Elevated CO₂ and Nanoparticles for the Management of Pulse Beetle, Callosobruchus chinensis L. in Stored Chickpea 

India is the largest producer and consumer of pulses in the world accounting for higher area (33% of the world area), production (25% of the total global production), consumption (27% of world consumption) and importer (14%) of pulses in the world. Gram or chickpea is the most dominant pulse having a major share under area sown 65% and production 72% followed by lentil and field pea (Phadtare et al., 2023). Poor storability and lack of improved storage facility is one of the important service constraints leading to post harvest losses in case of pulses to the extent of 25-50 per cent (Jeswani and Baldev, 1990).  Pulse beetle, Callosobruchus chinensis L. (Coleoptera: Bruchidae) is widely distributed and known as a major destructive insect of stored chickpea (Park et al., 2003; Aslam, 2004). The economic loss of this bruchid in various pulses ranged from 30-40 per cent within a period of six months and when left un-attended, losses could be up to 100 per cent (Dongre et al., 1996; Akinkurolere et al., 2006; Sharma et al., 2013). It is reported that the pulse beetle may cause 10-95 per cent loss in the seed weight and 45.5-66.3 per cent loss in protein content of the seeds under normal condition and the severity of damage increases with the duration of storage condition. The germination of pulse seed is also reduced to a great extent (Yadav, 1985). The infestation causes considerable losses both in terms of quality as well as quantity. Both the grubs and adults causes damage and the endosperm were eaten by the grubs leaving only the thin outer covering or thin film of seed coat making them completely unfit for human consumption (Nishad et al., 2020; Krishnavedi et al., 2020; Dwivedi and Devi, 2019). Therefore, now-a-days, eco-friendly, non-chemical and bio-rational approaches in insect pest management during storage are being considered as effective alternative to the hazardous synthetic chemicals and have assumed greater significance (Balikai and Neenu, 2018; Neenu and Balikai, 2019). Botanicals, plant oils, nano emulsions, carbon dioxide treatments etc. are quite effective in keeping the pest damage under control, which possesses no harmful effect on the stored chickpea kept for consumption and seed purposes.

Effect of elevated levels of CO₂ on Callosobruchus chinensis
 
The experiment on modified atmosphere studies using elevated levels of CO₂ was carried out against C. chinensis in the Department of seed science and technology, OUAT, Bhubaneswar in specially designed airtight containers and the required concentration of CO₂ was released into these containers with the help of carbon dioxide cylinder fitted with an outlet tube containing a nozzle and a needle. Five pairs of freshly emerged adults were transferred to airtight plastic containers of 250 g capacity separately and directly exposed to five different concentrations of CO₂ viz., 20%, 40%, 50%, 60% and 80% with six different exposure periods viz., 30, 60, 90, 120, 150 and 180 minutes and each treatment was replicated thrice (Hasan et al., 2020). The chickpea seeds were observed after 2, 4 and 6 months of treatment for each concentration of CO₂ and the data on fecundity, adult emergence, seed damage and weight loss due to damage, moisture content and germination per cent were recorded and analysed statistically. An untreated check was also maintained by following the same procedure adopted for CO₂ studies without exposing the seeds to CO₂ concentrations.
 
Effect of nano particles against Callobruchus chinensis in stored chickpea
 
The experiment was conducted to evaluate the toxicity of nanoparticles on chickpea pulse beetle using dry dust application. 100 g thoroughly dried chickpea seeds of genotype JG 16 were taken and were treated with the nanoparticles and 5 pairs of freshly emerged pulse beetle adults were released on the treated pods at four different concentrations viz., 250 ppm, 500 ppm, 750 ppm and 1000 ppm per kg of seeds (Kawadkar et al., 2011). An untreated check was also maintained simultaneously. The study was carried out in a completely randomized design and each treatment was replicated thrice. Adult mortality was observed at 1, 3, 5, 7 and 10 days after exposure to the treated seeds. After studying the adult mortality, the chickpea seeds were placed in the petridishes and kept undisturbed till the emergence of F₁ progeny and the numbers of eggs laid and the number of adults emerged from each treatment were also noted. The observations on the fecundity, adult emergence, seed damage and weight loss due to damage, germination per cent was recorded and statistically analysed after appropriate transformation.

