African Crop Science Journal, Vol. 9. No. 2, pp. 421-430
The effect of seed coat on the susceptibility of faba bean to Callosobruchus chinensis L.
Kemal Ali and R. H. Smith1
(Received 5 October, 1999; accepted 11 December, 2000)
Code Number: CS01024
In an earlier study, Kemal Ali and Smith (1996) demonstrated that the larvae of Callosobruchus chinensis L. suffer high mortality in most faba bean (Vicia faba L.) varieties tested. It was also evident that the percent adult emergence was a sensitive index for measurement of seed resistance. As hatched larvae have to physically penetrate the seed, the seed coat surrounding the grain could be a significant barrier to the insect and therefore provides useful source of resistance. In some cases features of the seed coat have been associated with difficulties experienced by insects in entering the seeds. For instance, Podoler and Applebaum (1968) observed that the thickness of seed coat of broad beans was the main factor limiting penetration by the larvae of C. chinensis. Brewer and Horber (1986) also reached a similar conclusion. Boughdad et al. (1986) reported that the first larvae died due to condensed tannins present in the tegument of V. faba.
The inability of C. chinensis to develop in soybeans is attributed mainly to the high protein carbohydrate ratio of the seed (Applebaum et al., 1968) and, in part, to its saponin content (Applebaum et al., 1965). The failure of this species to develop in haricot beans is partly due to the toxic pentosans present (Ishii, 1952). However, faba beans have been shown to be free of the neurotoxic amino acid B-lyanoalanine and its glutamyl peptide, which are common in many other species of Vicia (Bell and Trimanna, 1965), and lack a trypsin-imhibitor activity as well (Brochers et al., 1974).
This investigation was designed to enhance further understanding of the mechanism of resistance exhibited by faba bean varieties to seed penetration by C. chinensis and to investigate the role of the seed coat in resisting larval penetration.
MATERIALS AND METHODS
Two strains of C. chinensis were used in experiments collected in Kenya and Indonesia respectively. The initial stocks of the insects used in this study were obtained from the Natural Resources Institute (NRI) storage laboratory at Slough, UK. Four faba bean varieties were received from the Ethiopian Agricultural Research Organisation, 4 from Shambat Research Station, Sudan and 4 from Field Crops Research Institute, Egypt. These varieties are listed in Table 1.
The experimental grains were disinfested at a temperature of - 18°C for a week (Brewer and Horber, 1986). Grain moisture content and temperature were allowed to equilibrate for two weeks prior to the experiment in a constant temperature and humidity (CTH) room at 30°C and 70% relative humidity. At the end of this period the moisture content was determined by "one stage oven determination method" (Anon., 1980).
The rearing procedures of insects were similar to that of Giga and Smith (1983). The insects in stock culture were reared on an unknown faba bean variety obtained from a local food store, previously sterilised by freezing at 18°C. About 100-200 unsexed adult insects were placed in 850-ml jars containing approximately 400 g of seeds. The jars were sealed with filter paper stuck down with paraffin wax and kept in a thermo-statically controlled Griffin cooled incubator at 30°C.
Under this condition a new generation emerged within four to five weeks. Following the same procedure, new stock cultures were started as soon as each new generation of adults emerged. Newly emerged adults (0-24 h) were used in the experiment. In order to obtain the adults, the culture medium was sieved to remove the beetles therein and the insects that emerged the following day were collected for the experiment.
Three healthy seeds of each variety were exposed each day to a single mated female beetle for four days. Each day, adults were transferred to fresh seeds. After four days, the parental insects were removed and the seeds were kept in a CTH room at 300C and 70% RH. This experiment was replicated two times. Five days after infestation when the eggs had hardened, the total number of eggs laid on each seeds in each vial was counted using a Wild binocular microscope at a magnification of x12. Unhatched and excess eggs were removed from the seeds, so that each seed bore ten or less hatched eggs. This action was intended to prevent larval competition when crowded on a single seed. The remaining hatched eggs left on the seeds constituted the potential number of adults expected to emerge in each vial.
When emergence windows were seen, daily observation was made for progeny emergence. Number of eggs laid, time taken for development, number of adults emerging, and adult beetle body weight were recorded. Fifty days after infestation, the experiment was discontinued to avoid the possibility of including second generation insects. Based on cumulative total adults emergence and the initial number of eggs the percentage adult emergence was computed. The mean period for development was also calculated.
