Directory UMM :Journals:Journal of Stored Products Research:Vol 36.Issue1.Jan2000:

Journal of Stored Products Research 36 (2000) 89±99
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Chemical basis of resistance in pulses to Callosobruchus
maculatus (F.) (Coleoptera: Bruchidae)
S. Ignacimuthu*, S. Janarthanan, B. Balachandran
Entomology Research Institute, Loyola College, Chennai-600 034, India
Accepted 1 July 1999

Abstract
Seeds of various wild and cultivated pulses express varied degrees of resistance to damage by
Callosobruchus maculatus, a major pest of stored pulses. Seeds were analysed for trypsin/chymotrypsin
inhibitors (qualitatively and quantitatively) and protein pro®les (SDS-polyacrylamide gel electrophoresis)
to study the chemical basis of resistance to bruchid infestation. Seeds of the wild pulses Dunberia
ferruginea, Neonotonia wightii, Tephrosia cararensis, Cajanus albicans and Vigna bourneae di€er from
other wild germplasms as well as cultivars in having characteristically higher levels of trypsin/
chymotrypsin inhibitors. SDS-PAGE analyses of seed proteins of Dolichos lablab, Lablab purpureus and
Rhyncocea cana reveal the presence of high levels of non-nutrient chemicals such as vicilins, a-amylase
inhibitors, and lectins in quantities that may impart resistance to C. maculatus. The importance of these
protease inhibitors and other non-nutrient chemicals are discussed with reference to resistance to C.
maculatus infestation. # 1999 Published by Elsevier Science Ltd. All rights reserved.

Keywords: Pulses; Trypsin inhibitor; Chymotrypsin inhibitor; SDS-PAGE; Bruchid; Resistance

1. Introduction
Pulses are a good source of dietary proteins and other essential nutrients. However, postharvest insect infestation severely a€ects quality and storability of produce (Singh, 1978; Steele
et al., 1985). Pulses are known to contain a variety of non-nutrient compounds such as lectins,
proteinase inhibitors, a-amylase inhibitors and other compounds which could be part of an
array of constitutive defences against attack by stored grain pests, particularly those belonging

* Corresponding author. Tel.: +91-44-826-5542; fax: +91-44-826-5544.
E-mail address: erilc@tn.nic.in (S. Ignacimuthu)
0022-474X/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 2 - 4 7 4 X ( 9 9 ) 0 0 0 3 1 - 4

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to the family Bruchidae (Janzen et al., 1986; Gatehouse et al., 1990). While the exact nature of
resistance in certain pulses to Callosobruchus maculatus (F.) remains largely unknown, some
recent studies emphasize the presence of certain non-nutrient (storage) proteins in seeds of

resistant varieties as the basis of resistance (Macedo et al., 1993). In addition, trypsin and
chymotrypsin inhibitors, besides their well-known function as storage proteins, are also
important in imparting resistance (Liener and Kakade, 1980; Ryan, 1990). The ecacy of any
secondary (non-nutrient) compound is indicated by biological assay against the insect pest
(Agarwal et al., 1988). Several studies suggest the importance of biological assay to assess the
potential of such compounds to impart resistance (Gatehouse et al., 1989; Cardona et al., 1990;
Xavier-Filho, 1991; Pusztai et al., 1993; Macedo et al., 1995; Fory et al., 1996). In view of the
variation observed in damage due to feeding by C. maculatus on various wild as well as
cultivated pulses, the present study was planned to identify chemical factors that are
responsible for imparting resistance.

