Introduction

The genus Pisum L. has recently been classified into four distinct taxa of Pisum sativum subsp. sativum L., Pisum sativum subsp. abyssinicum A. Braun., Pisum sativum subsp. elatius M. Bieb. and Pisum fulvum Sibth. & Sm. (Smykal et al. 2015). The first two taxa are under cultivation as ‘garden pea’ or ‘field pea’ and ‘dekoko’ or ‘Abyssinian pea’, while the last two taxa include wild species (Smykal et al. 2015; Ladizinsky and Abbo 2015; Gebreslassie and Abraha 2016). The cultivated peas are important cool-season legumes and low cost protein sources compared to many animal products. Besides, they can fix atmospheric nitrogen to soil interacting with Rhizobium leguminosarum bv. leguminosarum as high as 51–71 kg per ha (Jensen 1996; Hauggaard-Nielsen et al. 2001), and they are crucial for rotation as cover and intercrop (Hauggaard-Nielsen et al. 2001). Cultivated peas are grown for their dry seeds and fresh-shelled green peas as food legumes, fresh vegetable as snap pea and fodder crop for animal feeding (Davies 1993). In 2016, the cultivated pea occupied a sowing area of 8.1 million ha and supplied dry seed production of 16.2 million tons, while 20.7 million tons were produced as a fresh vegetable. The dry and fresh seed yields were given as 1991 kg and 7755 kg per ha, respectively (FAOSTAT 2019).

Many biotic and abiotic stress factors cause yield reduction in peas (Muehlbauer et al. 1993; Kraft and Kaiser 1993; Duan et al. 2014; Toker and Mutlu 2011). Pulse beetles are important biotic stress factors leading to substantial damage in stored peas (Smith 1990; Clement et al. 2002). In a typical life history of Bruchidae, larvae puncture into pea seeds after hatching and feed in the seed. Developing larvae pupate in the seed and emerge as adults to find a new host (Tuda et al. 2004). Callosobruchus chinensis L. is an aggressive bruchid species which attacks many legume plants, e.g., chickpeas (Sharma et al. 2013; Raghuwanshi et al. 2016; Eker et al. 2018), mung beans (Liu et al. 1998; Lee et al. 2000; Maharjan et al. 2018), faba bean (Podoler and Applebaltm 1968), and peas (Bhagwat et al. 1995; Singh et al. 2001; Umrao and Verma 2002; Duan et al. 2014; Sharma et al. 2016). The larvae of this species use a variety of dried legume seeds as host. Females lay their eggs directly on the surface of pea seeds singly and move on to either a different part of the seed or else to a different one depending on the pea compactness and competition among other females to lay more eggs. Each female can lay as many as 90 eggs during its oviposition period (Varma and Anadi 2010). The eggs usually hatch after 3–5 days and the first instar larvae burrow and feed on the endosperm, mining tunnels in the seed until pupation. Mature adults emerge from the pea seed, biting a neat circular exit from the seed approximately 30 days after hatching. The adult beetles live for up to approximately 2 weeks after emerging from the pupa (Varma and Anadi 2010; Neog 2012).

The beetle can be controlled with some fumigant and contact pesticides, biological and cultural applications although pesticide residues can remain on the seeds threatening human health and can also cause a decrease in germination rates (Shaheen and Khaliq 2005). The use of resistant varieties is an effective, healthy and low-cost way to control this insect. Resistant varieties are also much more useful for farmers than other pest control methods such as the use of pesticides, biological and cultural practices. Most recent studies have focused on searching for resistant plant varieties playing a pivotal role in pest management (Brewer and Horber 1984; Shaheen et al. 2006). Therefore, some studies have been conducted to assess the seeds of many leguminous species for resistance to different bruchid species (Bhagwat et al. 1995; Umrao and Verma 2002; Shaheen et al. 2006; Erler et al. 2009; Rajasri and Rao 2012; Sarwar 2012; Duan et al. 2014; Sharma et al. 2016; Raghuwanshi et al. 2016; Eker et al. 2018). In addition, some cultivated peas have also been tested for resistance to pulse beetles, but resistant sources were limited (Bhagwat et al. 1995; Singh et al. 2001; Umrao and Verma 2002; Duan et al. 2014; Sharma et al. 2016). However, wild species in the genus Pisum have not been evaluated for resistance to the pulse beetle and compared to the cultivated species. Hence, the aim of the present study was to evaluate different pea accessions of P. sativum, P. abyssinicum, P. elatius and P. fulvum for resistance to C. chinensis.

