The Inheritance of Seed Resistance to Callosobruchus rnaculatus F. in ...

Aust. J. Agric. Res., 1983, 34, 681-95

The Inheritance of Seed Resistance to Callosobruchus rnaculatus F. in Cowpea (Vigna unguiculata L. Walp.). I Analyses of Parental, F,, F,, F, and Backcross Seed Generations R. J. ReddenA, P. DobieB, and Angharad M. R. GatehouseC AInternational Institute of Tropical Agriculture, P.M.B. 4320, Ibadan, Nigeria; present address: Hermitage Research Station, Warwick, Qld 4370. BTropical Products Institute, London Road, Slough SL3 7HL, U.K. CUniversity of Durham, Department of Botany, South Road, Durham, D H I , 3LE, U.K. Abstract Bioassays for bruchid emergence on cowpea seeds were conducted on the parent, hybrid F,, F1, Fj,and backcross seed generations of five crosses between susceptible cowpea lines and the resistant accession TVu 2027. The study was repeated with reciprocal F, hybrid seeds, and reciprocal backcross hybrid seeds. For the latter, the mean day of bruchid emergence was also measured. F, seed from individual F, plants of one reciprocal cross were also bioassayed. Trypsin inhibitor determinations were made on the parental, reciprocal F, and reciprocal and non-reciprocal Fj seed generations. The results indicated: (1) that the expression of resistance is mainly determined by the maternal genotype; (2) that resistance may be inherited as a major gene effect, with resistance recessive, and with the presence of modifier genes of different effects in different crosses; (3) that a cytoplasmic effect appears to be absent; (4) that a paternal or embryo genotypic effect on seed resistance can be detected in a certain backcross combination; (5) that trypsin inhibitors are partly associated with expression of resistance, but are uncorrelated in F, segregation, and appear to be quantitatively inherited and variable between crosses. Thus additional mechanisms so far undetected may also be implicated in the expression of seed resistance to bruchids.

Introduction Cowpea (Vigna unguiculata) is one of the principal grain legumes of West Africa and the north-east of South America, and a minor crop in Southern Asia, East Africa, the Caribbean and Central North America. In Australia cowpeas have been bred as a cover crop for sugar cane areas (Skinner 1968), and are expected to have increased use as a green manure and fodder crop in moisture limiting situations elsewhere in Queensland (Imrie and Bray 1983). Grain losses in storage due to the bruchid beetle Callosobruchus maculatus F. occur throughout the world, and up to 100% of the seed may be damaged after 5 months of storage (Singh 1978). Bruchid infestation begins in the field, particularly on pods damaged by boring pests, causing infestation of about 2% of the seed (Booker 1967). Although several bruchid species infest the seeds, only Callosobruchus maculatus continues to multiply in storage. The adults mate within 1 day after emergence from cowpea seed and the female proceeds to glue eggs onto the seed surface with a peak at 2-3 days (Booker 1967). Within 5 days the larvae bore directly into the seed and proceed through five instars while feeding on the interior of the seed. Prior to pupation a 'window' is made just below the outer seed wall to facilitate the emergence of adults, which perform a mating function only. The optimum conditions of 32.5"C and 90% relative humidity allow completion of a life cycle

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in 22 days (Howe and Currie 1964). However, bruchid development is temperature dependent and may take up to 38-6 days at 21°C (Booker 1967). When infestation is severe there may be up to 50% loss in seed weight (Booker 1967; Inyang, unpubl. data). Monthly surveys of 40 markets in northern Nigeria showed mean levels of bruchid-damaged seed rising from 14% of seeds infested at harvest in November to a maximum of 37% in July (Caswell 1956). However, not only is the total weight loss of seed important. Bruchid attack also affects the aesthetic and food value of the seed, and reduces the viability of seed used for the next annual crop (Booker 1967). Comparison of cowpea stored for 9 months showed that, whilst damage for unthreshed seed was 32%, for threshed seed it was as high as 87'70, and although many Nigerian farmers store seed unthreshed, the critical build-up of bruchid damage appears to be after threshing for sale to the market. Cowpea is usually marketed in small lots in open containers under conditions where pest control using pesticides would be difficult to carry out and seed resistance would be a more practical solution. Only one cowpea accession, TVu 2027, which originated from a market sample in Nigeria (M.W. Steel, personal communication), has shown resistance to this bruchid, with less than 20% survival from eggs laid on the seed at 25-30°C (Singh 1978). This contrasts with a survival range of 70-100% in almost all others of the 6000 accessions screened in 1974, the exceptions being in the 50-70% range. The level of resistance in TVu 2027 reduces the natural rate of increase of the bruchid population, thereby keeping the population and seed damage down to a low level in storage (International Institute of Tropical Agriculture (IITA) 1980~).This variety is therefore of significant value provided that the resistance can be successfully transferred genetically to more suitable agronomic types in a breeding program. The seed resistance exhibited by TVu2027 was shown to be associated with its relatively high levels of trypsin inhibitors which are present at about two to three times the levels found in susceptible accessions (Gatehouse et al. 1979). The use of a high trypsin inhibitor level in semi-artificial diets resulted in larval mortality similar to that of TVu2027 and, in the absence of any other class of toxic compound, it was concluded that the resistance expression of TVu2027 was determined, at least in part, by the abnormally high levels of trypsin inhibitors present (Gatehouse et al. 1979). At the Tropical Products Institute (TPI), Southgate (personal communication) has demonstrated that the penetration of the cowpea testa by newly hatched first instar bruchid larvae is mechanical. This and other evidence (Redden and McGuire 1983) suggest that testa characteristics are unimportant in a no-choice situation and that the resistance factor(s) is carried in the cotyledon (and embryo) of the seed. Control procedures, which may involve the use of insecticides, fumigation, oil treatment, or hermetic storage, are normally beyond the means of both farmers and market dealers in developing countries who typically operate on a small scale (O'Dowd 1971). Trypsin inhibitors are highly heat labile (Gatehouse, personal communication). Since all cowpea recipes for human food apparently involve cooking (Dovlo et al. 1976), the TVu2027 form of resistance appears likely to be an effective and inexpensive control measure, free of health hazards. This situation could be different for direct animal consumption, and further tests would be required to assess possible harmful effects. However, genetic resistance would be useful in seed production.