2. Materials and methods
2.1. Seeds
Seeds of the wild pulses were collected from the Palney Hills of the Western Ghats,
Tamilnadu, India (latitude 108; longitude 788; altitude 2010 m from MSL), whereas some
cultivated seeds (Cajanus cajan (L.) Millspaugh ICP14770 and ICP14990; Cicer arietinum (L.)
COG29 and CO3; Vigna unguiculata (L.) Walp. CO4 and V. mungo (L.) Hepper CO5) were
obtained from the Department of Plant Breeding, Tamil Nadu Agricultural University,
Coimbatore, India. The other cultivated seeds namely, V. unguiculata VCP8; V. mungo VBN1;
V. radiata (L.) Wilczek K851 and UGG4 were obtained from National Pulses Research Centre,

Vamban, Tamil Nadu, India.
2.2. Chemical analysis
Trypsin and chymotrypsin inhibitors were estimated following the methods described by
della Gatta et al. (1989) and Erlanger and Frances Edel Cooper (1996). They were expressed in
terms of units/g seed materials (a trypsin or a-chymotrypsin unit (TU or CU) is de®ned as the
amount of trypsin or a-chymotrypsin that catalyses the hydrolysis of 1 mol of substrate per
min. A trypsin or chymotrypsin inhibitor unit (TIU or CIU) is the reduction in activity of the
respective enzymes by one unit, i.e. TU or CU respectively.
2.3. Electrophoretic studies on trypsin and chymotrypsin inhibitors
Seeds were milled to a ®ne powder (after removal of the seed coat) and protease inhibitors
were extracted by incubating overnight in Tris±HCl±CaCl2 bu€er, pH 8.1 (at a ratio of 1:10 w/
v) at 48C. The homogenate (the ®ne powder with the extraction bu€er) was subsequently
centrifuged at 12,000 rpm at 48C for 15 min. The supernatant collected was used for
electrophoretic analysis with polyacrylamide gel electrophoresis (Native PAGE) as described by

S. Ignacimuthu et al. / Journal of Stored Products Research 36 (2000) 89±99

91

Kollipara et al. (1991). At the end of the run, the stacking gel was removed and the separating

gel stained for trypsin and chymotrypsin inhibitor activities based on Uriel and Bergas (1968)
with some modi®cations as described by Kollipara and Hymowitz (1992).
2.4. Seed protein pro®le
To analyse proteins of both wild and cultivar pulses, SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed according to the method of Laemmli (1970). Proteins were
extracted from powdered pulses with 0.1 M Tris±HCl bu€er, pH 8.0 (at a ratio of 1:10 w/v)
using sterilised glass powder in a mortar and pestle for 15 min at 0±58C (on ice). Extracts were
centrifuged at 15,000 g for 10 min at 58C. Aliquots of supernatant were used to quantify the
proteins using the Bradford method with BSA as standard (Bradford, 1976). An equal quantity
of each of the protein samples (100 mg) was applied to the gels. Standard molecular marker
proteins (Sigma) were simultaneously run for comparison.
Table 1
Trypsin and chymotrypsin inhibitor contents (units/g) in wild and cultivated germplasms1
Varieties

Trypsin inhibitors

Chymotrypsin inhibitors

29602 05.7a

27742 04.2b
27002 09.0b
18862 07.2c
10352 04.1d
8902 14.7e
5232 09.0f
4402 04.8g
3382 06.5h
3002 12.2i
1342 03.2j

4122 34.9a
4252 16.3a
3852 13.1b
2692 14.8c
1482 06.5d
1252 04.9e
1362 11.4de
62 2 14.7f
46 2 07.3gf

43 2 05.7g
22 2 07.3h

12382 02.5a
12332 36.2a
10752 33.6b
7002 25.3c
5752 16.3d
4182 01.2e
4082 10.7e
3352 01.8f
3332 18.0f
2892 09.8g

1532 6.0a
1772 5.6b
1522 3.4a
60 2 7.3c
82 2 2.5d
60 2 2.5ec

58 2 5.8ec
48 2 4.9f
0 2 0.0
41 2 6.5f

Wild pulses
Cajanus albicans
Dunberia ferruginea
Neonotonia wightii
Vigna bourneae
Tephrosia cararensis
Lablab purpureus(brown)
Rhyncocea rufescens
Rhyncocea cana
Dolichos lablab
Lablab purpureus (black)
Canavalia virosa
Cultivars
Cicer arietinum CO3
Cicer arietinum COG 29

Vigna unguiculata CO4
Vigna unguiculata VCP8
Cajanus cajanICP 14770
Vigna radiataUGG4
Cajanus cajan ICP 14990
Vigna mungo VBN1
Vigna radiata K851
Vigna mungo CO5

1
Values represent mean 2SE of four replications. Data analysed using Student's t-test (P < 0.01). Values in the
same column with the same superscript letters are not signi®cantly di€erent.

