Influence of Physico-chemical Traits of Chickpea Genotypes on Preference and Non-preference of Pulse Beetle, Callosobruchus maculatus (Fab.)

Chickpea (Cicer arietinum L.) is one of the most important pulse crops widely grown in rabi season under dry and rainfed areas of India, accounting for roughly 70 per cent of total area and 67 per cent of total production and also grown in approximately 57 countries across the globe under diverse agro-climatic conditions (Siddique et al., 2000). During 2020-21, a total of 119.11 lakh tonnes of chickpea was produced in India from an area of 99.96 lakh ha with a productivity of 1192 kg ha^-1. It is the most popular food legume with shelf life of more than one year and high in vitamins, minerals, fibre and also contain beneficial phyto-chemicals. The total losses of chickpea produce at the national level during harvest and post-harvest handling was 8.41 per cent, with an estimated monetary loss of Rs. 2453 crore, including 1.18 per cent loss with bruchids (Jha et al., 2015), accounting for the majority of storage losses. Callosobruchus maculatus (Fab.) is a cosmopolitan insect pest that can cause significant losses in stored chickpeas, even up to 100% per cent in tropical countries like India, rendering the grain unfit for food or seed within 4-6 months (Wolfson et al., 1991).
 
Host plant resistance (HPR), one of the most effective methods, being adopted for decades to identify the traits in the host plants that confer resistance against the insect pests. As the chickpea seeds are vulnerable in both field and storage to pulse beetles, there is a need to identify the bruchid resistant chickpea sources through proper screening methods and develop cultivars with resistance to bruchids through proper breeding methods. Breeding legume crops with identified resistant sources against storage insect pests is an environmentally benign technology that offers a simple and economical, approach to minimize bruchid damage (Swamy et al., 2019). The grain physical characters, nutritional and anti-nutritional factors of the legumes will also affect the development of bruchids to a greater extent (Rekha et al., 2017). Association between insect growth, oviposition preferences and grain parameters would assist in identifying the aspects that should be prioritized during crop breeding programmes (Swamy et al., 2020). Assessment of biochemical components in legumes is required to identify the key components aid in resistance against bruchids. Thus, legume cultivars with favourable components can be genetically engineered. In light of these facts, the current investigation was carried out to screen various chickpea genotypes against the pulse beetle, C. maculatus and to comprehend the impact of chickpea genotype physico-chemical traits on the growth and development of the pulse bruchid under laboratory storage conditions.

Twenty pre released and released chickpea genotypes including thirteen desi type viz., NBeG 3 (Nandyal Sanaga 1), NBeG 47 (Dheera), NBeG 49 (Nandyal Gram 49), NBeG 452 (Nandyal Gram 452), ICC 86111, JG 11, NBeG 699, NBeG 776, NBeG 779, NBeG 857 (Nandyal Gram 857), NBeG 1129, NBeG 1137 and NBeG 1174; and seven kabuli type viz., NBeG 119 (Nandyal Gram 119), NBeG 810 (Nandyal Gram 810), KAK 2, Vihar, NBeG 440, NBeG 789 and NBeG 833 procured from Regional Agricultural Research Station (RARS), Nandyal andhra Pradesh were screened against C. maculatus under free-choice conditions during 2020-21 (Plate 1). In addition to the screening experiment, the chickpeas’ physical characteristics and biochemical parameters that might affect the preference and population growth of the pulse beetle were examined. Later, correlation and regression analyses were used to determine how much each parameter affected the growth and development of the pulse bruchid, both individually and together. Before experimentation, in order to reduce any hidden infestation, the test genotypes were subjected to disinfestation by being fumigated for seven days with aluminum phosphide tablets @ 3 tablets per ton. To get rid of phosphine residues, the grains were then thoroughly aerated.


 
Rearing of the test insect
 
The insect culture was established on chickpea by introducing a few pairs of pulse beetles collected from the post-harvest technology centre in Bapatla andhra Pradesh, as described by Andrewartha (1961). C. maculatus mother culture was grown in the laboratory on a locally available chickpea variety, JG-11.
 