Inheritance of Resistance of Cowpea to Bruchids. I

The characteristics of the cultivar TVu 2027 which are of value to a breeding program include: bruchid resistance, a desirable large, white, rough testa seed, and resistance to cowpea aphid-borne mosaic virus. It is an unimproved cultivar of spreading habit, low-yield potential, high susceptibility to most of the principal humid zone diseases, especially cowpea yellow mosaic virus (CYMV), and its flowering is very sensitive to a short day requirement (IITA 1980b). An important objective of the IITA program was to transfer this resistance into an improved genetic background for further breeding work. Simultaneously, the inheritance of seed resistance to bruchid was investigated. In order to utilize this resistance in the IITA breeding program, it was important to investigate the heritability of this character and its possible genetic relationship with high levels of trypsin inhibitor. Materials and Methods Materials Hybridization with TVu2027 was begun at IITA in 1978 (series 1). Three high yield potential breeding lines - TVx's 1193-9F, 1193-7D, and 289-46 and one CYMV-resistant accession, TVu 410 - were each initially crossed as male parents with TVu 2027, plus one reciprocal combination with TVx 1193-7D as female parent. The hybrid seed from the maternal parent (F, seed), F, seed from naturally selfed F, plants, F, seed from individual F2 plants and the first backcross generation hybrid seed (F, used as female) were produced. These same series of combinations \%ereagain made at IITA in 1979 (series 2) to provide for assay reciprocal F, hybrid seed, non-reciprocal F, seed, and with TVu410 and TVx 1193-9F seed of the four reciprocal combinations of backcrosses involving F,'s in which TVu2027 was the male parent. For the parents TVu2027 and TVx 1193-7D only, all eight possible combinations of reciprocal backcrosses were made in 1980 taking account of all male and female designations (series 3). In 1979, TVu 2027 was crossed reciprocally with TVx 1193-7D, TVx 1193-9F, and non-reciprocally as female parent with TVx289-4G and TVu412 (series 4). At IITA F, plants (20-30 each) were harvested for each combination, including reciprocal crosses. Bruchid Strain Different strains of Callosobruchus maculatus F. were maintained in stock cultivars in excess of 1000 each in the IITA and the TPI laboratories. These strains are referred to as the IITA and Campinas strains. The latter originated from Campinas, Brazil. A comparison of the resistance of TVu 2027 to the IITA and Campinas strains is shown in Fig. 1. Overall percentage survival of the IITA strain (41%) was much higher than that of the Brazilian strain (1 1910). The median development period of the IITA strain was longer than that of the Brazilian strain (35.4 days and 33.0 days). However, the estimate of the median development period of the Brazilian strain was based upon only 12 surviving insects, and is therefore not very reliable. Survival of the IITA and Brazilian strains on the black-eyed pea control was very high and median development periods were short (25.6 and 25.8 days). Both strains laid fewer eggs on the TVu2027 than on the black-eyed peas (6.0+ 0 . 5 (s.e.) and 0 . 7 for the IITA strain; 4.5 0 . 6 and 6 . 9 1 0.7 for the Brazilian strain). 7.6 Thus assay conditions differed between the two laboratories both in terms of the strain of bruchid, and of environmental conditions. These were closely regulated at TPI, but fluctuated with ambient temperatures and relative humidities at IITA. Criteria for Bruchid Resistance in Seed Bioassay The seed resistance of TVu 2027 is able to suppress the build-up of a bruchid population on stored cowpeas (IITA 1 9 8 0 ~ ) Increased . generation time, high mortality of developing larvae, reduced body weight of bruchids, and reduced rate of natural increase per generation are manifestations of the resistance of TVu 2027. Various criteria for assessment of resistance are examined in a separate paper (Redden and McGuire 1983). The two most useful criteria were (a) the mean development time of emerged bruchids dated from termination of egg laying, and (b) the total percentage of adults produced from eggs laid within a specified time period. The time period which best separates the expressions of seed resistance and seed

R. J. Redden et al.

susceptibility has been found empirically to coincide with twice the initial emergence period on susceptible seed (any later time risks the appearance of a second generation of bruchids). This cut-off point also coincides with 20-30% of potential emergence of bruchids o n resistant seed. Criterion (a) was the one used at TPI and preferred at IITA since it allowed the experiment to be terminated at 45-50 days instead of the 65-70 days required for estimate of mean development time of first generation bruchids due to delayed emergence on resistant seed. The cut-off time for estimate of percentage emergence is affected by temperature and humidity and for a particular study is specified, e.g. VoE45 refers to percentage of adult emergence after egg laying at 45 days. At TPI the assay was always terminated at 42 days since conditions were standardized, whereas at IITA the termination time ranged from 42 to 50 days. Although percentage emergence could be used with just one count of emerged insects at date of assay termination, adults were removed daily and recorded after emergence began. This provided a closer monitor of the assay, and reduced possible errors in recovery of adults which have a short life span of up to about 7 days. Black- Eyed peas (b)

Fig 1. Daily emergence of (a), (b) Nigerian (IITA) and (c), (4 Brazilian (Campinas) strains of C, maculatus developing from eggs laid o n cowpea variety TVu 2027 and black-eyed peas. Survival: (a) 41 %, (b) 79'70, (c) 11%, (d) 95%.

