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a USDA–ARS Plant Germplasm Introduction and Testing Research Unit, Washington State Univ., Pullman, WA 99164-6402
b Entomology Section, Dep. of Agriculture Western Australia, 3 Baron-Hay Court, South Perth, WA, 6151 Australia
* Corresponding author (slclement{at}wsu.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: GRIN, Genetic Resources Information Network
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONREFERENCES |
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Worldwide, pea producers rely mainly on contact insecticides to control adults in pea fields before females lay eggs on pods (Horne and Bailey, 1991; O'Keeffe et al., 1992; Clement et al., 2000). However, timing chemical applications to coincide with female egg laying is difficult. More than one application may be required if weevil invasions continue for 2 to 4 wk in a pea field (Michael et al., 1990). The development and use of cultivars with pod and seed resistance to B. pisorum would reduce control costs and provide an environmentally safer option than contact insecticides for adult weevil control.
Some P. sativum lines with the Np gene respond to the presence of pea weevil eggs on pods by forming callus (neoplastic pod trait) that reduces larval entry into the pod (Hardie, 1990; Berdnikov et al., 1992; Doss et al., 2000). In a field trial, this pod-based resistance was responsible for a lower rate of weevil infested seed (62.2%) in Np plants compared with that in a susceptible line (85.4%) (Doss et al., 2000). In addition, plant biotechnology has the potential to protect peas from B. pisorum damage, as evidenced by the development of transgenic P. sativum for resistance to Callosobruchus weevils (Shade et al., 1994) and B. pisorum (Schroeder et al., 1995; Morton et al., 2000). This resistance is based on the insecticidal activity of the 
-amylase inhibitor in seeds of bean, Phaseolus vulgaris L., which blocks the action of the starch-digesting enzyme -amylase and thus prevents weevil larvae from digesting starches in seed. More research is required before weevil-resistant transgenic peas can be released to growers (Morton et al., 2000). Moreover, any concerns about the biosafety of genetically engineered peas, as voiced by consumer and environmental groups for transgenic crops (Stewart et al., 2000), must be addressed by researchers and producer groups before transgenic peas are commercially deployed for weevil protection.
An alternative to genetically engineered crops is the development and deployment of crop cultivars with natural insect resistance from primary and secondary gene pools. In the absence of seed resistance to pea weevil in the Pisum primary gene pool (P. sativum and all subspecies) (Hardie, 1990; Clement et al., 1994), searches for resistance were expanded to the secondary gene pool in the late 1980s, resulting in the discovery of seed resistance in P. fulvum during field evaluations (Hardie et al., 1995; 1999). This wild species from the eastern Mediterranean and Near East areas, where pea domestication took place (Zohary, 1973) and where B. pisorum is native (Clement et al., 1999), is the only species in the secondary gene pool (Muehlbauer et al., 1994). Because P. fulvum is cross-compatible with P. sativum when it is used as the pollen parent (Muehlbauer et al., 1994), conventional plant breeding could potentially transfer P. fulvum resistance traits to domesticated pea. Further consideration of P. fulvum as a potential source of weevil resistance genes for cultivar development requires detailed knowledge of the level and extent of seed resistance and identification of pod-based resistance in this species.