Screening of chickpea genotypes through free choice test
 
Relative preference of pulse bruchid to the different chickpeas genotypes was studied under free-choice test, conducted in artificially designed ‘circular screening containers’ developed by following modifications over earlier descriptions (Duraimurugan et al., 2014). The circular containers (80 cm diameter and 20 cm H) were made up of a card board with wooden circular base, covered with a transparent polythene cover. The inner flat bottom of the container included 20 plastic cups (5 cm diameter and 2 cm H) arranged at equal distance, where seeds of the genotypes were placed. A single pipe (2.5 cm diameter, 30 cm H) having holes at the bottom was inserted in the center of the flat bottom for insect release. Hundred grains of each chickpea genotype were placed in plastic cups and the circular container was closed with transparent polythene sheet and secured tightly by using cello tape (Plate 2). The pulse beetles (One day old) (80 No.) were released in the middle through the pipe inserted to choose the grains of their choice and the pipe was removed after insects reached their preferred genotypes and closed the hole with cello tape immediately. The adults were carefully removed after five days and the grains were then put into separate plastic containers and left undisturbed until adult emergence. This experiment was replicated thrice, with three circular containers. The observations on number of eggs laid was recorded at five days after release of insects while the adult emergence and grain damage were recorded from the samples after 40 days of insect release. The experiments were conducted in completely randomized block design replicating thrice. The obtained data were subjected to suitable transformations and analysed for comparison.

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Physical parameters of chickpea genotypes
 
Physical parameters of the grains viz., type of grain (desi/ kabuli), test weight (100 grain weight) and seed coat thickness were recorded. Test weight of each genotype was measured with the help of analytical balance and expressed in grams and for the thickness of seed coat, the grains were picked at random from each genotype in three replications and soaked for two hours. After soaking, seed coat was removed carefully and dried at 50°C for 24 hours by using hot air oven. Then, it was measured by using digital Vernier’s scale. The mean of thickness of seed coat of ten grains was calculated and expressed in millimeters.
 
Biochemical components in chickpea genotypes
 
The chickpea genotypes were analysed for the biochemical components, viz., total soluble sugars, total phenols, tannins the protocols given by Yemm and Willis (1954), Malik and Singh (1980) and Schanderi (1970) were followed, respectively. The data were subjected to ANOVA by Completely randomized design, with three replications. Impact of the physical and biochemical parameters was expressed by correlating them separately and also in combination with number of eggs laid, adult emergence and grain damage using the SPSS statistical software version 21.0.

The data on number of eggs laid, pulse beetle adults and grain damage of chickpea genotypes under free choice conditions indicated that there were significant differences in insect preference and non-preference towards the genotypes of chickpea grains (Table 1). NBeG 452 was found superior over other genotypes by recording least number of eggs (24.33) which was on par with NBeG 776, NBeG 1129, ICC 86111 and NBeG 49 which recorded 25.33, 25.33, 25.67 and 27.67 eggs, respectively and the genotype, NBeG 789 recorded higher number of eggs (90.33), which significantly differed with all the genotypes. Similarly, NBeG 1129 was found superior over other genotypes by recording the lowest number of adult emergence (19.00) and was on par with NBeG 452 (19.67), NBeG 49 (20.67), ICC 86111 (20.67) and NBeG 776 (21.00) and significantly different from other genotypes, while NBeG 789 recorded with higher adult emergence (84.33). Lowest per cent grain damage was recorded by NBeG 452 (17.75%) by weight method, on par with NBeG 1129 (18.18%), NBeG 776 (19.22%), NBeG 49 (19.44%) and ICC 86111 (19.89%), while, the per cent grain damage was observed highest in NBeG 789 (65.27%) followed by NBeG 440 (52.99%) and NBeG 833 (51.60%), by differing significantly with the remaining genotypes.


 
Thus, the genotypes NBeG 452, NBeG 1129 and ICC 86111 were categorised as less susceptible, whereas the genotypes NBeG 440, NBeG 789 and NBeG 833 were placed in the highly susceptible group, based on oviposition, adult emergence and grain damage. Smaller grains and a thicker seed coat characterized the genotypes that were less vulnerable. The results of the biochemical studies revealed substantial differences between the test genotypes of chickpea in the amounts of total soluble sugars, total phenols and tannins.
 