Days after oviposition Methods Series 1 From 8-25 seed of most crosses plus 19-40 seed of each parent, and 50-160 F, seed, and 25 F3 seed per F, plant, were bioassayed both at TPI, U.K., and at IITA for percentage adult emergence per number of eggs laid. At T P I the bioassay was conducted with each seed in individual tubes in which a single gravid female of between 24 and 48 h old (Nwanze and Horber 1975; P . Dobie, unpublished data) infested the seed for 5 h. The number of eggs laid were counted and adult emergence was noted separately for each seed. After bioassays were completed at TPI, the same seed was assayed for trypsin inhibitor at the University of Durham. The trials were carried out under controlled conditions of 27k 1°C and 70 f 5% relative humidity. At IITA, F3 seed of each genotype was bulk infested in small plastic boxes (5 by 5 by 2 cm), using a ratio of one adult pair per 10 seed, then egg number per seed was standardized by removing excess eggs to leave two per seed, and the mean value of bruchid emergence was recorded. The parental F, plants were grown in pots in the open at IITA, and were naturally infested with the beetle transmitted by cowpea yellon mosaic virus (CYMV). All associated pots of TVu 2027 parent were infested, and the F, plants were rated for resistance to CYMV.


Series 2 Seed from two to five individual plants of each parent and two to seven individual pods of each hybrid were separately bioassayed at TPI, and then trypsin inhibitor content was analysed. Series 3 These hybrid seeds, plus reciprocal hybrid F, and reciprocal F, seed from F, plants, were bioassayed at IITA in 1981, using six replicates and 2-20 seeds per experimental unit. Total seed numbers were 24-40 per entry except the F, and the parents with 78-105 seed, and backcross TVx 1193-7D/(TVx 11937D x TVu 2027) with 13 seed. The latter had 12 eggs laid per seed, while the range was 298-9.8 for other entries, with fewer eggs per seed as seed number increased. Seed of each experimental unit (a plastic box 5 by 5 by 2 cm) were bulk infested using a ratio of one adult pair per 10 seed. The ambient temperatures for this assay were 22-24OC. Serles 4 F, seed from F, plants were bioassayed as individual seed at T P I , along with the parent seed in the fully randomized design. For most F, plants, and the parents, 25 seeds were assayed. The F, seed of reciprocal cross TVx 1193-7D x TVu 2027 were nor bioassayed. Biochemical Assays Simultaneously with the bioassay at T P I , trypsin inhibitor assays were conducted on all series 4 combinations on separate samples mostly of 25 F, seeds per F, plants, but occasionally fewer (3-15). The trypsin level was estimated as the percentage by weight per gram of seed (Gatehouse and Gatehouse 1979). Trypsin inhibitor analyses were carried out by electrophoresis of total seed extracts on agarose gels containing immobilized trypsin (Gatehouse and Gatehouse 1979). Seed extracts were prepared by extracting finely ground meal overnight at a concentration of 20 mg/ml in 0.05 u CaCCI,, p H 8.2 at 4°C. The samples were then centrifuged at 9000g for 5 min and 10 ml aliquots applied to the gel. Electrophoresis was carried out at 100 V.

Results Parental Lines As shown in Table 1, the bruchid adult emergence on seeds of cowpea TVu 2027 was significantly less than any other of the parental lines used. This verified that all parents except TVu2027 were susceptible to bruchids. Differences in adult emergence between the susceptible parental lines were observed, but these were significantly less than the difference between any susceptible parental line and the resist'ant line. The IITA data were in agreement with those of TPI in assigning resistant and susceptible classifications, although a generally lower level of adult emergence at TPI than at IITA was obtained. The trypsin inhibitor assays of the parental lines showed that TVu 2027 had a significantly higher level of trypsin inhibitor than any of the susceptible parental lines, although variation from one susceptible parental line to another was observed.

F, Seed When F, seeds from reciprocal crosses of the susceptible parental lines with TVu 2027 were examined, a pronounced maternal effect on the inheritance of resistance was observed. In all cases the bruchid adult emergences on F, seeds of the RQ x sd cross were significantly lower than on those of the SQ x R$ cross (Table 1). Variations between crosses were observed, in that the cross of TVu 410 x TVu 2027 gave significantly higher levels of emergence on the RQ x S$ seeds than the other crosses. In the other crosses, the adult emergences of the RQ x ~dseeds were not significantly different from that on TVu 2027, and the emergences on SQ x ~d seeds were similar to those on the susceptible parental lines.