Effect of elevated levels of CO₂ on adult mortality
 
The mortality of C. chinensis adults exposed to various concentrations of CO₂ after 30 minutes of exposure period indicated that low concentration (20%) of CO₂ did not show any effect on adult mortality (Table 1). After 60 minutes of exposure, the mortality was found to be 13.33 per cent which substantially increased with increased exposure time. At 90, 120, 150 and 180 minutes of exposure, the mortality increased to 26.67, 43.33, 50.00 and 56.67 per cent respectively. Further, the elevated levels of CO₂ at 60 and 80 percent for 150 and 30 minutes respectively resulted in cent percent mortality of the bruchids. The present findings are partially in agreement with the results found by earlier researchers (Jyothsna, 2014; Kumar et al., 2017; Krishnaveni, 2012; Haile, 2015).


 
Effect of elevated levels of CO₂ on fecundity and adult emergence of Callosobruchus chinensis
 
It is evident from the results presented in Table 2 that among all the concentrations of CO₂, higher concentrations were very much lethal to the chickpea pulse beetle and exhibited in high mortality and inhibited egg laying during six months of storage. The lowest concentration (20%) of CO₂ was least effective and recorded 5.33, 21.67 and 49.67 eggs after 2, 4 and 6 months of storage respectively while at 20 per cent CO₂ concentration recorded 2.67, 12.33 and 31.67 eggs after 2, 4 and 6 months after storage respectively. The data pertaining to the effect of elevated levels of CO₂ on adult emergence of C. chinensis are presented in Table 3. The results indicates that higher CO₂ concentration of 50 per cent was effective initially for 2 months of storage with 0.67 number of adult emergence but it was further increased to 8.67 after 6 months of treatment. However, the untreated control recorded 41.67, 82.33 and 105.67 adult insects after 2, 4 and 6 months after treatment (MAT), respectively and all the treatments were significantly superior to control. The results are in line with the findings of Krishnaveni (2020) and Rustamani et al., (1985), who did not notice any fecundity and adult emergence of C. chinensis in pigeonpea seeds treated with 40, 60 and 80 per cent CO₂ during 6 months of storage.




 
Effect of elevated levels of CO₂ on chickpea seed damage and weight loss (%) infested by Callosobruchus chinensis
 
The observations pertaining to the studies on the effect of elevated levels of CO₂ on seed damage to stored chickpea subjected to artificial infestation with C. chinensis are presented in Table 4. From the data it isclear that higher concentrations of CO₂ viz., 60 and 80 per cent did not record any seed damage even after six months of storage, whereas in untreated control the seed infestation was found to be 28.31 per cent after 2 months of treatment and increased to 41.32 per cent after 4 months and further increased to 57.61 per cent at 6 months after treatment. The chickpea seeds subjected to infestation by C. chinensis and exposed to different concentrations of CO₂ were observed for weight loss after 2, 4 and 6 months of treatment (Table 5) which exhibited the similar trends as recorded with seed damage. The CO₂ concentration of 80 per cent was found to be lethal to the pulse beetle infestation and did not record any weight loss up to six months of storage. The lower CO₂ concentration of 20 and 40 per cent recorded 4.37 and 2.11 per cent weight loss after 2 months of storage, respectively. It was further increased to 11.63 and 7.22 per cent after 6 months of treatment, respectively which indicated that the lower concentrations of CO₂ were ineffective in protecting the stored chickpea for prolonged period. The results are in conformity with Kumar et al., (2017) who stated that high concentrations of CO₂ viz., 50, 60, 70 and 80 per cent did not witness any pod damage and weight loss even after 9 months of treatment. Similarly, Divya et al., (2016) reported that CO₂ concentration of 50 per cent and more not only checked seed infestation by C. chinensis but also reduced the weight loss of seed compared to the normal atmosphere.




 
Effect of elevated levels of CO₂ on germination (%) of chickpea
 
Table 6 indicates the chickpea seeds subjected to infestation by C. chinensis and exposed to different concentrations of CO₂ and were observed for germination after 2, 4 and 6 months of treatment, which exhibited the similar trends as recorded with seed damage and weight loss. The CO₂ concentration of 60 and 80 per cent were found to be comparatively better treatments and recorded 84.00 and 86.33 per cent germination of chickpea seeds at 2 months of storage. The germination observed in the untreated control was found to be 70.00 per cent after 2 months which further decreased to 54.67 and 42.33 per cent after 4 and 6 months of treatments, respectively. Similar results were also reported by various researchers (Raghupathi et al., 2021; Shivaraja et al., 2012; Paikaray et al., 2022).