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2.5. Bioassay
A stock culture of C. maculatus was reared on green gram (V. radiata K851 cultivar) seeds
at 282 18C in glass jars covered with muslin cloth. For feeding bioassays, seeds were

conditioned at 282 18C and 70% r.h. for a period of 10 days before use. Seeds of each
cultivar/wild germplasm were kept in glass vials covered with muslin cloth and a single pair of
newly emerged adults of C. maculatus from the stock culture was introduced into each of six
vials for oviposition. Six replicates were maintained for each variety. Vials were maintained at
282 18C and 70% r.h. After 5 days, the numbers of eggs laid on the seeds of each variety were
counted. Such seeds were observed for a maximum duration of 60 days and the numbers of
emerging adults were counted for a duration of 8 days with adults being isolated to prevent
further breeding. Developmental period, percentage adult emergence and percentage loss in
weight [(initialÿ®nal)/initial seed weight  100] were calculated to evaluate developmental
performance of C. maculatus on cultivars and wild germplasm of pulses.
2.6. Statistical analysis
All data were subjected to Student's t-test and the level of signi®cance was tested at
P < 0.01. Each value was statistically compared with other values of the same parameter.

3. Results
Seeds of wild germplasms generally were found to contain higher amounts of trypsin and
chymotrypsin inhibitors than cultivated seeds (Table 1). Maximum levels of trypsin inhibitors

Fig. 1. Electrophoretogram showing trypsin inhibitor bands in the wild germplasms. Note strong reaction at the
region of Rf 0.60±0.80. Wild pulses: (a) Rhyncocea rufescens, (b) Rhyncocea cana, (c) Canavalia virosa, (d) Lablab

purpureus (black), (e) Lablab purpureus (brown), (f) Tephrosia cararensis, (g) Dolichos lablab.

S. Ignacimuthu et al. / Journal of Stored Products Research 36 (2000) 89±99

93

(TI) were observed in Cajanus albicans (Wight and Arnott) Maesen with 2960 units/g followed
by seeds of Dunberia ferruginea Wight and Arnott and Neonotonia wightii (Wight and Arnott)
Lackey which exhibited 2774 units/g and 2700 units/g, respectively. The seeds of a brown
variety of Lablab purpureus (L.) Sweet were found to contain a signi®cantly (P < 0.01) higher
amount of TI than the black variety. A signi®cant (P < 0.01) di€erence in TI content was
noticed at intraspeci®c levels between cultivars (Table 1) except in Cicer arietinum where no
signi®cant di€erences were observed. The amount of TI was always greater than the amount of
chymotrypsin inhibitor. Among wild germplasms the amount of chymotrypsin inhibitors (CI)
were higher in D. ferruginea (425 units/g), C. albicans (412 units/g) and N. wightii (385 units/g)
(Table 1). Interestingly, CI was absent from the V. radiata K851 cultivar. Although the range
of inhibitors in both the cultivated and wild accessions overlapped, overall the levels were
signi®cantly higher in the wild accessions.
Electrophoretograms (Fig. 1) showed prominent bands of TI in the seeds of L. purpureus
(black) at Rf values 0.75 and 0.80, whereas L. purpureus (brown) showed two successive bands

at the same region with Rf values 0.75 and 0.77, respectively. Although the other wild
genotypes such as Rhyncocea rufescens (Willdenow) de Candolle and D. ferruginea reveal a
clear TI band at Rf value 0.60, the seeds of V. bourneae Gamble, on the other hand, exhibited
three bands at Rf values 0.50, 0.60 and 0.70. Among the various cultivars, gel electrophoresis
revealed a TI band at Rf value 0.70 in the cultivated seeds of V. radiata (K851 and UGG4)
and C. arietinum (COG 29 and CO3) (Fig. 2). Interestingly, the other cultivated seeds did not
reveal any inhibitor bands.

