Resistance to Callosobruchus maculatus among cowpea (Vigna unguiculata (L.) Walp) genotypes

ABSTRACT Callosobruchus maculatus is an important field-to-store pest that causes close to 100% losses in stored produce. This study aimed at assessing the resistance of cowpea genotypes to C. maculatus infestation. Thirteen cowpea advanced breeding lines pre-selected on the basis of their high grain yield, and three released varieties (Padi-tuya, Kirkhouse Benga and Wangkae) were evaluated in a no-choice experiment. The experiment was laid out in a completely randomized design with four replications under laboratory conditions. Data were collected on mean number of eggs per seed (MEPS), days to first emergence (DFE), adult bruchid emergence (ABE), percentage weight loss (PWL), percentage seed damage (PSD) and median development period (MDP). Dobie’s susceptibility index (DSI) was computed and used to classify the genotypes as resistant or susceptible. Significant differences were found among the genotypes for the measured traits, except for DFE and MDP. IT13K-1070-2 had the lowest MEPS, ABE, PWL and PSD. C. maculatus, with high ABE, developed best on Kirkhouse Benga and Wangkae. Wangkae had 100% seed damage at the end of the experiment. Based on the DSI score, IT13K-1070-2 was found to be moderately resistant; the rest of the genotypes were susceptible. Among the susceptible genotypes, IT10K-817-3, IT07K-303-1, SARI-2-50-80, SARI-3-11-100, IT10K-837-1, IT13K-1424-12 and SARVX-09-004 had PWL of less than 16%, indicating tolerance to bruchid infestation. DSI was significantly and positively correlated with ABE (r = 0.81), PSD (r = 0.93) and PWL (r = 0.79). DSI had a significant negative correlation with the final seed weight (r = −0.83). IT13K-1070-2 could serve as a useful source of resistance to the cowpea bruchid in cowpea breeding programs or released as a variety for cultivation.


Introduction
Cowpea is grown for its multipurpose uses, such as food for humans, fodder for livestock and atmospheric nitrogen fixation. It plays a critical role in the lives of millions of people in Ghana and other developing countries, where it is a major source of dietary protein that nutritionally complements lowprotein staple cereals and tuber crops. It is also a valuable and dependable commodity that provides income for farmers and traders (Singh et al. 2002;Langyintuo et al. 2003).
The mean yield of cowpea (492 kg/ha) on farmers' fields is lower than its potential yield. This is because of a host of biotic and abiotic factors, such as diseases, insect pests, parasitic weeds, poor soils and inappropriate plant density (Togola et al. 2017). Cowpea is attacked by numerous insect pests at every growth stage. The cowpea weevil (Callosobruchus maculatus) is a field-to-store pest of cowpea. Damage by this pest is caused by oviposition on the surface of the grains and subsequent larval penetration into the grains. The attack causes grain weight loss, a decrease in retail, quality and nutritional value, a reduction in the level of product hygiene and seed germination rate (Faroni and Sousa 2006). Because of the short life cycle and reproductive capacity of the insect, the losses could be up to 100% within six months of storage in untreated produce (Mkenda, Mtei, and Ndakidemi 2014). Hence, a need exists to manage weevil infestation.
The growing concern about the possible harmful effects of pesticides, such as toxicity to applicators, environmental pollution, and the presence of residues in foods, has necessitated studies on alternative control strategies for this pest, including the use of genetically resistant cultivars (Lara 1991;Panda and Khush 1995). The use of resistant genotypes is a promising strategy for the management of C. maculatus. This is because it maintains the population of C. maculatus below the economic damage threshold without any environmental effects. It does not require specific knowledge by the farmer, it is cost-effective, and it is compatible with other strategies of control. Furthermore, it is in accordance with the integrated pest management philosophy (Smith 2005; Vendramim and Guzzo 2009) Efforts have been made to identify cowpea bruchid-resistant genotypes from existing cowpea germplasm. The International Institute of Tropical Agriculture (IITA) screened 8000 germplasm accessions, out of which only three (0.004%) were found to be resistant (TVu-2027, TVu-11,952 and TVu11953) (Singh, Singh, and Adjadi 1985). Castro et al. (2013) identified three resistant genotypes, namely, IT85-F-2687, MN95-841-B-49 and Sanzi Sambili, in a screening exercise of cowpea genotypes in Brazil. An evaluation of accessions in India revealed the presence of two resistant lines (IC107466 and IC106815) (Tripathi et al. 2012). Similarly, 145 cowpea accessions were subjected to a no-choice bioassay in Uganda and some resistant genotypes were identified (Miesho et al. 2018). Identification of bruchid-resistant cowpea will be an important step toward the development of cowpea varieties with resistance to this insect pest. Candidate cowpea genotypes are rarely screened for resistance to C. maculatus before release. It is therefore important to screen cowpea genotypes for resistance to this post-harvest insect pest to inform decisions on post-harvest storage of cowpea to prolong its shelf life. Hence, the objectives of this study were to identify cowpea genotypes that are resistant to C. maculatus and to determine the relationships among C. maculatus resistance traits.