Data from life tables (Poole, 1974, p. 11–15; Southwood, 1978, p. 366–369) may provide insight into the genetics of plant resistance to insects if the resistance through high insect mortality is expressed by different plant parts. Indeed, the expression of resistance by different plant parts (i.e., a pod factor and seed antibiosis) can be under the control of different genes (Rusoke and Fatunla, 1987; Kennedy and Barbour, 1992). The objectives of this research were (i) to determine the extent of pod and seed resistance to pea weevil in P. fulvum accessions, and (ii) to use the life table format to characterize weevil stage-specific mortality and survivorship on P. fulvum accessions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONREFERENCES |
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Field-infested pea seeds were harvested at Farmington, WA, USA (46°43' N, 117°9' W) and York, WA, Australia (31°52' S, 117°48' E) and stored at 4°C (Pullman) or 10°C (South Perth), for 7 to 12 mo before greenhouse trials were conducted at each location. Adults emerging from infested peas were sexed using the dimorphic character described by Pesho and Van Houten (1982). Five to ten pairs were placed in cages and allowed to feed on pollen, which was in the form of pellets from commercial honey bee hives (Healthy Life Carousel, Cannington, WA, Australia) in South Perth cages (130 x 200 x 80 mm3 clear plastic). Fresh flowers of greenhouse-grown P. sativum ‘Alaska 81’ provided pollen in Pullman cages (125 x 175 x 45 mm3 clear plastic) at a rate of 10 to 15 flowers every 2 to 3 d for 10 to 14 d per cage. After 7 to 10 d, two or three flat or swollen pea pods from greenhouse-grown P. sativum were placed each day in the Pullman (Alaska 81) and South Perth (‘Pennant’) cages as oviposition substrates. In both environments, mated females began to lay fertile eggs on excised pea pods after feeding on pollen for 7 to 14 d. Cages were held at 25 ± 2°C and a 12- to 14-h photophase during feeding, reproduction, and egg laying on pea pods.
Experimental Protocol
In Pullman, seeds of wild P. fulvum accessions were germinated following methods described by Kaiser et al. (1997) for wild Cicer seed. Seeds were scarified by scoring testa with sandpaper before they were placed in labeled cheese cloth bags in 1000-mL beakers filled with distilled water that was aerated with laboratory-supplied air. Every 2 d, the water in each beaker was changed and any germinated seeds were removed for planting. P. sativum ‘Alaska 81’ seeds were germinated in a similar fashion except they were not scarified.
Newly germinated seeds were planted individually in 15-cm pots containing a commercial soil mix and grown in a greenhouse between March and July 1995 and 1996, without any fertilizer. No pesticides were applied. The 1995 and 1996 trials evaluated 22 and 12 P. fulvum accessions, respectively. However, a total of 31 accessions were evaluated because three accessions were included in both trials. A pea weevil-susceptible cultivar (Alaska 81) of P. sativum (Clement et al., 1996; Hardie et al., 1999) was included in each trial. Entries (PI accessions and susceptible control) were arranged on two greenhouse benches in a completely randomized design with five replications (potted plants). Both greenhouse trials were conducted under prevailing natural light/dark cycles and 15 to 21°C nighttime and 23 to 28°C daytime temperatures.
Mature weevil eggs (dark brown head visible through chorion) were transferred from oviposition cages with a moistened fine-tipped brush to surfaces of pods in the late flat and early swollen pod stages (Meichenheimer and Muehlbauer, 1982) on potted test plants in Pullman and South Perth trials using procedures in Hardie and Clement (2001). Eggs were oriented ventrally so hatching neonate larvae could burrow directly into the pod wall.
In Pullman, each pod received two eggs placed 
15 mm apart for a total of 10 eggs per plant and 50 eggs per entry (2 eggs per pod x 5 pods per plant x 5 plants per entry). Infested pods were observed daily and the number of hatching eggs was recorded. Pods were harvested at maturity and seeds were placed in labeled petri dishes (100 x 15 mm) on a laboratory bench (25°C) and observed for adult emergence every 3 to 4 d. Newly emerged adults were weighed and sexed. After 90 d, seed with feeding scars, circular windows on testa, and with adult exit holes were split and scored for damage on a scale of 1 to 5, where 1 = feeding scar or puncture on testa, 0 to 1% cotyledon tissue eaten or damaged, dead first instar; 2 = 2 to 5% cotyledon tissue eaten or damaged, dead first instar; 3 = >5% cotyledon tissue eaten, dead second to fourth instar; 4 = extensive damage, dead prepupa or adult; and 5 = extensive damage, live adult, or adult emergence (Hardie and Clement, 2001). The larval stage was determined by head capsule width (Brindley, 1934).