Materials and methods

Pisum taxa

In the present study, seeds of four Pisum taxa, two accessions of P. sativum L. (ACP 11 and ACP 15), two accessions of P. elatius (AWP 442 and AWP 449), two accessions of P. fulvum (AWP 600 and AWP 601) and one accession of P. abyssinicum (ACP 100), were evaluated for resistance to C. chinensis using both free-choice and no-choice test methods under laboratory conditions. The abbreviations in the names of accessions, for instance ACP and AWP, come from Akdeniz University cultivated Pisum and Akdeniz University wild Pisum, respectively. Detailed information on pea accessions is presented in Table 1. The reason for a small number of accessions in this study was that 210 accessions of four Pisum taxa had already been screened for resistance to pea weevil (Bruchus pisorum L.) under field conditions. This study was carried out only to compare resistant and susceptible accessions to pea weevil. Prior to screening, all of the accessions were maintained for 2 days in an incubator at 26 ± 2 °C and 65 ± 5% relative humidity (RH).

Table 1 Specific characteristics of seeds among Pisum taxa
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Callosobruchus chinensis

The insects were obtained from a laboratory culture of C. chinensis maintained for approximately 2 years in the Department of Plant Protection, Akdeniz University, Antalya, Turkey. Insect rearing was made with susceptible pea (P. elatius) seeds, which were the most preferred host, at 26 ± 2 °C and 65 ± 5% RH in complete darkness. To obtain the fresh adults of the beetle of the same age, seeds containing eggs were placed in clean jars filled with a large number of pea seeds. The jars were checked every day and adults of the beetles were collected for use in the experiment.

Resistance tests

Under laboratory conditions, the pea accessions were tested for resistance to the pulse beetle using two tests (free-choice and no-choice). The methods were previously reported by Raina (1971) and Dahms (1972) and subsequently elaborated by Erler et al. (2009) and Eker et al. (2018). In the free-choice test, all the accessions were exposed to pulse beetle adults freely in 20-L rectangular plexiglas cages. One side of the cages was covered with a gauze cloth to introduce the insects and to allow air circulation. Seven Petri dishes, each containing ten seeds of each separate accession, were put into each cage. A total of 140 insects (70♀ and 70♂) of 0–24-h-old adults collected from the maintained culture were released in each cage. Each cage was considered as one replicate and three replications were used for each accession in the free-choice test. The adult insects were allowed to oviposition for 1 week, and then the insects were removed. Oviposition in each Petri dish was checked under a stereo-microscope (25×) and the eggs laid were recorded for each accession separately. The cages were checked daily for adult emergence throughout 30 days, and emerging adults were recorded daily and removed from the respective cages. Counts lasted until the adult emergence ceased.

In the no-choice test, seeds of each accession were put in a separate glass jar (1 L) and ten seeds were used for each accession. Ten pairs (10♀ and 10♂) of 0–24-h-old adults of the beetle were released into each jar, and then they were covered with a gauze cloth to prevent the insects from escaping and to allow air circulation. The insects were not given any choice in the selection of an eligible host and were forced to feed only on the seeds of one accession. Each jar was considered as one replicate and three replications were used for each accession. After a 1-week oviposition, the adult insects were carefully removed. Then after, the same procedures were followed as in the free-choice test.

Data collection

Resistance to the pulse beetle was assessed by recording the number of eggs per seed (oviopozition), number of holes per seed (adult emergence), percentage of seed damage (damaged seed rates) and seed weight loss in each accession in both free-choice and no-choice tests. Damage to seeds was detected by the round holes with the ‘flap’ on testa (seed coat) done by the emerging adult beetles. Seed damage was counted as the damaged seeds for each accession in both free-choice and no-choice tests, and then converted into percentage as damage incidence according to Khattak et al. (1995):

$$ Damage\,incidence\,(\% ) = (Number\,of\,seeds\,damaged/Total\,number\,of\,seeds) \times 100 $$

The damage incidence was classified according to Eker et al. (2018): 0% = completely resistant or immune (no holes observed), 1–9% = resistant, 10–69% = moderately susceptible, 70–99% = highly susceptible, 100% = completely susceptible.

To determine weight loss caused by the beetle in the seeds of each accession, the initial weight of seeds (n2) prior to the tests and the weight of the damaged seeds (n1) after the tests were calculated using the following formula (Khattak et al. 1995):

$$ Total\,loss\,(\% ) = (n_{2} {-}n_{1} )/n_{2} \times 100. $$

Data analyses

The data were performed to analysis of variance (ANOVA) using MINITAB 16 software. For each parameter, significant differences between the accessions were determined using the Tukey’s test, and a probability value of P ≤ 0.05 was accepted as statistically significant.