Fig. 2. Electrophoretogram showing trypsin inhibitor bands. Note the absence of reaction in cultivated germplasms.
Wild pulses: (a) Cajanus albicans, (b) Dunberia ferruginea, (c) Vigna unguiculata, (d) Neonotonia wightii. Cultivars:
(e) Vigna radiata K851, (f) Vigna radiata UGG4, (g) Cicer arietinum COG29, (h) Cicer arietinum CO3.

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S. Ignacimuthu et al. / Journal of Stored Products Research 36 (2000) 89±99

Four intense CI bands at Rf values 0.65, 0.75, 0.77 and 0.80 were observed in L. purpureus
(black) while the brown variety of L. purpureus showed three intense bands with Rf values
0.75, 0.77 and 0.80 (Fig. 3). The seeds of Canavalia virosa (Roxburgh) Wight and Arnott had
two intense bands at Rf values 0.45 and 0.75, whereas Rhyncocea rufescens had one prominent
band at Rf value 0.60 and two more bands at Rf values 0.77 and 0.80. Two bands at Rf values
0.77 and 0.80 were observed in the seeds of R. cana (Willdenow) de Candolle while only one
band at Rf value 0.60 was observed in the seeds of Dolichos lablab L. The electrophoretogram
showed no CI bands in the cultivated seeds.
In the present investigation, trypsin and chymotrypsin inhibitors varied in both number and
migration patterns among various wild germplasms of pulses. There was also variation in
intensity of these bands and some seeds showed no bands (Figs. 1±3). This could be due to
lower concentration of an inhibitor protein or lower anity (weaker inhibition) for bovine
trypsin or a-chymotrypsin. Inhibitor contents relative to wild germplasms and cultivar pulses
are presented in Table 1. A broad variability of activities among samples was observed for all
tested inhibitors. Wild varieties of pulses, D. ferruginea, N. wightii, C. albicans, R. rufescens
and V. bourneae used in this study were characterised by higher values of both TI and CI
(Table 1). Among them, C. albicans, D. ferruginea, R. rufescens were found to be infested by
the bruchid, C. maculatus (see results of bioassay studies, Table 2) or, in other words, the
susceptible varieties (reported above) showed higher inhibitor activity against trypsin and
chymotrypsin. The other wild germplasms exhibiting lower inhibitor activity against trypsin
and chymotrypsin were found to be susceptible (except the genus Lablab, in which the gels
showed more intense TI and CI bands).
SDS-PAGE seed protein pro®les showed di€erences in banding patterns among various wild
germplasm lines (Fig. 4). Major polypeptide components at 94, 56 and 52 kD regions were
identi®ed in wild accessions of L. purpureus (brown), R. cana and D. lablab. All the other wild

Fig. 3. Electrophoretogram showing chymotrypsin inhibitor bands in the wild germplasm. Note strong reaction at
the region of Rf 0.45±0.80. Wild pulses: (a) Rhyncocea rufescens, (b) Rhyncocea cana, (c) Canavalia virosa, (d)
Lablab purpureus (black), (e) Lablab purpureus (brown), (f) Tephrosia cararensis, (g) Dolichos lablab and (h) Cajanus
albicans.

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S. Ignacimuthu et al. / Journal of Stored Products Research 36 (2000) 89±99
Table 2
Biological performance of Callosobruchus maculatus on di€erent species of wild and cultivated pulses1
Varieties

Fecundity
(No. of eggs/female)

Adult emergence
(%)

Developmental period
(days)

Seed damage
(%)

45 2 3.6a
Nil2
Nil2
Nil2
Nil2
Nil2
41 2 5.8a
20 2 5.2b
57 2 7.2c
Nil2
±

37 2 1.6a
±
±
±
±
±
32 2 1.4b
34 2 3.0ab
38 2 0.7a
±
±

19 2 1.7a
±
±
±
±
±
14 2 2.8b
4 2 1.4c
15 2 3.4a
±
±

39 2 1.4a
47 2 2.6b
Nil2
2
Nil
69 2 5.9c
63 2 4.2cd
75 2 6.8ce
70 2 5.3cf
77 2 1.5e
72 2 4.2ef