In South Perth, scarification involved scoring seed testa with a pair of pliers, followed by soaking the seed in a 10% solution of sodium hypochlorite for 5 min and washing in boiled water. After washing, seed were placed on moistened filter paper in petri dishes (100 x 15 mm) on a laboratory bench (24°C) for germination. Seed of Pennant, a weevil-susceptible P. sativum (Hardie et al., 1999), was not scarified but was germinated on moistened filter paper. Five germinated seeds per entry were planted in a 25-cm pot containing washed river sand and top dressed with a slow release granular fertilizer (Osmocote, The Scotts Company, Marysville, OH)1. No pesticides were applied. One trial was conducted in 1993 with 23 P. fulvum accessions (also in Pullman trials) and the Pennant control. The 24 pots were randomly arranged on a greenhouse bench. The greenhouse trial was conducted under the prevailing natural light/dark cycle and temperatures ranged from 10 to 32°C.
Each pod on South Perth plants received eight mature eggs, but a variable number of pods per entry were infested (5 to 28 pods per entry from 1 to 6 pods per plant). Pods were harvested at maturity and stored at room temperature in labeled seed envelopes. After 60 d, when adult weevils began emerging from Pennant seeds, all seeds were inspected and assessed for adult emergence. A seed was classified as infested if a live pupa or adult was found in the seed or an adult exit hole was found.
Data Analysis
A few eggs on pods did not hatch in the Pullman trials, leaving a starting cohort of 7 to 10 on plants (range of 46 to 50 eggs per entry). The absence of feeding and penetration marks on testa was used to calculate the number of larvae dying en route to the outer surfaces of developing seeds inside green pods. Larvae on each infested pod were categorized as 0, 1, or 2 dead if feeding damage was recorded on 2, 1, or 0 seeds, respectively. Within-seed mortality of B. pisorum was compartmentalized, as follows: first instars consuming 0 to 5% of the cotyledon tissue (Damage Ratings 1 and 2); second to fourth instars consuming >5% of the cotyledon tissue (Damage Rating 3); and development to prepupa or adult with extensive damage to seed contents (Damage Ratings 4 and 5).
The life table format (Poole, 1974, p. 11–15; Southwood, 1978, p. 366–369) was previously used to record numerical changes in arthropod populations on cotton, Gossypium hirsutum L., (Trichilo and Leigh, 1985) and cowpea, Vigna unguiculata (L.) Walp., cultivars (Messina, 1984) and was used to quantify pea weevil mortality and survival on P. fulvum accessions and a susceptible pea cultivar (Alaska 81) in the Pullman trials. Parameters estimated in the life table analysis for pea weevil on each plant (replicate) of each entry were: lx, the number of individuals entering stage x; dx, the number of individuals dying in stage x; 100qx, the percentage mortality in stage x; and 100Sx, the percentage surviving stage x. Instead of presenting these values in complete life tables for all pea weevil-entry interactions, tables are presented for B. pisorum on three PI accessions of P. fulvum in which values were generated from starting entry-cohorts of 48 or 50 neonate larvae (Table 1) . This approach was used to conserve space while illustrating the structure of three representative life tables. To simplify the presentation of all life table results and to facilitate assessment of pod and seed resistance among entries, entry means of three parameters were statistically analyzed: (i) mortality [100qx] of first instar larvae before reaching seed; (ii) mortality [100qx] of all weevil stages within seed; and (iii) survival [100Sx] to the adult stage (Tables 2 and 3) .
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONREFERENCES |
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On the basis of within-seed mortality rates of larvae, seeds of all P. fulvum accessions except PI 560065 (1995 trial) were significantly more (P < 0.05) resistant to larval feeding than seeds of Alaska 81. Pea weevil larvae sustained 88.4 to 100% within-seed mortality on 17 of 22 P. fulvum accessions in 1995 and 85.3 to 100% on all accessions in 1996. This high level of seed resistance in P. fulvum is also revealed by the seed damage ratings in both trials, which for all accessions except PI 560065 (1995 trial) were significantly lower (P < 0.05) than the average ratings of 4.8 (1995) and 5.0 (1996) for Alaska 81. Seed damage ratings averaged less than 3.0 for 17 P. fulvum accessions in 1995 and for all accessions in 1996 (Tables 2 and 3).