Physical parameters of chickpea genotypes
 
The test (100 grains) weight of kabuli type chickpeas ranged from 17.27 (ICC 86111) to 44.60 g (NBeG 833) and was significantly greater than that of desi types. The genotype with the smallest test weight (17.27 g) was ICC 86111. A larger grain size is indicated by a higher test weight, which also guarantees a greater supply of food to feed growing insects. The thickness of the seed coat varied among the chickpea genotypes, ranging from 0.060 mm in NBeG 440 to 0.210 mm in NBeG 776.
 
Biochemical parameters of chickpea genotypes
 
Higher quantities of total soluble sugars (TSS) were found in the kabuli genotypes; NBeG 440 (75.36%), NBeG 789 (73.65%) and NBeG 833 (72.53%). JG 11 has the highest quantity of carbohydrates (71.66%) of the desi types. The TSS concentrations were significantly lower in the genotypes NBeG 452 (55.94%), NBeG 1129 (59.98%) and NBeG 776 (60.11%). The largest amount of total phenol was 119.06 mg CAE 100 g^-1 in NBeG 452, while the lowest amount was 74.25 mg CAE 100 g^-1 in NBeG 789.
 
Correlation analyses
 
Physical parameters of chickpea grains Vs insect preference and non-preference
 
Test weight and seed coat thickness of chickpea grains were correlated to the insect response, which included oviposition, the number of adults that emerged and grain damage (Table 2). A substantial positive correlation between test weight and oviposition, adult emergence and percent grain damage was found (r= 0.502, 0.504 and 0.498, respectively). With regard to oviposition (r= -0.577), adult emergence (r= -0.583) and percentage of grain damage (r= -0.607), the seed coat thickness was inversely associated.


 
Biochemical parameters of chickpea grains Vs insect preference and non-preference
 
With respect to oviposition (r= 0.730), adult emergence (r= 0.734) and percentage of grain damage (r= 0.745), total soluble sugars exhibited a strong positive correlation. In contrast, there was a strong negative association between total phenols and tannins and oviposition, adult emergence and the percentage of grain damage (r= -0.838, -0.798).
 
Regression analysis
 
Physical parameters of chickpea grains Vs insect preference and non-preference
 
A multiple linear regression equation was fitted to the physical characteristics of the several chickpea genotypes, including test weight and seed coat thickness, adult emergence and percent grain damage (Table 3). The physical characteristics of chickpea grains were found to have a 39.6% influence on grain damage (R2= 0.396), a 37.9% influence on adult emergence (R2= 0.379) and a 37.3% influence on oviposition (R2= 0.373).

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Biochemical parameters of chickpea grains Vs insect preference and non-preference
 
Similarly, a multiple linear regression equation that took into account oviposition, adult emergence, per cent grain damage and total soluble sugars, total phenols and total tannins of the genotypes of chickpea was fitted (Table 4). The biochemical factors had an impact on grain damage to the amount of up to 74.7% (R2= 0.747), adult emergence to the extent of 69.5 (R2= 0.695) and oviposition to the extent of 67.5 (R2= 0.675).

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Physical and biochemical parameters Vs insect preference and non-preference
 
Multiple linear regression was used to quantitatively analyze the combined impact of physical attributes and biochemical components of chickpea grains from various genotypes on the preference and growth of bruchid insects. According to Table 5, the oviposition was strongly influenced by the physical characteristics and biochemical components of chickpea grains to the extents of 70.1% (R²= 0.701), 72.2% (R²= 0.722) and 76.6% (R²= 0.766).