Experimental site and Callosobruchus maculatus culture
The experiment was carried out at the Entomology Laboratory of the Council for Scientific and Industrial Research-Savanna Agricultural Research Institute (CSIR-SARI), Nyankpala, Ghana. Insects were reared following the procedure described by Swella and Mushobozy (2007). Briefly, adult insects were obtained from an infested sample of cowpea. Prior to infestation, grains were frozen for 48 h and oven-dried at 60°C for 24 h. Several adult males and females were placed in jars containing 500 g of the cowpea grains to allow for rapid increase in bruchid culture. Adults were removed after two weeks and the jars containing cowpea with eggs were incubated at a temperature of 27 ± 3°C and 50% -70% relative humidity. After 30 days, the young C. maculatus cohorts were sieved out and used for the trial.

Experimental design and treatment
The experiment was laid out in a completely randomized design (CRD) with four replications (Table 1). The treatments were the sixteen (16) cowpea genotypes described in Table 1. Seed grains of each cowpea variety were infested with five males and females of adult C. maculatus in each setup.

Experimental procedure
One-hundred grams (100 g) of each cowpea variety were weighed to obtain their initial weight and were placed in each experimental jar before infestation. Prior to sampling, grains were frozen for 48 h and oven-dried at 60°C for 24 h. Each set-up was infested with C. maculatus obtained from the stock culture. Five (5) pairs of newly emerged adult males and females were selected and introduced into each set-up. A duration of 48 h was allowed for mating and oviposition on the seeds, after which adults were removed with the aid of a fine-mesh sieve. Individual insects were picked and placed in the experimental jar containing the grains using a fine camel hairbrush. After infestation, the experimental glass jars were covered with perforated lids lined with a piece of fine nylon mesh cloth. Each experimental jar was tightly closed and well labeled. They were placed under laboratory conditions and maintained at a constant temperature of 27 ± 3°C and 50% -70% relative humidity.

Data collection
Before infestation of the seeds with C. maculatus, the initial seed weight of the cowpea genotypes in each replicate was recorded. One week after infestation, 10 seeds were randomly selected, and the number of eggs laid was counted and recorded as described by Lambert et al. (1985). The set-ups were examined daily and the number of days to first emergence (DFE) recorded. The number of adults emerged from the eggs was recorded weekly for eight weeks; each experimental unit was sieved with a fine-mesh sieve into a large container. The sieved weevils were frozen for three to five minutes to immobilize them for easy counting. Inactive weevils were divided into 10 equal proportions and 3 parts, counted three times. The mean was multiplied by the total number of divisions to obtain the total population. Weight loss as a result of bruchid damage was calculated using the following formula: Grain weight loss = Initial weight -Final weight. This was recorded every week till the end of the experiment. At the end of the experiment, 10 seeds were randomly selected from each treatment and the numbers of damaged and undamaged grains were recorded. The percentage seed damage (PSD) was calculated using the formula: Percentage seed damage (PSD) = Nd NdþNu ð Þ ×100 where Nd = Number of damaged grains Nu = Number of undamaged grains Dobie Susceptibility Index (DSI) was calculated for each genotype using the data on adult bruchid emergence at the end of the experiment and median development period (MDP) using the formula: where Log e = Natural logarithm of numbers; F 1 = total number of emerged adults and MDP = median development period in days (Dobie 1974). The following susceptibility index rating was used in categorizing the cowpea genotypes into resistant/susceptible classes, where 1-5 = resistant; 6-10 = moderately resistant; 11-15 = susceptible; and >16 = highly susceptible (Chakraborty, Mondal, and Senapati 2015). A repeated measures analysis (RMA) was undertaken using SAS analytical software (Version 9.4-SAS Institute, Cary NC). The Proc mixed statement was used. Treatment means were separated using Duncan Multiple Range Test at 5% probability level. A Pearson's correlation analysis was carried out using R version 4.0.3 (RStudio Team 2021) to determine the nature of association among the measured traits.
Percentage seed damage (PSD) was significantly influenced by genotypes (p < 0.05) ( Table 3). The released varieties Wangkae, Kirkhouse Benga and Padi-tuya had high percentage seed damage (Table 7). IT13K-1070-2 had the lowest PSD, which differed significantly from that of the other genotypes (p < 0.05). MDP among the genotypes was statistically similar (p > 0.05) ( Table 2) with values ranging from 27 to 32 days ( Figure 1) The distribution of the DSI scores is presented in Figure 2. DSI among the genotypes was statistically dissimilar (p < 0.05). IT13K-1070-2 had the lowest DSI score that differed significantly from that of the other genotypes. Wangkae, Kirkhouse Benga and Padi-tuya had relatively higher DSI scores than the other evaluated genotypes (Figure 2). The DSI scores of the evaluated genotypes ranged from 6.95 to 26.65 (Figure 2).