The effect of the seed coat on C. chinensis was assessed by taking off the seed coat from all the varieties. Decortication of the grains was done with the help of a sharp scalpel and a pair of forceps. Seed coat thickness was measured with a micrometer on five representative samples of the testa and these were further divided into five subunits from each representative sample of each variety. The insects were weighed accurately (±0.005 mg) on electronic balance (Cahn Model G.).
The susceptibility index (Dobie, 1974) was computed using the following formula:
I = LogeY x 100 t
Data collected were subjected to analysis of variance using the Statistical Analysis System (SAS Institute Inc., 1988).
RESULTS AND DISCUSSION
Total number of eggs laid. The differences in oviposition were not statistically significant among varieties (P<0.05) nor between the two strains (P<0.05). In general, the Indonesian females had higher fecundity than the Kenyan strain (means of 53.3±3.01 and 36.4±2.30 eggs per female, respectively). The oviposition of C. chinensis strains during the four days with and without seed coat is shown in Table 2.
The removal of the testa of faba beans increased the fecundity of the Kenyan strain and the differential response on whole beans was non-significant in all varieties when decorticated. In the present study, the decorticated grains of all the varieties (except Giza 402) proved to be more preferred by the Kenyan strain for egg laying than the respective whole grains. On average, the Kenyan strain laid a mean of 24.5 eggs on whole beans and this was increased to 34.6 eggs when offered decorticated seeds. These results are in contrast to those of Singh et al. (1980), who reported that among 56 varieties of 7 pulses, whole grains were more preferred by C. chinensis and C. maculatus for egg laying than decorticated grains. Similarly, Gokhle and Srivastava (1973) reported that C. maculatus laid 13 times more eggs on the whole grains than on decorticated ones. El-Sawaf (1956) reached similar conclusions, indicating thereby that the testa was the most important factor for egg-laying stimulus by both species.
The Indonesian strain behaved differently and showed no preference except for faba bean varieties Kuse-227-33 from Ethiopia and Giza 402 from Egypt for coatless seeds and more eggs on seeds with intact seed coat (Table 2). This strain laid on average 53.3 eggs on whole grains and 34.9 eggs on decorticated seeds of faba beans.
F1 adult emergence. The analysis of variance (square root transformation) showed no significant difference between C. chinensis strains and there was no indication of any interaction between varieties and strains. But there were significant differences (P< 0.01) among the decorticated faba bean varieties in terms of the number of emerging adults.
The highest number of adults of the Indonesian strain emerged from var. Giza 402 (mean 27.0±1.10) and the lowest from Selaim (mean 3.7±0.53). For the Kenyan strain, var. Giza 402 (mean 27.5±1.64) had the second highest number of adult emergence and correspondingly, the fewest adults were recorded from Selaim. Among the decorticated faba bean varieties, Kasa, Hudieba-72, Bf 2/2, Giza 3, Giza 402 and 123A 45/76 were relatively susceptible to both strains. The remaining varieties showed varying levels of resistance (Table 3).
When the percentage survival from egg hatch to emergence is considered on different decorticated varieties, no difference was detected among the two strains. Mean percentage emergence on each variety, with and without seed coat, of the two strains is given in Table 4. Both strains exhibited <30% survival to adult emergence on faba bean varieties, whereas >50% larvae survived in Californian cowpeas used as a susceptible check (Kemal Ali and Smith, 1996). Thus, it appears that neither strain is well adapted to survive on the faba bean varieties tested. However, irrespective of the strain, the percentage of larvae entering faba beans that completed their development and emerged as adults was greater in decorticated seed strains compared with whole seeds. The role of the seed coat in influencing the penetration of larvae is demonstrated by the following: seeds without testa were suitable while whole grains restricted the entry of the first instar larvae. Similarly, the decrease in emergence in whole seeds reveals that the larvae were unable to penetrate as a result of either seed coat thickness or the presence of suppressive compounds.
Sixty four percent of all hatched eggs of the Kenyan strain yielded adults in the decorticated seeds compared with only 6.4% on whole seeds. The corresponding figures for the Indonesian strain were slightly higher at 70% and 14.2%, respectively (Table 4).