R. J . Redden et at.

A reasonable correlation with trypsin inhibitor content was obtained in that the RQ x sd seeds from all crosses, except TVu2027 x TVu410, had trypsin inhibitor contents significantly higher than those of the corresponding SQ x R$ seeds (Table 1). The inhibitor contents of the RQ x ~d seeds were similar to that of TVu 2027, but those of the SQ x ~d differed (significantly at the 5 % level) from those of the susceptible parents, though apparently in a random manner. In the cross TVu 2027 x TVu 410 the RQ x ~d seed, although its inhibitor content was higher than SQ x R$ seed, did not differ significantly from either the susceptible parent or SQ x ~d seed, and was significantly lower than that of TVu 2027. Bruchid bioassay and trjpsin inhibitor level of parents and F, generation seed of cowpea Series 1: crosses between resistant cowpea line TVu 2027 and four susceptible lines R, resistant parent; S, susceptible parent. At least 20 seeds were used in each assay

Table 1.

R TVu 2027 TVx 1193-9F

TVu 410

S TVx 1193-7DTVx 289-4G

R TI (%)* % emergenceB RQ x s6 TI (%) To emergence S g x ~6 TI (To) % emergence S TI (%) To emergence *Mean of per cent trypsin inhibitor per seed weight. Standard error of estimate 5 kO.O8%. Mean per cent adult emergence per egg laid on seed, at 42 days after infestation (data from TPI). S.e. of estimate 5 F 4 % .

Backcross and F2 Seed Backcrosses of F, plants with both susceptible and resistant parents were carried out for crosses TVu 2027 x TVx 1193-9F, and TVu 2027 x TVu 4120, to allow the expression of resistance in backcrossed and F, seeds to be compared. Results are given in Table 2. Generally, bruchid adult emergences were not significantly different from each other, and were similar to those of the susceptible parental lines in all crosses except when the resistant parental line was used as female parent, where the adult emergence was significantly lower, and similar to TVu 2027. Adult emergence on seeds of the cross (TVx 1193-9F x TVu 2027) Q x TVu 2027 d was lower than the other 'susceptible' seeds, but still significantly higher than TVu 2027 and the backcross with TVu2027 as female parent. Trypsin inhibitor levels in the crosses involving TVu2027 and TVx 1193-9F showed some correlation with resistance in that the inhibitor level in the backcross involving TVu 2027 as female parent was significantly higher than those of the other backcrosses or the F, seed. Further, in the backcross (TVx 1193-9F x TVu 2027) Q x TVu2027 d the inhibitor level was significantly higher than the other


Table 2. Bruchid bioassay and trypsin inhibitor level of parental backcross (B.C.) and F2 generation seed of cowpea Series 2: crosses between resistant cowpea line TVu 2027 and the susceptible lines TVx 1193-9F and TVu 410 R, resistant parent; S, susceptible parent. At least 18 seeds were used in each assay

S R = TVu 2027

TVx 1193-9F

TVu 410

% emergenceB B.C. (S x R) 9 x ~6 TI (%) % emergence B.C. SQ x (S x ~ ) 6 T I (%) % emergence B.C. RQ x (S x R) 6 TI (Yo) % emergence F, (S x R) T I (%) % emergence

Mean of per cent trypsin inhibitor per seed weight; s.e. of estimate ~0.07%. Mean per cent adult emergence per egg laid on seed, at 42 days after infestation (data from TPI); s.e. of estimate 5 5.2%.

A

Table 3.

Bruchid hioassa) of parent, F,, F, and reciprocal hackcross seed generations of cowpea for cross TVu 2027 x TVx 1193-7D (Series 3)

Seed genotypeA

TVu 2027 TVx 1193-7D F , (TVu 2027 x TVx 1193-7D) F, (TVx 1193-7D x TVu 2027) F, (TVu 2027 x TVx 1193-7D) F, (TVx 1193-7D x TVu 2027) B.C. TVu 2027/(TVu 2027 x TVx 1193-7D) B.C. TVx 1193-7D/(TVu 2027 x TVx 1193-7D) B.C. TVu 2027/(TVx 1193-7D x TVu 2027) B.C. TVx 1193-7D/(TVx 1193-7D x TVu 2027) B.C. (TVu 2027 x TVx 1193-7D)/TVu 2027 B.C. (TVu 2027 x TVx 1193-7D)/TVx 1193-7D N.C. (TVx 1193-7D x TVu 2027)/TVu 2027 N.C. (TVx 1193-7D x TVu 2027)/TVx 1193-7D

Per cent emergenceB

Mean days to emergenceC

9.8 46.4 15.5 63.9 42.6 50.2 18.4 55.8 15.7 51 . O 32.4 51.4 53.3 50.7

49.9 40.2 48.8 40.1 43.2 43.6 46.5 41.7 48.5 40.5 46.5 43.8 41.4 41.3

Female parent listed first; B.C. indicates backcross. Per cent adult emergence per egg laid on seed, at 45 days after infestation (data from IITA); s.e. of estimate S *7%. Mean number of days from oviposition to adult emergence on those seeds where adults emerged (up to 62 days: no emergence was observed after this time); s.e. of estimate 5 k 1 . 5 days. A

R. J. Redden et al.

'susceptible' backcrosses and the F, seeds, corresponding to the greater resistance of this backcross noted above. However, in the crosses involving TVu 2027 and TVu 410, only the backcross (TVu 410 x TVu 2027) Q x TVu2027 d had significantly higher trypsin inhibitor level than the other backcrosses and F, seeds, and this did not correlate to decreased adult emergence. The overall correlation (both sets of backcrosses) between trypsin inhibitor level and adult emergence was intermediate (0.58), indicating an incomplete correspondence between the antimetabolite and resistance.