Seed of P. fulvum accessions in both Pullman trials weighed significantly less (P < 0.05) than seed of Alaska 81 (Tables 2 and 3). However, resistance in small seeded P. fulvum accessions did not appear to be the result of limited food for larval development, as evidenced by nonsignificant (1995: r = 0.29, P = 0.2105; 1996: r = 0.40, P = 0.1994) correlation coefficients between P. fulvum seed weights and damage ratings and nonsignificant (1995: r = -0.25, P = 0.2692) or weakly significant (1996: r = 0.57, P = 0.0507) correlation coefficients between seed weights and within-seed mortality rates. Moreover, 81.8% (n = 768) and 87.5% (n = 384) of the first instar larvae that reached seed of P. fulvum entries in 1995 and 1996, respectively, died before consuming >5% of the cotyledon tissue of seed and before developing into second instars. This is further evidence that starvation was not responsible for high mortality of first instars on P. fulvum seeds of weights that averaged 49.4 to 94.9 mg in 1995 and 52.1 to 87.9 mg in 1996. We speculate that insecticidal properties are responsible for seed resistance, although the absence of -amylase inhibitor in seed of P. fulvum (Schroeder et al., 1995) precludes associating this seed protein with weevil toxicity. Schoonhoven et al. (1983) speculated that factors other than seed size and weight were responsible for resistance in small seeded and uncultivated bean accessions to the bean weevil, Acanthoscelides obtectus (Say), and Mexican bean weevil, Zabrotes subfasciatus (Boheman) (Coleoptera: Bruchidae).
Mean adult emergence values (100Sx) from P. fulvum were significantly lower (P < 0.05) than those from Alaska 81, which had emergence values of 86% (1995) and 94% (1996). However, emergence rates were relatively high (32.4 to 60.4%) for five P. fulvum accessions. Adults that emerged from P. fulvum weighed significantly less (P < 0.05) than those from Alaska 81 seed in both Pullman trials (Tables 2 and 3), prompting us to speculate that these very small adults would have reduced survivability and reproductive fitness. No large departure from a 1
:1
sex ratio was recorded for adults emerging from Alaska 81 in 1995 and 1996 (49:51%) and from all P. fulvum accessions in both trials (52:48%).
In the South Perth trial, 15 P. fulvum accessions produced no adults (PI 595939, PI 560064, PI 595942, PI 595948, PI 560061, PI 595932, PI 595937, PI 595946, PI 595934, PI 595935, PI 595933, PI 560062, PI 560067, PI 595940, PI 560063). However, adults emerged from eight accessions with values averaging between 4 and 6% for three accessions (PI 595936, PI 595944, PI 343955) and between 15 and 47% for five accessions (PI 595938, PI 595941, PI 595943, PI 595945, PI 595947). Adult emergence from the susceptible P. sativum control (Pennant) averaged 85%. There was good concordance between the Pullman and South Perth results. A few adults emerged (2 to 8%) from seven accessions (PI 595939, PI 560064, PI 595942, PI 595948, PI 560061, PI 595932, PI 595937) in Pullman (Table 2), but none emerged from these entries in South Perth, and a few adults emerged (4 to 15%) from three accessions (PI 595947, PI 595944, PI 595936) in South Perth, but none emerged from these entries in Pullman (Tables 2 and 3).
Any variation among weevil populations (including B. pisorum) for virulence to resistant genotypes could minimize the ability of a resistant cultivar developed in one country to confer resistance to weevil populations in other countries (Dick and Credland, 1986; Shade et al., 1999). However, differences in adult emergence rates between Pullman and South Perth trials were not of a magnitude for us to conclude that significant variability exists among pea weevil populations in eastern Washington and Western Australia for virulence to resistant P. fulvum accessions. Thus, the resistant accessions in this study could be used to develop pea cultivars in different geographical areas with durable pea weevil resistance.