The Principal Components Analysis (PCA) was subjected to determine the distribution of accessions based on the three parameters (number of eggs per seed, percent seed damage and weight loss) because number of holes or emergence of adults per seed was significantly correlated with percent seed damage and weight loss using XLSTAT.

Results

The ANOVA results for almost all traits tested showed that there were statistically significant differences between the pea accessions tested in the free- and no-choice tests (Fig. 1). However, no significant differences were detected for number of eggs per seed among the accessions in the no-choice test (Fig. 1a).

Fig. 1
figure 1

Number of eggs (a) and holes (b) per seed, percent weight loss (c) and seed damage (d) by C. chinensis in seeds of accessions in Pisum taxa in both free-choice and no-choice tests. Upper-case letters indicate compare the accessions in free-choice test (black bars) whereas lower-case letters indicate compare the accessions in no-choice test (white bars). Bars indicate means with standard errors and different upper- or lower-case letters are statistically significant (Tukey’s test; P < 0.05)

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Number of eggs per seed

Number of eggs (oviposition by the beetle on the seeds of pea accessions) was shown in Fig. 1a. In the free-choice test, cultivated peas generally had more number of eggs than the wild peas. The cultivated accession, ACP 15 had the highest number of eggs with 16.17 eggs per seed, followed by the other cultivated accessions, ACP 11 and ACP 100 (12.07 and 8.57 eggs per seed, respectively). The accessions of P. elatius were the second most preferred pea type by the beetle for the number of eggs; AWP 442 (8.14 eggs per seed) and AWP 449 (7.87 eggs per seed). The P. fulvum accession, AWP 601 had the lowest number of eggs with 3.84 eggs/seed, followed by the other P. fulvum accession, AWP 600 (4.97 eggs per seed).

In the no-choice test, the cultivated accession, ACP 100 had the highest number of eggs with 20.43 eggs/seed and followed by the P. elatius accession, AWP 442 (19.47 eggs per seed). The other accession of cultivated pea (ACP 15), and accession of P. elatius (AWP 449) had 16.57 eggs and 14.43 eggs per seed, respectively. While the two P. fulvum accessions (AWP 600 and AWP 601) had less number of eggs with 11.13 and 10.63 eggs per seed, respectively. The cultivated accession, ACP 11 had the less number of eggs with 9.0 eggs per seed (Fig. 1a).

Number of holes on seeds

In free-choice test, the highest number of holes per seed or adult emergence occurred in the P. elatius accession, AWP 449 with 0.4. On the contrary, accessions that no adult emergence occurred as follows: one accession of P. elatius (AWP 442), one accession of P. sativum (ACP 11) and two accessions of P. fulvum (AWP 600 and AWP 601). As for no choice test, the results had showed parallelism with of free choice test (Fig. 1b).

Seed weight losses

The highest weight loss was seen in the P. elatius accession, AWP 449 with 15.35% and 11.39% in the no-choice and free-choice tests, respectively. The second highest weight loss occurred in ACP 100 with 11.38% and 2.02% and followed by ACP 15 (2.29% and 2.96%) in both tests. As there was no adult emergence, there were no weight losses in the remaining four accessions in both tests (Fig. 1c).

Seed damage

The percent seed damage of the pea accessions tested was parallel to the number of adult emergence holes on their seeds in both the free-choice and no-choice tests (Fig. 1d). The highest damage occurred in the P. elatius accession, AWP 449 with 20% in both tests. The accessions, ACP 15 and ACP 100, had the same seed damage rate (3.3%) in the no-choice test, although in the free-choice test, ACP 100 exhibited a more seed damage (13.33.60%) than ACP 15 (10%). When the accessions were categorized according to the seed damage rates, the accessions AWP 449, ACP 100 and ACP 15 were determined as moderately susceptible due to having seed damage rates between 10 and 69% (Fig. 1d). Both accessions of P. fulvum (AWP 601 and AWP 600) were observed to be completely resistant to C. chinensis as they had less number of eggs and no seed damage. ACP 11 and AWP 442 were found to be resistant to C. chinensis with seed damage of 0% (Fig. 1d).

Principal components analyses (PCA)

According to the results obtained from the PCA analyses, accessions were definably divided into two groups as resistant and susceptible accessions in both of free- and no-choice tests (Fig. 2). When number of eggs per plant, percent of weight loss and seed damage were considered together, resistant accessions of P. fulvum and P. elatius had the lowest values in free-choice test (Fig. 2a), while resistant accessions of P. fulvum and P. sativum had the lowest values in no-choice test (Fig. 2a).