Clearly, the 5- and 10- fold difference in percent emergence between whole and decorticated faba bean varieties infested by the Indonesian and Kenyan strains, respectively, demonstrate the role of the seed coat in the resistance of faba bean seeds to C. chinensis infestation. The comparative resistance of these varieties can be attributed to the seed coat, as development was very successful on decorticated seeds. Although in our case the seed coat has been shown to be detrimental to larval development of C. chinensis, it does not account for the total lack of larval development in some varieties of beans (e.g., Variety Selaim), which still showed a high level of resistance without seed coat. However, the most remarkable results concern the resistance of vars. NC-58 and CS-20DK (both from Ethiopia) to the Kenyan strain in the whole seeds not a single larva completed its development but on the decorticated seeds, 45.0 % and 76.6% of the larvae completed their development and emerged as adults (Table 4). This is in notable contrast to the report by Singh et al. (1980) that the removal of testa in red gram slowly brought down the development and growth of C. chinensis. However, the results of the present study collaborate those of Podoler et al. (1968), Gokhale (1973) and Brewer and Horber (1983) who documented varietal difference in the susceptibility of decorticated faba beans to damage by C. chinensis and observed better growth of this species in coatless varieties than whole seeds.
The lack of significant correlation (r = -0.11, P= 0.001) between seed coat thickness and percent adult emergence as a result of the Kenyan strain infestation is contrary to other findings (Podoler and Applebauem, 1968; Nwanze and Horber, 1976). This may be due to biochemical antibiosis of coats, but not thickness, which governs the mechanism of partial resistance in faba beans. The correlation coefficient (r =-0.816) between seed coat thickness and percent emergence was negative and statistically significant for the Indonesian strain.
The factor conferring antibiotic effect of seed coats merits further investigation. Features of the seed coat have been associated with difficulties by C. chinensis in entering seeds. Nwanze and Horber (1976) showed that the orientation of cells in testa of cowpea affected the ability of hatching C. maculatus larvae to bore into the seeds and the orientation of these cells varied among different cowpea varieties.
Developmental period. The developmental period (time taken from oviposition to adult emergence) was significantly different between C. chinensis strains ( P<0.001), faba bean varieties and interaction between the main effects (P<0.001). On average, the time from oviposition to emergence in decorticated faba bean seeds was 26.5 days (range 25-29). Although there was a difference between the two strains, the difference was small and probably of little importance. The developmental period of Indonesian strain was fastest on varieties NC-58, Kuse-227-33 and Kasa and slowest on Selaim, Giza 2 and Giza 3. The development of the Kenyan strain took significantly longer (mean 31.2±2.01 days) on Selaim than on any of the other 11 varieties. For the Indonesian strain, mean development time ranged from 24-28 days while it was 26-31 days for the Kenyan (Table 5).
The emergence was completed in 32 days and over 62% of the adults emerged within the first 4 days for the Indonesian strain. On the other hand, the emergence of the Kenyan strain was extended up to 35 days and less than half (38%) of the adults emerged within the first four days. The results of this experiment showed that the development of Indonesian strain was faster on decorticated grains than on whole seeds, which is in contrast to Singh et al. (1980) who reported that decortication of red gram resulted in an increase of six days in the development period of C. chinensis. Dina (1971) also observed prolonged development time of this pest after decortication. In general, beetles of the Indonesian strain consistently required a lower amount of time than the Kenyan to emerge from both whole and decorticated grains of faba beans. The extent of damage to stored seeds depends upon the number of emerging adults during each generation and the duration of each life cycle. Therefore, seeds permitting more rapid and higher level of adult emergence will be more extensively damaged.
Progeny adult body weight. The mean female and male adults body weight of C. chinensis for each variety and strain are shown in Table 6. There were highly significant differences between strains (P<0.001), between varieties (P<0.001) and their interaction (P<0.001). The female beetles of the Indonesian strain were heavier than the Kenyan strain with average weights of 3.67 mg and 2.95 mg, respectively.