Reciprocal F, Seeds, Backcross Seeds and F, Seeds As a check on the resistance data obtained above, bruchid adult emergence was assayed on seeds of two parental lines, TVu 2027 and TVx 1193-7D , reciprocal F, seeds, F, seeds, and all possible backcross combinations. In addition, to provide another parameter of resistance, the number of days required for insect development was measured by estimating the mean number of days from oviposition to adult emergence. Data for percentage adult bruchid emergence (Table 3) showed that TVu2027 and all F, and backcross hybrid combinations involving it as the female parent had significantly lower values than combinations involving either TVx 1193-7D or either F, genotype as female parent. Adult emergence from seeds of the backcross (TVu2027 x TVx 1193-7D)/TVu2027 is low, although within the range of significance from the TVx 1193-7D parent, and could be regarded as intermediate. The mean number of days from oviposition to emergence is nearly 10 days greater for TVu 2027 than for TVx 1193-7D. The accurate separation of entries reinforces the results for percentage emergence and also places the backcross (TVu 2027 x TVx 1193-7D)/TVu 2027 in the resistant category. The corresponding backcross with TVx 1193-7D as male parent and both F, combinations also have later emergences than the susceptible parent at the 5% significance level only, and may be regarded as moderately susceptible. F3 Seed Bioassays for bruchid resistance on F, seeds from the first set of crosses of the resistant cowpea line, TVu 2027, with other lines (series I), gave a reasonable correlation (0.5) between results obtained at two independent laboratories (TPI and IITA), although a lower mean bruchid emergence level was obtained at TPI (49%) than IITA (63%). There was a generally close agreement between the resistant low emergence scores, below 50% at IITA and below 10% at TPI, but for intermediate to high levels of emergence the agreement was relatively low. The bruchid emergence data from IITA appears to be continuous, with a highly skewed distribution towards susceptibility of high adult emergence (Table 1). The dominance of susceptibility in the F, is consistent with the similar results for the F, generation. If all values of 50% emergence and less are regarded as resistant, the overall ratio is 44 resistant : 95 susceptible (Fig. 2), suggesting a mainly monogenic control of a recessively expressed resistance to bruchids. The fit to a 1 : 3 ratio also holds for individual crosses (x2 0.4-1.5, P > 0.25) with the exception of the TVx 1193-7D combination ( X 2 20.2, P < 0-005), which had a relatively high number (16/32) of resistant values. At TPI, classification of F, plants into resistant, intermediate and susceptible categories was possible through application


Percentage adult emergence

Fig. 2. F, segregation for resistance to bruchids in F3 seed bioassays over crosses between TVu2027 and five susceptible lines. Total numbers of F, plants, 139.

0

5

0

'-R-'

g

-s-

Percentage emergence

Percentage emergence

Fig. 3. Bioassay of F, generation plants of cowpeas in five crosses, with classification by percentage emerge of bruchids per eggs laid u p to 45 days after infestation. TVu 2027, resistant parent (0-20% emergence) all other parents susceptible with 50-95% emergence. x, mean emergence per cross. S, s.e. of mean. n, number of F, parents. R, resistant; I, intermediate; S, susceptible.

of the Newman Keuls confidence intervals (Fig. 3). The upper limit for resistance varied from 25 to 38% according to the cross, and the segregation ratios ranged from 1 R : 2s to 1 R : 4s.

R. J . Redden et al.

Over all the crosses, with division of the intermediate category, the segregation ratio is 60.5 (R): 117.5 (S) which fits a 1 : 3 ratio at the 5% but not at the 1% significance level. The tendency for segregation patterns to vary with crosses, and the presence of an intermediate class in two crosses are suggestive of minor gene effects superimposed on an apparent major gene control of resistance through the maternal genotype. The expression of resistance is largely recessive. It is to be noted that there are some differences in bioassays between locations, with relative excess of resistant values for the TVx 1193-9F cross at TPI, and for TVx 1193-7D at IITA, as well as relatively few resistant values for the TVx 289-40 cross at IITA. Therefore the observation of a major gene effect must be regarded as tentative. At IITA, the F, plants were classified for CYMV resistance over all crosses as 43 resistant : 96 susceptible (x2 1:3 2.61, probability (0.1-0.25). For individual crosses the x2 values for 1 CYMV resistant : 3 susceptible ranged from 0 . 3 to 0.8 ( P > 0.05), except TVx 1193-9F x TVu 2027 with 12 resistant : 15 susceptible, for which the x2 = 5.44 ( P < 0.05). The X2 value for heterogeneity of 1 : 3 segregation within 10 percentile classes of adult emergence was 16.83 (probability 0.05-0. I), suggesting that CYMV and bruchid resistance tend to be genetically independent.

Reciprocal Cross F, Seed Bioassays for reciprocal combinations of F, plants (F3 seed, series 4) indicated no differences between reciprocals for the TVx 1193-9F x TVu 2027 crosses (Table 4). Thus expression of bruchid resistance appears to be genetic, with little or no cytoplasmic component. Table 4.