27 2 2.2a
25 2 1.6a
±
±
28 2 4.1a
27 2 3.5a
24 2 1.5a
24 2 1.3a
25 2 2.5a
26 2 0.5a

39 2 2.5a
24 2 3.1b
±
±
43 2 3.5ac
37 2 1.6a
38 2 1.4a
37 2 2.0a
44 2 4.5ac
42 2 1.7c

Wild pulses
Cajanus albicans
Dolichos lablab
Vigna bourneae
Lablab purpureus (brown)
Canavalia virosa
Lablab purpureus (black)
Dunberia ferruginea
Rhyncocea cana
Rhyncocea rufescens
Neonotonia wightii
Tephrosia cararensis

96 2 6.3a
76 2 5.4b
71 2 5.6bc
70 2 0.7cd
68 2 4.0cde
59 2 9.0e
34 2 8.3f
27 2 6.4fg
25 2 4.6g
14 2 2.3h
±

Cultivars
Cajanus cajan ICP14770
Cajanus cajan ICP14990
Cicer arietinum COG29
Cicer arietinum CO3
Vigna unguiculata CO4
Vigna unguiculata VCP8
Vigna mungo CO5
Vigna mungo VBN1
Vigna radiata K851
Vigna radiata UGG4

1202 8.8a
1132 9.5a
27 2 2.1b
19 2 3.6c
85 2 5.8d
97 2 4.6e
58 2 6.2f
49 2 3.4g
76 2 3.5h
88 2 6.5id

1

Values represent mean 2 SE of six replications. Data analysed by Student's t-test (P < 0.01); ±, not tested.
Values in the same column with the same superscript letters are not signi®cantly di€erent.
2
No adult emergence was observed.

germplasms showed variations in polypeptides of low molecular weights in the range of 20±45
kD. All cultivar accessions show similar banding patterns except Cicer arietinum (Fig. 5). In
general, polypeptide components of 94, 66, 56, 52 and 30 kD were identi®ed in all accessions
of cultivars together with some minor bands.
Developmental performance of C. maculatus on various varieties of wild germplasms has
been summarized in Table 2. Maximum numbers of eggs (96) were laid on the seeds of C.
albicans, whereas eggs were not laid on the seeds of Tephrosia cararensis de Candolle.
Variation in percentage adult emergence was observed in both cultivated and wild germplasms
with observed higher values in wild germplasms. Percentage adult emergence was found to be
41, 45, 57 and 20 in the wild seeds of Dunberia ferruginea, C. albicans, R. rufescens and R.
cana, respectively (Table 2). Adult emergence was not observed from other wild germplasms.
Accordingly, they supported lower levels of infestation by the bruchid as is re¯ected by

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S. Ignacimuthu et al. / Journal of Stored Products Research 36 (2000) 89±99

Fig. 4. SDS-PAGE pro®le of wild germplasms. (a) Lablab purpureus (brown), (b) Lablab purpureus (black), (c)
Canavalia virosa, (d) Cajanus albicans, (e) Dunberia ferruginea, (f) Tephrosia cararensis, (g) Vigna unguiculata, (h)
Rhyncocea rufescens, (i) Rhyncocea cana, (j) Neonotonia wightii and (k) Dolichos lablab.

percentage seed damage. Minimum percentage of seed damage was recorded in R. cana (4%)
and maximum percentage of seed damage was recorded in C. albicans (19%). The di€erence in
percentage seed damage was statistically signi®cant (P < 0.01) among infested seeds of wild
germplasms (Table 2).
Oviposition, percentage adult emergence and percentage seed damage from cultivated seeds
showed interspeci®c as well as intraspeci®c variation. Callosobruchus maculatus laid the
maximum number of eggs on the seeds of C. cajan ICP 14770 (120), while the minimum
number of eggs (27, COG29 and 19, CO3) was observed on the seeds of C. arietinum (Table 2).
However, in contrast to the wild germplasms, successful adult emergence was observed from all
the cultivated seeds except for C. arietinum (COG 29 and CO3) (Table 2). Likewise, percentage
adult emergence was higher in various species of genus Vigna than Cajanus, with the maximum
being observed from the seeds of V. radiata K851 (77%). Nevertheless, intraspeci®c variations
were also observed. Developmental period of C. maculatus in cultivated seeds was shorter than
in wild seeds within the same genus (Table 2). Seed damage or loss in seed weight in insect
infestation studies (bioassay studies) was also carried out. Among cultivars, seeds of C. cajan
ICP 14990 showed lesser loss in grain weight (24%) when compared to other cultivar varieties
(Table 2).

