotypes tested. The number of eggs deposited by Callosobruchus spp. was affected by seed size, curvature, colour, thickness of the seed coat and smoothness of the seeds (Mphuru, 1981). Oviposition is a paramount behavior exhibited by an insect for continuation of its race and establishment of their population (Sehgal and Sachdeva, 1985). The ovipositional responses of C. chinensis seem to be governed by several biotic and ecological factors. The differential preference for oviposition of C. chinensis on different accessions might be due to odour of the seed which could emanate from its chemical constituents, may provide the stimulus for oviposition (Howe and Curie, 1964).
 

 
Number of exit holes on seeds
 
There were significant differences in mean number of holes per ten grains among the genotypes. The lowest mean number of holes were observed in ICPW 68 (3.00), followed by ICPL 20186 (5.67) and ENT 11 (6.00) while highest mean number of holes were observed in ICPL 161 (12.33) (Fig 3). The results of the present studies are in agreement with the reports of Kuldeep et al., (2015) who recorded minimum number of emergence holes of C. chinensis in Pusa Komal (18.33) and maximum in IC313300 (84.67) followed by IC326996 (78.67).
 

 
Adult emergence %
 
The per cent adult emergence was found to be lowest on ICPW 68 (6.25), while the highest adult emergence percent was observed on ICPL 161 (31.44) (Fig 3). These findings are similar to Rosemond and Khan (2013) who reported that the adult emergence per cent ranged from 90.20 in B26 to 23.88 in B59. They screened eighteen cultivars for resistance to C. chinensis and reported that B26PL2, B59, B6PL2 and B194 exhibited less than 50% adult emergence. B26PL2, B59, B6PL2 and B194 did not differ significantly from each other (z-test, P>0.05). Low adult emergence coincided with low egg to adult survival on these varieties.
 
Percent weight loss
 
All the pigeonpea genotypes differed significantly in terms of percent weight loss due to C. chinensis. The data revealed that genotype ICPW 68 recorded significantly lowest average percent grain weight loss (2.01). ICPL 161 recorded highest average percent weight loss (24.39) (Fig 3). The findings of the present study are in corroboration with the findings of Alok et al., (2005) who studied the weight loss due to C. chinensis on green gram which incurred highest weight loss (22.88) followed by arhar (20.79), peas (15.19), Vijay (13.14), Vishal (13.03) and Kabuli (11.71). While in kidney bean no weight loss was observed as there was no infestation.
 
Total flavonoids content
 
ICPW 68 recorded high flavonoid content (1.28 mg/g) followed by ICPL 87091 (1.21 mg/g), ENT 11 (1.19 mg/g) and ICPL 20103 (1.18 mg/g). The lowest amounts of total flavonoids were recorded in ICPL 161 and ICPL 332 (0.40 and 0.36 mg/g respectively) (Table 1). Similarly, Ahmad et al., (2019) reported that flavonoids content of different chickpea varieties ranged from 0.283 to 0.466 mg/g. Rani et al., (2014) observed  that flavonoids content in whole seed extract was (8.65±0.6 mg/g ) in pigeonpea.
 

 
Total tannin content
 
The total tannin content in pigeonpea genotypes ranged from 9.69 mg/g to 16.75 mg/g with a mean value of 12.57 mg/g. The highest tannin content (mg/g) was found in ICPW 68 (16.75) followed by ICPL 20186 (15.00), ICPL 20103 (14.38) and ENT 11 (14.06). ICPL 161 recorded lowest tannin content (9.69) (Table 1). Singh and Jambunathan (1981) reported that the tannin content in pigeonpea genotypes ranged from 4.3-11.4 mg/g. Tannin content in chickpea has been reported to range from 1.9 mg/g to 6.1 mg/g (Singh et al., 1982) and in lentils from 0.80 mg/g to 6.48 mg/g (Ahuja et al., 2015).
 
Phenol content
 
The average content of total phenols was found to be higher in ICPW 68 (1.38 mg/g), followed by ICPL 87091 (1.05). ICPL 161 recorded lowest phenol content (0.44), followed by ICPL 332 (0.46), ICPL 86022 (0.48) and ICPL 88034 (0.48) (Table 1). Phenolic compounds have been reported to lower the activity of digestive enzymes such as amylase, trypsin and chymotrypsin and could also damage the mucosa of the digestive tract. The present results are in corroboration with earlier (Japjot et al., 2017) studies where in the wild type species (C. scarabaeoides, ICP15683/W15) with smallest seed size exhibited highest phenol content. Nwosu et al., (2013), reported the total phenol content value of 1.6 mg/g in pigeonpea.
 
Total soluble sugars
       
ICPL 161 (47.25 mg/g) had maximum amount of sugars (mg/g) followed by ICPL 332 (45.22) and ICPL 86022 (43.09). The lowest sugar content was recorded in ICPL 20186 (27.00) followed by ENT 11 (32.14) and ICPW 68 (35.41) and these genotypes were statistically superior to the rest of the genotypes. Similarly, Japjot (2017) categorized the sugar content higher than 52.23 mg/g under higher total soluble sugars group, values between 38.44-52.23 mg/g under medium total soluble sugars group and lower than 38.84 mg/g under low total soluble sugars group (H ≥ 52.23; L ≤ 38.44). High combined larval and pupal mortality reduced adult eclosion in some cultivars and this might be due to biochemical composition of the seeds which also appear to play a role in reducing populations of bruchids on C. cajan.
 
Association of biochemical components with expression of resistance to C. chinensis using principal coordinate analysis
 
Principal coordinate analysis placed the test genotypes into different clusters. The resistant genotype ICPW 68 was placed distinctly in cluster A. The moderately resistant genotype ICPL 20186 was placed in cluster B, while the susceptible genotypes were placed in clusters C, D and E. (Fig 4) suggesting the possibilities for developing lines resistant to C. chinensis.
 

 
Correlation studies of pigeonpea genotypes resistant to bruchids
 
The correlation studies between per cent seed damage and biochemical parameters of pigeonpea genotypes revealed that percent seed damage in both multi-choice and single choice conditions exhibited significant and negative correlation with flavonoids (r = -0.752 and r = -0.776), phenol (r = -0.942 and r = -0.921) and tannins (r = -0.751 and r = -0.832). A significant positive correlation was observed with total soluble sugars (r = 0.479 and r = 0.461) (Fig 5). Similarly, Ambidi et al., (2022) reported that phenolic content and pod damage by pod borer complex showed highly significant negative association with r-value (-0.729**). This clearly indicates that high phenol content played a critical role in resisting pod borers under field conditions.
 

The results revealed that pigeonpea genotypes and their wild relatives varied significantly in terms of high anti nutritional contents, which exhibited greater resistance to the C. chinensis. Hence, promising genotypes could be used in breeding programs to develop hybrids/ cultivars resistant to C. chinensis.

The authors are thankful to the CGIAR-CRP-Grain Legume and Dry land Cereals for funding the research activities and Indira Gandhi Krishi Viswavidyalaya, Raipur for granting scholarship during research period.

All authors declared that there is no conflict of interest.