Bruchid resistance analysis of parental F,, F, and F, seed generations of cowpea crosses between susceptible parentsA and TVu 2027 (Series 4, location TPI) Frequency of F, plants (F, seed) listed by bruchid emergence class

Cross and seed generation

F, F, F, F,

Number Per cent emergence of bruchid adults per egg l a ~ don seed: of seed per F 10.1-20 30.1-40 50.1-60 70.1-80 90.1-100 Total plantB 0-10 20.1-30 40.1-50 60.1-70 80.1-90 F, plants

(TVu 2027 x TVx 1193-9F) 25 (TVx 1193-9F x TVu 2027) 25 (TVu 2027 x TVu 410) 25 (TVu 2027 x TVx 289-4G) 7-23

2 1

1 I

3

6

2 1

1 1

2

2

1 9 6

1

2 4 25

1

8

3 3 1 7

1 1

22 21 34 38

Apescentage emergence of parents were 2 . 5 % for TVu2027, 81.9% for TVx 1193-9F, 65.8% for TVu 410, and 71.5% for TVx 289-40. Female parent listed first.

If all F, plants giving F, seed with a bruchid emergence below 50% are regarded as resistant, analysis of the pooled combination TVx 1193-9F x TVu 2027 shows 6 (R) : 37 (S); and cross TVx 289-46 x TVu 2027 has 9 (R) : 29 (S), both of which fit 1 : 3 (x2 2.8, P > 0.1) and 3:13 (x2 0.6, P > 0.1) ratios. The cross TVu2027 x TVu 410 does not show any segregation for resistance to bruchids.

Trypsin Inhibitor Trypsin inhibitor assays (Table 5) on a duplicate set of the same series 4 materials were not consistent with the bioassays. The correlation in the F, plant


data (F, seed) for the two characters was +0.25. No marked differences in segregation pattern were noted between the reciprocal combinations for TVx's 1193-7D and 1193-9F. The F2 distributions for trypsin inhibitor appear to be continuous but non-normal and irregular, and are thus different from the skewed distribution of the bioassay. The segregations tended to distribute about the midparental values, and the ranges tended to fall between parental values with the exception of the cross TVu 2027 x TVu 410. This cross showed a wide distribution for trypsin inhibition values, although no segregation for bruchid resistant seed was detected. Some F, plants had trypsin inhibitor levels in excess of TVu2027; however, the latter had a lower assay value in this set than in the F, series (Table 1). Table 5.

F, segregation pattern for trypsin inhibitor level assayed on F, seeds of various crossesA involving TVu 2027 Trypsin inhibitor as per cent of seed weight, tabled in class intervals of 0.5%. Frequency of F2 plants tabled for classes of trypsin inhibitor Seed materials F, T V x 1193-9F x T V u 2027 F, T V u 2027b x TVx 1193-9F F, T V x 1193-7D x T V u 2027b F, T V u 2027 x TVx 1193-7D F, T V u 2027b x T V x 289-4G F, T V u 2027b x T V u 410

&umber ofF, plants

0.3-0.34 0.40-0.44 0.50-0.54 0.60-0.64 0.70-0.74 0.80-0.84 0.90-0.94 1.0-1.04 0.35-0.39 0.45-0.49 0.55-0.59 0.65-0.69 0.75-0.79 0.85-0.89 0.95-0.99

22 22 27 33 38 34

ATrypsin inhibitor levels of parents: TVu2027, 0.82; TVx 1193-9F, 0.42; TVx 1193-7D, 0.37; TVx289-46, 0.61; TVu 410, 0.62.

Discussion Bioassay of F, and Backcross Generations The most noticeable feature of the bioassay for adult emergence is the highly consistent maternal control of seed resistance. The data obtained for inheritance of adult emergence (Tables 1 and 2) can be fitted only to a model that assumes that the phenotype of the seed follows the genotype of the maternal parent plant, and will not agree to models assuming that the phenotype of the seed is determined by the genotype of the embryo (i.e. cotyledons plus embryonic axis). This effect applies only to the immediate seed generation. Thus TVu2027 confers resistance as maternal parent on hybrid seed, but the subsequent seed generation, whether F, or backcross, was susceptible (with one exception) for the derived F, used as female parent. This implies that the expression of susceptibility is dominant, so that the F, seed and backcross seed with the F, as female parent also largely express the F, genotype (susceptible). It may be concluded that resistance to bruchid attack appears to be a characteristic of the maternal plant rather than the seeds. Greater discrimination between 'resistant' and 'susceptible' genotypes was obtained with series 3 by the mean number of days from oviposition to emergence than by the percentage adults emerged. As shown in Table 3, this provides evidence

R. J. Redden et al.

either that the dominance of susceptibility to bruchid attack is incomplete or that the maternal genotypic expression is modified by a small genotypic effect. The backcrosses involving F1 (TVx 1193-7D x TVu2027) as female parent are fully susceptible, but the reciprocal F, seeds with Fl's as female parents both appear only moderately susceptible. In addition, the backcross (TVu 2027 x TVx 1193-7D)/TVu 2027 is moderately resistant. If strict maternal determination of seed resistance held, all these seeds, since they have a common maternal genotype, should be equally susceptible. Thus it would appear that the embryo genotype is making a minor contribution to seed resistance which is normally masked by the maternal contribution.