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The results of the current study are consistent with those of earlier studies, which found that differences in the morphological and physical characteristics of different legume seeds, such as seed size and shape and seed coat thickness, affect resistance to bruchid infestation. Test weight of the grains showed a positive correlation with oviposition and adult emergence, while seed coat thickness showed a significant negative correlation with oviposition, adult emergence and grain damage (Eker et al., 2018 and Swamy et al., 2020). As per the Neog and Singh (2011), total soluble sugars had positive influence on oviposition of pulse beetle. Sharma and Thakur (2014) reported that high amounts of carbohydrates were present in the susceptible genotypes. Swamy et al., (2020) stated that the chickpea genotypes with higher total soluble sugars were susceptible to bruchids. The total soluble sugars favoured the successful development of pulse beetle (Senthilraja and Patel, 2021). In contrast, total phenols and tannins showed significant negative correlation with oviposition (r= -0.783, -0.756), adult emergence (r= -0.800, -0.772), grain damage (%) (r= -0.838, -0.798). The current research findings are in line with Patel et al., (2003), reported negative correlation between egg laying behaviour and phenol content of chickpea grains. Swamy et al., (2020) reported that the chickpea genotypes with lower phenols were highly susceptible to bruchids. It was reported that significant negative correlation was there in between seed damage and phenol content (Tripathi et al., 2020). High phenols and tannins were detrimental to the growth and development of C. maculatus (Senthilraja and Patel, 2021). Kaur and Gill (2016) reported that of antinutritional factors such as tannins inside pulses offered resistance against C. maculatus development. Kaur et al., (2016) reported that the tannins were negatively correlated with adult emergence of C. maculatus.
 
According to Patel et al., (2003), more adult emergence and grain damage by the pulse beetle were caused by high test weight and thinner seed coat and the egg-laying behaviour of C. chinensis on pulses was inversely correlated with phenol content. Kamble et al., (2016) noticed resistance in genotypes with lower test weight. Chickpea genotypes with lower test weight and grain size and more seed coat thickness adversely affected the oviposition, grub penetration and growth of C. maculatus as there is hard barrier to enter larvae in to the grain and less space for their development inside the grain (Eker et al., 2018 and Swamy et al., 2020). Genotypes possessing higher amounts of total soluble sugars had a significant positive effect and phenols and tannins have significant negative effect on the infestation by pulse bruchid (Tripathi et al., 2020). According to Swamy et al., (2020), resistant varieties had higher phenol and tannin contents and lower TSS content and stated that physical and biochemical parameters of chickpea seed together influenced growth and damage parameters of C. maculatus to the greater extent.

According to current research findings, no single physical or biochemical parameters of chickpea is responsible for imparting resistance/susceptibility to the C. maculatus. Preference/ non-preference of chickpea genotypes to C. maculatus is determined by collective effect of different physical and biochemical factors of chickpea genotypes. The test weight and seed coat thickness showed some influence on damage caused by pulse beetle but mostly the biochemical parameters like total soluble sugars and antinutritional factors like phenols and tannins contributed to the tolerance of chickpea genotypes against C. maculatus damage and development.
 
NBeG 452, NBeG 1129 and ICC 86111 were among the 20 chickpea genotypes tested for resistance to C. maculatus, found to be less susceptible due to lower numbers of eggs and adults per 100 grains, a lower percentage of grain damage. The resistance of chickpea to C. maculatus was influenced by genotypes with lower test weight, thicker seed coats, lower total soluble sugar levels and greater levels of phenols and tannins.
 
It is very helpful for post-harvest management and for subsequent genetic development of the genotypes to screen host grains of various genotypes for their resistance to stored grain pests. It is clear from the research that the physical characteristics of grains, such as a thicker seed coat and a smaller size, had a greater impact on the pulse bruchid resistance reaction than the biochemical components. Therefore, combining both antixenosis and antibiosis mechanisms of resistance genetically could be a feasible technique for reducing bruchid infestations in stored grain legumes. 

The authors would like to thank Dr. V. Jayalakshmi, Principal Scientist (Chickpea), Regional Agricultural Research Station, Nandyal, Andhra Pradesh for providing the genotypes to conduct these experiments. The authors are thankful to Acharya N. G. Ranga Agricultural University, Guntur, Andhra Pradesh, India for providing necessary facilities to undertake the studies.

All authors declared that there is no conflict of interest.