 
Effect of nano particles on adult mortality of Callosobruchus chinensis
 
The data presented in Table 7 reveals that among the nano particle treatments, nano silica @ 1000 ppm was the most effective treatment which registered 72.67 per cent adult mortality after 1 day of treatment and cent per cent mortality was obtained 5 days after treatment (DAT). Likewise, nano zinc oxide @1000 ppm recorded 65.00 per cent mortality 1 DAT and complete adult mortality was observed at 7 DAT. Lower concentration of nano zinc oxide @250 ppm was least effective and recorded 21.93 per cent mortality at 1 DAT which increased to 68.67 per cent after 10 days of treatment. Similarly, nano silica @ 250 ppm recorded 27.33 per cent mortality which was further enhanced to 82.67 per cent at 10 DAT. Treatment with nano silica and nano zinc oxide @750 ppm manifested 95.67 and 90.67 per cent mortality at 7DAT, respectively and both the nano particles exhibited cent per cent mortality at10 DAT. The results are in line with Jyothsna (2014) who recorded nano silica 1000 ppm and 500 ppm to be highly effective and caused cent per cent mortality at 1 day after treatment.


 
Effect of nano particles on the fecundity and adult emergence
 
The data presented in Table 8 revealed that the higher concentrations (1000 and 750 ppm) of both the silica and zinc nano particles resulted in complete mortality of the adults within 2 to 6 months after treatment (MAT). The egg laying and emergence of adults were not observed from the above treatments. The lower concentrations of nano silica and nano zinc @ 250 ppm resulted in very few eggs laying (6.00 and 5.00 eggs respectively) by the pulse beetle at 2 months after treatment which further enhanced to 10.67 and 13.33 eggs at 6MAT respectively. The eggs laid by the test insect also could not properly develop in to adults. Treatment with nano silica @ 500 ppm recorded 4.67 eggs, whereas nano zinc oxide at the same concentration registered 6.00 eggs during 6 months of storage. Both the nano particles at 500 ppm were also found to be effective in preventing the development of the insect thus reducing the adult emergence at 6 MAT. These nano treatments were significantly superior to control where the adult emergence at 2, 4 and 6 MAT was 40.00, 67.33 and 91.67 adults, respectively. Jyothsna (2014) reported the superiority of nano silica and nano zinc @ 1000 ppm and 500 ppm concentrations in reducing the fecundity and adult bruchid emergence.


 
Effect of nano particles on the seed damage (%) and weight loss (%) in chickpea seeds
 
It is evident from the data presented in Table 9 that nano silica and nano zinc oxide @ 1000 and 750 ppm did not record any seed damage even after 6 months of storage. Among the different nano particle treatments, nano zinc oxide at 250 ppm was comparatively less effective which recorded 5.00 per cent seed damage at 2 MAT followed by nano silica treatment (3.67%) at the same concentration. Nano zinc oxide at 500 ppm recorded 2.00 per cent seed damage which was at par with nano silica treatment at the same concentration (1.33%) at 2 MAT. Nano zinc oxide at 500 ppm resulted in 5.44 per cent seed damage which was at par with nano silica at 500 ppm (4.33%) after 6 months of storage. Similarly, higher concentration of nano silica and nano zinc both at 1000 ppm and 750 ppm did not exhibit any loss in seed weight up to 6 months of storage as no seed infestation or damage was noticed. Among the different nano particle treatments, nano zinc oxide at 250 ppm resulted in 9.44 per cent of seed weight loss after six months of storage which was at par with nano silica at the same concentration (7.96%). Malaikozhundan and Vinodhini (2018) reported minimum per cent pod damage by Caryedon serratus (4.34) with nano ZnO particle treatment.

The exposure of adult pulse beetle to 60% and 80% CO₂ for 180 minutes resulted in a complete mortality of adults within one or two days of treatment. Exposing the chickpea seeds to a higher concentration of CO₂ not only checked seed infestation but also prevented the progeny development up to 6 months of treatment but did not affect the seed germination. Management of pulse beetle by nanoparticles @ 250, 500, 750 and 1000 ppm per kg of seeds showed the superior performance of the higher concentrations (1000 ppm and 750 ppm) of nano silica and nano zinc over other treatments which completely protected the chickpea seeds from oviposition, development and damage by C. chinensis.