The female body weight of the Indonesian strain in different varieties ranged from 3.22 mg (var. CS-20DK) to 3.85 mg (var. Giza 2) while weights of the Kenyan strain ranged from 2.52 mg (var. Selaim) to 3.22 mg (var. Giza 2). Therefore, the adult females reared on var.Giza 2 were the heaviest for both strains (Table 6). The Indonesian male adults that developed on decorticated seeds were also bigger in size and heavier in weight compared with those of the Kenyan strain. In the case of adult male beetles, average weights of 2.68 mg and 1.95 mg were observed for the Indonesian and Kenyan strains, respectively. Of the varieties tested, var. 123A 45/76 (2.50 mg) and var. Giza 402 (1.79 mg) had the lowest progeny weight, and the adults reared on Giza 2 (2.86 mg) and Selaim (2.31 mg) produced the heaviest adult beetles of the Indonesian and Kenyan strains, respectively (Table 6).
Overall, the Kenyan strains reared on all varieties of decorticated seeds produced significantly lighter progeny weight. For the Indonesia strain, body weight was 2.69-3.28 mg (male) and 3.63-4.78 mg (female) on whole seeds, while it was 1.95-2.68 mg (male) and 3.22-3.85 mg (female) on decorticated grains. The weights of adult beetles, both male and female of the Indonesian strain were reduced in decorticated seeds, whereas there was no marked effect of decortication on weight of the Kenyan strain. A mean body weight of adult C. chinensis of 3.00 mg (male) and 3.85 mg (female) on whole and 3.13 mg (male) and 3.61 mg (female) on decorticated cowpea seeds were recorded by Singh et al. (1980). According to these authors, there is no appreciable difference in adult body weight on whole and decorticated grains. Their results agree with those for the Kenyan strain but contrast with those for the Indonesian strain in our experiment. These inconsistencies could be due to genetic differences between the strains. Yadav and Pant (1978) reported that high food utilisation value led to high weight of C. chinensis adults.
Susceptibility Index (SI). Table 7 shows the relative susceptibility of the 12 faba bean varieties with and without seed coat. Susceptibility indices increased from 7.2 to 12.3 (Kenyan) and 10.9 to 13.3 (Indonesian) when averaged over varieties. For the Indonesia strain, the effect of decortication on the SI was maximum in variety Giza 402 (16.1), Kuse-227-33 and Kasa (16.0). The minimum recorded for the same strain was 8.9 from variety Selaim and Giza 2, whereas decortication of variety Hudieba-72 and MB 9/3 had no effect on the SI indicating that these two varieties remained susceptible as both whole and decorticated grains. For the Kenyan strain, the lowest SI was 5.7 from variety Selaim and the highest was 14.6 from variety Giza 2, the remaining varieties had similar values. However, decortication of variety BF /2/2 and Giza 3 had no effect on SI.
The most remarkable differences were observed on NC-58, CS-20DK and Hudieba-72 where the SI for the Kenyan strain increased from zero to 12.6. Susceptability index of the Indonesian strain did not markedly improve on decorticated grains compared with whole seeds, especially when compared with the Kenyan strain. Therefore, the protection confered by seed coat is only effective against some strains of C. chinensis, but this is also dependent on faba bean variety.
Physical barrier in faba bean to C. chinensis attack provides efficient protection against population growth and dispersal. However, this depends on the strain and faba bean variety. The oviposition data obtained from this study indicated that the Indonesian strain was more fecund on coatless seeds than the Kenyan strain as indicated by the overall mean of 53.3 and 36.4 eggs per female, respectively. The removal of the testa increased the rate of egg lay of the Kenyan strain irrespective of the faba bean variety.
The results also demonstrated that the percentage of larvae entering faba bean varieties and completing their development and emerging as adults in decorticate seeds of both strains was greater than for the whole seeds. Therefore, the comparative resistance of these varieties might be due to the properties of seed coats or biochemical antibiosis as development was very successful on coatless seeds. There was minimum effect of decortication on the susceptibility index in the case of Indonesian strain whereas the removal of the testa increased the susceptibilty of faba beans to the Kenyan strain. Although the seed coat has been shown to be a factor of resistance in faba beans, it does not account for all the resistance to C. chinensis development since some varieties still showed a high level of resistance with or without seed coat.
The authors wish to thank Mr. Jonathan Levin for his advice on the statistical analysis. Financial support to the senior author was provided by ICARDA/IFAD, for which we are grateful. The faba bean varieties were provided by the Ethiopian Agricultural Research Organisation (Ethiopia), Field Crops Research Institute (Egypt) and Shambat Research Station, Sudan.