Association among C. maculatus resistance parameters
Using the eight resistance parameters, 28 associations with their Pearson's linear correlation coefficient were generated (Table 8). FSW was significantly negatively correlated with PWL, PSD, ABE, and DSI. PWL was positively and significantly correlated with PSD, ABE and DSI. PSD was positively and significantly correlated with ABE and DSI. ABE was significantly and positively correlated with DSI (Table 8).

Discussion
The significant differences with respect to oviposition among the cowpea genotypes are indicative of the existence of genetic variability in the evaluated genotypes and the insects' preference of some genotypes for oviposition. Low egg load was obtained for IT13K-1070-2, IT86D-610, SARI-3-11-100, SARI-6-2-6 and IT07K-303-1, which varied in seed size, seed coat color and texture. This was probably due to the presence of oviposition deterrent biochemical factors (Sharma and Thakur 2014;Cope and Fox 2003), which are the main factors of antixenosis. No relationship was found between seed coat color and oviposition, which affirmed the conclusions of Edde and Amatobi (2003) and Cruz et al. (2016), but it was contrary to the findings of Asiedu, Powell, and Stuchbury (2000) and Gbaye and Holloway (2011). This could be due to genotypic differences, insect population used and the  environmental conditions under which the experiments were conducted. Significant variation observed among genotypes with respect to oviposition suggested that some of the genotypes tested were able to deter egg-laying. Further work on the mechanisms involved is needed. IT13K-1070-2 and IT86D-610 had prolonged days to bruchid emergence. This according to Togola et al. 2017) is a property of antibiosis, where compounds, such as 7S vicilins, α-amylase inhibitor, E-64 cysteine protease inhibitors, retard the development of the insects, and delayed emergence. These genotypes probably contained these compounds.  Figure 2. Dobie's susceptibility score of the different cowpea genotypes. Significant variation among different genotypes of cowpea artificially infested with bruchid has been reported by several authors, reinforcing the point that different genotypes respond differently to C. maculatus infestation. The number of adult C. maculatus emerging from cowpea grains determines the extent of damage caused (Badii, Asante, and Sowley 2013;Mogbo, Okeke, and Akunne 2014). Cowpea genotypes with the highest adult emergence had the highest grain weight loss and susceptibility index in this study. The relatively high DSI scores recorded for most of the genotypes were indicative of the high susceptibility of the evaluated genotypes. Except for IT13K-1070-2, the remaining genotypes were suitable hosts for the bruchids. IT10K-817-3, IT07K-303-1, SARI-2-50-80, SARI-3-11-100, IT10K-837-1, IT13K-1424-12 and SARVX-09-004 had a PWL of less than 16% and did not significantly differ from IT13K-1070-2, which had a PWL of 0%. These genotypes could be considered tolerant to the bruchid, as explained in a study by Peterson, Varella, and Higley (2017).
In the present study, the highly significant positive relationship between DSI and ABE, PSD and PWL signified that the higher the ABE, PSD and PWL of a genotype the higher the susceptibility of the genotype to C. maculatus attack and vice versa. Amusa et al. (2014) and Tripathi et al. (2020) observed a similar pattern in their work. PWL, ABE and PSD can therefore be used as selection criteria for identifying cowpea genotypes resistant to C. maculatus.