Three distinct patterns of B. pisorum mortality and survival on the entries in the Pullman trials emerged from the construction of life tables, illustrated by the values for the weevil on PI 560065, PI 595942, and PI 595940 (Table 1). The PI 560065 life table characterizes pea weevil susceptibility in P. fulvum, a pattern that was exhibited by four other accessions (PI 595941, PI 595938, PI 595953, PI 595943) in the 1995 trial. These susceptible accessions had within-seed larval mortality rates of 14.4 to 59.5%, seed damage ratings >3.0, and adult emergence percentages of 32.4 to 60.4% (Table 2). Although the remaining accessions could not be separated into resistant classes on the basis of statistically different mean values for within-seed mortality and seed damage ratings (Tables 2 and 3), their adult emergence values yielded two distinct patterns. First, 13 P. fulvum accessions from the Pullman trails and two accessions from the South Perth trial produced a few adults, albeit only one or two small adults or emergence percentages of 8% or less (exemplified by PI 595942 in Table 1), and thus were classified as moderately resistant. Second, 14 accessions shared by the Pullman and South Perth trials produced no adults and were classified as resistant (exemplified by PI 595940 in Table 1) (Tables 2 and 3). A wide range in first instar mortality rates on pods (Tables 2 and 3) precluded the use of this parameter for classifying entries. Conclusions about the geographical distribution of susceptibility and resistance in P. fulvum accessions are not possible because of a paucity of passport information in GRIN.
Results from this study revealed the presence of low to complete levels of B. pisorum resistance in germplasm stocks of P. fulvum. This antibiosis resistance is based on the death of larvae on different plant parts (pod and seed) of P. fulvum, as revealed by life tables. Such patterns of insect resistance in plants can be complex in nature and controlled by more than one gene (Rusoke and Fatunla, 1987; Kennedy and Barbour, 1992). Indeed, this seems to apply to pea weevil resistance in P. fulvum because initial crosses between P. sativum and a weevil-resistant accession in Western Australia which produced resistant recombinant progeny indicate that resistance is likely controlled by two or more genes (Byrne et al., 2000). Success in transferring bruchid resistance in a wild crop relative to agronomically acceptable cultivars via traditional plant breeding has been achieved. In this example, single gene resistance in a wild mungbean (Vigna radiata L. Wilczek) accession of the subspecies sublobata (Kitamura et al., 1988; Fujii et al., 1989) was used to develop mungbean cultivars with resistance to Callosobruchus chinensis L. (Coleoptera: Bruchidae) in many countries (Kaga and Ishimoto, 1998).
Our findings identify sources of natural resistance in the Pisum genome to develop pea cultivars with pod and/or seed resistance to B. pisorum. The best sources of resistance are the resistant accessions, although some of the moderately resistant accessions should not be overlooked because they exhibited high within-seed larval mortality rates (>95%) and low seed damage ratings (<2.2) (Tables 2 and 3). With different P. fulvum parts and tissues expressing resistance, it may be possible to develop a multi-tiered defense against B. pisorum in one pea cultivar to provide long-term stability of resistance to the weevil. In addition, strong pod resistance from P. fulvum, or from P. sativum lines with the neoplastic pod trait (Doss et al., 2000), would prevent many larvae from reaching seeds and causing damage. Finally, P. fulvum resistance traits could be transferred into transgenic peas containing the -amylase inhibitor protein to provide durable protection against B. pisorum in one cultivar (Schroeder et al., 1995).
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ACKNOWLEDGMENTS |
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NOTES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONREFERENCES |
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Received for publication August 3, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONREFERENCES |
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-amylase inhibitor 1 in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions. Proc. Natl. Acad. Sci. USA 97:3820–3825.-amylase inhibitor confers resistance to the pea weevil (Bruchus pisorum) in transgenic peas (Pisum sativum L.). Plant Physiol. 107:1233–1239.[Abstract]This article has been cited by other articles:
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  R. Jing, R. Johnson, A. Seres, G. Kiss, M. J. Ambrose, M. R. Knox, T. H. N. Ellis, and A. J. Flavell Gene-Based Sequence Diversity Analysis of Field Pea (Pisum) Genetics, December 1, 2007; 177(4): 2263 - 2275. [Abstract] [Full Text] [PDF]
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