S. Ignacimuthu et al. / Journal of Stored Products Research 36 (2000) 89±99

97

Fig. 5. SDS-PAGE pro®le of cultivated seeds. (a) Vigna unguiculata VCP8, (b) Vigna unguiculata CO4, (c) Cicer
arietinum COG29, (d) Cicer arietinum CO3, (e) Vigna mungo CO5, (f) Vigna mungo VBN1, (g) Vigna radiata K851,
(h) Vigna radiata UGG4, (i) Vigna radiata CO3, (j) Vigna radiata CO4, (k) Cajanus cajan ICP 14770 and (l) Cajanus
cajanICP 14990.

4. Discussion
Identi®cation and analysis of sources of resistance in seeds provide us with opportunities to
identify and breed pulse varieties that can naturally resist bruchid attacks. The production of
grain legumes is devastated by C. maculatus and, with current limitations on chemical control
of stored grain pests, the probability of identifying the chemical basis of resistance in pulses,
speci®cally the enzyme inhibitors and some deterrent proteins, prompted us to pursue this
study.
Protease inhibitors are proteins or polypeptides which bind proteolytic enzymes and, in
plants which produce them, are thought to provide a form of natural defence against
herbivorous insects (Green and Ryan, 1972; Ryan, 1979; Broadway et al., 1986; Xavier-Filho
and Campos, 1989; Burgass et al., 1994). In the present investigation in cultivars, both
quantitative and qualitative analyses on TI and CI showed lower activities when compared to
those from their wild counterparts, except C. arietinum, and all were susceptible to C.
maculatus. It has been explained that, since a threshold value to discriminate between resistant
and susceptible lines cannot be established for any tested inhibitors, evaluating only one or two
antimetabolites cannot provide an e€ective way of checking the degree of resistance towards
storage pests (Piergiovanni et al., 1994). This conclusion agreed with previous studies where
several antimetabolites in a larger number of germplasms of pulses had been analysed (Baker
et al., 1989; Xavier-Filho et al., 1989; Cardona et al., 1990; Piergiovanni et al., 1991; Fory et

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S. Ignacimuthu et al. / Journal of Stored Products Research 36 (2000) 89±99

al., 1996). It seems more likely, in the light of these observations, that more antimetabolites
could be involved in the mechanism of bruchid beetle resistance in the case of the present pulse
varieties. Furthermore, antinutrients, ``like the vicilins (55 kD polypeptides), lectins (30 kD
polypeptides), and a-amylase inhibitors (13±17 kD polypeptides) which are reported to have a
role in imparting resistance to bruchids'' (Gatehouse et al., 1989; Pratt et al., 1990; Pereira De
Sales et al., 1992; Macedo et al., 1993, 1995; Fory et al., 1996), may also be involved in
imparting resistance in the present investigation. The present work on protein pro®les
enumerates the presence of bands of 56 and 52 kD in wild accessions of Dolichos lablab, R.
cana and L. purpureus under denaturing conditions. In addition, our results displayed several
minor bands of higher mobilities on SDS-PAGE gels which match well with the protein antimetabolites reported in earlier ®ndings (Liener and Kakade, 1980; Ryan, 1990; Macedo et al.,
1993, 1995).
Acknowledgements
The authors gratefully acknowledge the ®nancial assistance and support provided by a grant
to Fr S. Ignacimuthu from the Department of Biotechnology, Government of India, New
Delhi. We also thank Dr Alok Sen, Entomology Research Institute, Loyola College, Chennai,
for his fruitful and critical suggestions.
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