Bioassays of the F, Generation The sizes of the F, plant populations per cross tended to be low for an adequate study of F, segregation. Thus it is not possible to clearly distinguish between various alternative segregation ratios such as 1 resistant : 3 susceptible, or 3 resistant : 13 susceptible. The data do fit a 1 (R) : 3 (S) ratio in most cases, but the fit is not always at a high level of significance, and there are exceptions associated with the cross and location for bioassay. These effects are suggestive of modifying genes in addition to a major gene(s), as the simplest hypothesis. In particular, the low segregation for resistance in cross TVu 410 x TVu 2027, and the absence of segregation in the reciprocal cross, suggest a very marked modifer gene@) effect. An alternative consideration is a possible superimposition of a minor embryonic expression on the maternal expression. Since the bioassays for per cent adult emergence at 42/45 days after infestation differed to some degree between locations, it appears to be important to standardize both the bruchid strain and environmental conditions in order to obtain reproducible results. The effectiveness of the TVu 2027 resistance, and similarity of inheritance with respect to two bruchid strains of widely different origins, provides some evidence that this resistance might be generally effective world wide in cowpea production zones. The identical distributions for percentage emergence in the reciprocal F,'s confirm that the expression of resistance is largely, if not wholly, genetic, with little or no cytoplasmic component in the maternal genotype determination of expression. However, the data may not be sufficiently sensitive to detect possible combinations of maternal (cytoplasmic) or paternal effects at a minor level as suggested by series 3 backcrosses (Table 3). The data for F, and F, seed generations confirm that the major expression of seed resistance is controlled by the maternal genotype. Biochemical Assays The inheritance of trypsin inhibitor levels also follow the maternal genotype, at least in F, seeds of crosses of TVu 2027 with other lines (Table 1). However, results are not as consistent as those for bruchid adult emergence, and in one case (TVu 2027 x TVu 410) can be fitted to a model assuming that trypsin inhibitor level is a property determined by the embryo genotype. Results from backcrosses show a clear partial breakdown of the correlation of seed trypsin inhibitor level with maternal genotype (Table 2), since when F, plants are backcrossed with TVu2027 pollen, high trypsin inhibitor levels are obtained. The inheritance of trypsin


inhibitor levels thus follows a complex model, with contributions from both the maternal and embryo genotypes. The trypsin inhibitor assays on F, seed are complicated by the lack of distinction on the extent to which trypsin inhibitor are controlled by the maternal genotype, the embryo hybrid genotype, or both. Cytoplasmic effects appear to be unimportant, as shown by the F, of the reciprocal crosses. The lack of association at the F, level between trypsin inhibitor and bruchid resistance is consistent with the F, data and makes the role of trypsin inhibitors in resistance less clear. A difficulty arises over the maternal expression of biological resistance, in cotyledonous tissue of embryonic origin. The predominant determination of seed resistance to bruchids by the maternal genotype may be compared to the models for maternal inheritance proposed by Davies (1973). From these suggestions, three possible explanations for this pattern of inheritance may be put forward. Firstly, resistance may be due to a factor in the seed coat, which is derived from the maternal plant and expresses the maternal genotype. However, this is not very likely, since larvae are able to penetrate the seed coat in resistant seeds and resistance is still expressed in seed meals (Gatehouse et al. 1979). In cowpea the trypsin inhibitor level is negligible in the testa (A. M. R. Gatehouse, unpublished data), agreeing with the results of Beevers and Poulson (1972), who noted in Pisum sativum that albumin synthesis (i.e. including trypsin inhibitors) occurs within the cotyledon, though not necessarily to the exclusion of possible transport from maternal tissue. This agrees with the observation of Southgate that bruchid larvae mechanically penetrate the seed. Secondly, the factor causing resistance may be synthesized in the maternal plant and transported to the seed embryo. A resistance factor of this type is unlikely to be a protein for reasons given above. Thirdly, the level of expression of an embryonic gene may be regulated by the supply of nutrients and other substances from the maternal plant, e.g. in Lupinus angustifolias the levels of cotyledonary storage proteins are known to be affected by the availability of sulfur to the maternal plant (Blagrove et 01. 1976). This type of regulation is known to affect trypsin inhibitor levels in Pisum sativum (R. R. D. Croy, unpublished data). There are thus mechanisms to account for the strong maternal effect on the levels of trypsin inhibitors in the seed embryo and the determination of resistance by the maternal genotype. Janzen et al. (1977) argue that, since all cowpea contain trypsin inhibitors, then C. rnaculatus is obviously able to digest this class of compound. At the time of their studies a variety of cowpea resistant to C. maculatus was not available, and so they were unable to consider the possibility that the quantitative amount of trypsin inhibitor determined resistance. Moreover, in their feeding trials the authors looked at the effect of soybean and other non-cowpea trypsin inhibitors in cowpea meal and not the effect of cowpea trypsin inhibitor. Since trypsin inhibitors from different sources show marked species specificity in their inhibitory activity against trypsin-like enzymes (Laskowski and Sealock 1971), the non-toxicity of one (or more) trypsin inhibitor(s) towards a particular species cannot be extended to all trypsin inhibitors. It has recently been demonstrated that, whilst cowpea trypsin inhibitors are effective antimetabolites of C. rnaculatus, soybean trypsin inhibitors are not (Gatehouse and Boulter 1983). Certainly no other potentially toxic compounds besides trypsin inhibitors have been reported in cowpea (Janzen et al. 1977; Gatehouse et al. 1979), but as the present data suggest that other mechanisms

R. J. Redden et a/.

of resistance must be operative, the actual role of trypsin inhibitor requires further investigation and the presence of other antimetabolites cannot be excluded. Gatehouse and Boulter (1983) have suggested that resistance in TVu2027 may depend on availability of nutrients to the growing larvae, since this example of resistance can be overcome by sulfur amino acid supplementation.