Fig. 2
figure 2

Principal component analyses (PCA) for pea accessions in free-choice (left) and no-choice (right) tests. Each dot represents an accession in the genus Pisum L

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Discussion

Resistance to C. chinensis of seven pea accessions belonging to four different Pisum taxa was evaluated for the first time through free-choice and no-choice test methods. The mean number of C. chinensis eggs on the seeds of the accessions varied from 3.83 to 16.17 in the free-choice test and from 10.63 to 20.43 eggs per seed in the no-choice test, indicating that all seeds were preferred by the beetle. The cultivated species had more number of eggs per seed than that of the wild species (Fig. 1a). The number of eggs on seed of accessions depended on seed size because seed size of the cultivated species were bigger than that of the wild species (Table 1). Although eggs were laid on seed surface of all accessions tested by the beetle, the difference in the level of damage was significant in the tested accessions according to the scale described by Duan et al. (2014), categorizing the percentage of seed damage. Therefore, these two methods were convenient for evaluation of C. chinensis resistance of peas in this study, which will contribute to mass screening of pea accessions to C. chinensis.

The results of the present study revealed that all the pea accessions screened under laboratory conditions displayed significant variation in expression of resistance to C. chinensis. There were significant differences between the accessions in terms of the number of eggs laid, number of emergence holes and seed weight losses of C. chinensis on the pea accessions tested (Fig. 1). Similarly, Duan et al. (2014) reported that among the tested P. sativum varieties only three varieties were found to be completely resistant or immune to C. chinensis. It was also reported that 12% of these varieties were resistant while 82% were susceptible and highly susceptible to C. chinensis (Duan et al. 2014). Bhagwat et al. (1995) screened 30 genotypes of P. sativum under laboratory conditions and reported that only JP Batri-Br-3 and JP-Br-4 were resistant to C. chinensis. In another study by Singh et al. (2001), 20 P. sativum genotypes were evaluated in respect of resistance to C. chinensis, and the genotypes KPMR 186 and Rachna were found to be moderately susceptible. Teotia and Singh (1966) tested the development and oviposition rates of C. chinensis on varieties with different seed shapes. The varieties with a smooth seed shape were seen to be more preferred for oviposition (Teotia and Singh 1966). The results of the current study are in parallel with those findings in terms of oviposition preference of the beetle.

Another important point was that pea varieties with black and brown seed color had significantly lesser number of eggs per seed by C. chinensis than varieties with yellow, dun and green colored seeds (Table 1 and Fig. 1). In the present study, the number of eggs, number of holes, weight loss and seed damage were found to be significantly (P < 0.01) correlated to green seeds, while black and brown seeds were negatively correlated with the same characteristics (Data not shown). Similar findings were obtained in a previous study by Duan et al. (2014). More recently, Eker et al. (2018) reported that chickpea accessions with dark colored seeds were less preferred than those with white colored seeds by the C. chinensis, and they had significantly lower oviposition rates. The differences in the seed color of pea accessions can be considered to have affected oviposition of the bruchid and especially the “unsightly” seeds of black accessions prevented bruchid females from laying eggs. All these results indicate that dark seeds are more resistant to C. chinensis. According to PCA analyses, resistant and susceptible accessions were definably categorized in two groups (Fig. 2). In general, wild species especially all accessions of P. fulvum were found to be resistant to C. chinensis (Fig. 1) as reported in wild Cicer species (Talip et al. 2018; Eker et al. 2018).

Taking into consideration the seed damage rates, both accessions of P. fulvum (AWP 601 and AWP 600), one accession of P. sativum (ACP 11) and one accession of P. elatius (AWP 442) were completely resistant or immune to C. chinensis since no holes were observed on their seeds. Three accessions (AWP 449, ACP 100 and ACP 15) were found to be moderately susceptible (Fig. 1). The accessions ACP 11 and AWP 442 were resistant to C. chinensis with seed damage of 0% (Fig. 1). Although the accession, AWP 449, had a lesser number of eggs per seed with 7.87 eggs than above-mentioned two accessions, it was categorized as moderately susceptible with a seed damage rate of 20% (Fig. 1a, d).

In conclusion, of the seven pea accessions screened for resistance against C. chinensis, it was determined that four accessions were resistant. Two accessions of P. fulvum (AWP 601 and AWP 600) were found to be completely resistant to C. chinensis as they had both less number of eggs and no seed damage. In addition to these two accessions, ACP 11 (P. sativum) and AWP 442 (P. elatius) were found to be resistant to C. chinensis with seed damage of 0% although they had much number of eggs per seed. The wild species of P. elatius and P. fulvum were identified as completely resistant and could be used in breeding studies for the development of resistant cultivars, and P. sativum (ACP 11) could be stored without the need to use of insecticides under storage conditions.