Disease Resistance The apparent absence of genetic linkage between the genes for CYMV susceptibility and for bruchid resistance in TVu 2027 means that genes for the latter can be transferred to an improved disease resistant background using moderately large F2 populations (over 100 plants). Choice of Scale The pattern of bruchid emergence follows different distributions on the resistant and on the susceptible lines (Fig. 2). Individual seed data for percentage emergence also showed greater attenuation for TVu 2027 in comparison with susceptible lines (Dobie, personal communication). However, mean values for either percentage emergence to for number of days to emergence taken over 40 or more eggs were consistent for parental lines, under the same test conditions. By using 20-25 seed per genotype the desired egg number for a suitable assay was easily obtained, although especially for F,'s this number was sometimes below 20 eggs. Low bruchid survival on seed of resistant plants does reduce the accuracy of mean values for estimates of variables, thus the data are probably not homogeneous. Further detailed investigation of this point is required; however, because of both repeatability of the results and the capacity to separate classes, it was decided to continue with standard analyses of the data on an untransformed scale. Acknowledgements The assistance of Drs S. R. Singh and M. J. Lukefahr with bioassays at IITA and Miss L. Fairbank and K. Streatfield at TPI is gratefully acknowledged. We would also like to thank Dr J. A . Gatehouse for his aid in preparing the manuscript and Professor D. Boulter for his support of this work. References Beevers, L., and Poulson, R. (1972). Protein synthesis in cotyledons of Pisum sativum L. .Plant Physiol. 49, 476-81. Blagrove, R. J., Gillespie, J . M., and Randall, P . J. (1976).Effect of sulphur supply on the seed globulin composition of Lupinus angustifolias. Aust. J. Plant Physiol. 3, 173-84. Booker, R. H. (1967). Observation o n three bruchids associated with cowpea in Northern Nigeria. J. Stored Prod. Res. 3, 1-15. Caswell, G. H. (1956). The storage of cowpea in the Northern Nigeria. Samaru Res. Bull. No. 120. Davies, D. R. (1973). Differential activation of maternal and paternal loci in seed development. Nature (New Biol.) 245, 30-2. Dovlo, F. E., Williams, C. E., and Zoaka, L. (1976). Cowpeas. Home preparation and use in West Africa. Int. Devel. Res. Coop. Can. 0 55e. Gatehouse, A. M . R., and Boulter, D. (1983). Assessment of the antimetabolic effects of trypsin inhibitors from cowpea (Vigna unguiculata) and other legumes on development of the bruchid beetle Callosobruchus maculatus. J. Sci. Food Argic. (in press). Gatehouse, J . A., and Gatehouse, A. M. R. (1979). Two simple quantitative assays for trypsin inhibitors using immobilised trypsin. Anal. Biochem. 98, 438-44.


Gatehouse, A. M. R., Gatehouse, J . A., Dobie, P., Kilminster, A. M., and Boulter, D. (1979). Biochemical basis of insect resistance in Vigna unguiculata. J. Sci. Food Agric. 30, 948-58. Howe, R. W., and Currie, J. C . (1964). Some laboratory observations on the rates of development, mortality and oviposition of several species of bruchids breeding in stored pulses. Bull. Entom. Res. 55, 437-77. Imrie, B. C., and Bray, R. A. (1983). Estimates of combining ability and variance components of grain yield and associated characters in cowpea. Proc. Aust. Plant Breeding Conf., pp. 202-4. (Ed. C. J. Driscoll.) (University of Adelaide.) International Institute of Tropical Agriculture (1977). Annu. Rep. International Institute of Tropical Agriculture (1980~).Annu. Rep. International Institute of Tropical Agriculture (1980b). Res. Highlights. Janzen, D. H . , Juster, H . B., and Bell, E. A . (1977). Toxicity of secondary compounds to the seed eating larvae of the bruchid beetle Callosobruchus maculatus. Phytochemistry 16, 223-7. Laskowski, Jr, M., and Sealock, R. W. (1971). Protein proteinase inhibitors - molecular aspects. In 'The Enzymes'. Vol. 3, pp. 375-473. (Ed. P . Boyer.) (Academic Press: New York.) Nwanze, K. F., and Horber, E. (1975). Laboratory techniques for screening cowpeas for resistance to Callosobruchus maculatus F . Environ. Entom. 4 , 415-19. O'Dowd, E. T . (1971). Hermetic storage in Nigeria using weldmesh silos lined with butyl rubber. Inst. Agric. Res. Samaru, Zaria, Nigeria, Misc. Pap. No. 3. pp. 1-21. Redden, R. J., and McGuire, J. (1983). The genetic evaluation of bruchid resistance in seed of cowpea. Aust. J. Agric. Res. 34, 707-15. Singh, S. R. (1978). 'Resistance to Pests of Cowpeas in Nigeria.' (Ed. S. R. Singh, H . F. van Emden, and T. A. Taylor.) pp. 267-97. (Academic Press: New York.) Skinner, J. C. (1968). Cowpea breeding by the Bureau. Cane Growers Quart. Bull. 32, 24-30.

Manuscript received 28 January 1983, accepted 15 July 1983