Progress 10/01/00 to 09/30/05
Outputs
The long-term objectives of this project have been to 1) identify insecticidal proteins having an impact on insect pest population dynamics (especially proteins routinely consumed by humans and other animals), 2) develop germplasm release strategies for enhancing gene durability, 3) develop improved biological assay systems for identifying insecticidal proteins, and for studying insect-gene-product interactions, 4) study bruchid population dynamics in general (Coop.: L. Murdock and B. Pittendrigh). 1A) Avidin (chicken egg-white cystatin) previously shown to have insecticidal activity against some stored grain pests, was lethal to cowpea beetle and common bean weevil larvae at 20-25 ppm. Sub-lethal doses contributed to a slower expansion of cowpea beetle populations. A High insecticidal activity at a low dose makes Avidin a candidate for transforming cowpea (and other legumes) with a gene for bruchid resistance. 1B) VSP (a common protein in many plants) was active
against the cowpea beetle at 3000-4000 ppm (patent disclosure document filed from TX A & M) (Coop.: K Salzman from TX A & M). 2A) Experiments have been initiated for testing the hypothesis that gene durability is greater when genes are pyramided rather than when released sequentially . 3+1A) The Purdue Insect Feeding Monitor (PIFM) / artificial seed bioassay was modified for studying insect gene regulation in response to specific insecticidal proteins. By monitoring larval feeding, larvae can be transferred from artificial seeds containing non-toxic proteins to seeds containing insecticidal proteins at any stage of larval development. The changes in feeding behavior in response to insecticidal proteins can described in detail using the PIFM. 3+1B) The development of a fruit fly bioassay is nearly completed. Because fruit fly genetics are far more advanced than genetics of any other insects, the fruit fly bioassay is being used to study cowpea beetle-protein interactions (gene
regulation) at the gene level. 4A) When Wolbachia-infected C. chinensis males mate with cured females the incompatibility results in 100% egg mortality. Incompatibility as a potential agent for reducing C. chinensis populations in stored azuki bean seeds, an azuki bean / C. chinensis bioassay was developed and used to measure the impact of male and female incompatibility on C. chinensis population dynamics. While certain levels of incompatibility reduced C. chinensis populations by over 90%, the practicality of using Wolbachia to manage insect pests of stored cowpeas is questionable (Coop.: R. Giordano at the Univ. of Vermont). 4B) Experiments to date have shown that the metabolic heat generated by cowpea beetle larvae feeding on susceptible cowpea seeds can reduce generation time from 40 to 30 days at 26C and 50% RH. However, the slower larval development on resistant cowpea TVu 2027 did not generate a significant amount of metabolic heat. Consequently, the differences in generation
time between larvae feeding on resistant and susceptible is greater than expected, and the value of using resistant TVu 2027 greater than expected.
Impacts
Provide an in vivo bioassay system for the relatively rapid identification of insecticidal proteins, or other insect-active compounds. In general, the management of certain insect pests of stored grain without introducing toxic chemicals into the environment. Specifically, manage bruchid beetle pests of stored cowpeas without introducing toxic chemicals into the environment
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
(A) Prospecting for insect resistant genes (Coop: L.L. Murdock): Using the cowpea bruchid / artificial seed model bioassay system, research over the past two years has shifted from gene prospecting to understanding the interaction between previously-identified, insecticidal proteins and the cowpea bruchid (Callosobruchus maculatus). Dose response experiments conducted on three plant proteins showed that (based on their level of insecticidal activity) they remain candidates for conferring plant resistance to the cowpea bruchid via plant transformation. (Coop: K. Zhu-Salzman, Texas A&M): The model bioassay system was used to identify active forms (from site-specific mutations) of the cowpea bruchid-active soybean cysteine protease inhibitor (SCPI). Also, the combination of SCPI and the cowpea bruchid-active lectin (Griffonia simplicifolia) were evaluated. (B) Modification of the cowpea bruchid / artificial seed model bioassay system: This project interacts with a
project focusing on understanding insect / plant interactions at the molecular level (Coop: B.R. Pittendrigh & L.L. Murdock). One modification of the bioassay system (which incorporates the Purdue Insect Feeding Monitor) continues to be used for attempting to identify cowpea bruchid genes that are being regulated when challenged by specific insecticidal proteins. The modified bioassay system provides detailed feeding behavior information on cowpea bruchid larvae when challenged by specific gene products that can be correlated with insect gene regulation. (C) Impact of the cowpea bruchid-resistant seed on the population dynamics of the cowpea bruchid (Coop: L.L. Murdock). Research results in 2001 were difficult to interpret because seed-heap temperatures generated by insect metabolic heat were much higher than expected in susceptible seeds compared to resistant seeds. The results of a second experiment in 2002 show that (because of metabolic heat) cowpea bruchid-resistant cowpea
variety TVu 2027 may provide a greater protection against the cowpea bruchid than previously thought. Also, metabolic heat is a factor that will have to be accounted for in future insect population dynamics studies conducted in stored grain heaps.
Impacts
Provide an in vivo bioassay system for the relatively rapid identification of insecticidal proteins, or other insect-active compounds. In general, the management of certain insect pests of stored grain without introducing toxic chemicals into the environment. Specifically, manage bruchid beetle pests of stored cowpeas without introducing toxic chemicals into the environment
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
The long-term objectives of this research project are 1) identifying genes whose products have a detrimental impact on insect pests, 2) developing strategies for releasing insect-resistant genes, and 3) developing biological assay systems that improve the efficiency of identifying insect-resistant genes. (A) Prospecting for insect resistance genes: The research focus has switched from mass screening crude plant extracts to screening purified fractions of crude extracts (identified in our lab as having a high level of activity against the cowpea weevil), and proteins having insecticidal activity. Avidin and Streptiavidin were found to have activity against the cowpea weevil at < 25 ppm. (Coop: L.L. Murdock) (B) Population dynamics of insect populations associated with insect resistance: When the population dynamics of the cowpea weevil were compared on susceptible California Blackeye No. 5 (CB5) and resistant TVu 2027 cowpea varieties, larval metabolic heat was a
significant factor in population expansion. Larval feeding generated sufficient metabolic heat to reduce generation time by 10 days on CB5 and 3 days on TVu 2027. This is a critical factor in comparing the benefits of resistant cowpea weevil resistant lines, and in modeling the expansion of cowpea weevil populations. (Coop: L.L. Murdock) (C) Modification of the cowpea weevil, in vivo, model bioassay system: The model bioassay system was coupled to the Purdue Insect Feeding Monitor so that larvae of a specific physiological age could be identified and challenged by selected plant defensive compounds for specific periods of time. The larval response to these compounds was accurately described by larval feeding behavior. Analyses of larval RNA sampled prior to and during the period of challenge was used to identify insect genes activated by plant defensive compounds. (Coop: B.R. Pittendrigh and L.L.Murdock)
Impacts
The potential impact of the novel bioassay will be an increased discovery rate of genes whose products have a detrimental impact on insect pests of crop plants. In the long-term, this means an increased number of genes available to plant breeders for developing crop plants which resist insect attack.
Publications
- Hisashi, K., M.P. D'Urzo, I. Machleidt, K. Zhu-Salzman, R.E. Shade, H. An, L.L. Murdock, W. Machleidt, R.A. Bressan and P.M. Hasegawa. 2001. Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of cysteine proteinase. Plant Journal, 27 (5): 383-391.
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Progress 10/01/99 to 09/30/00
Outputs
(A) Prospecting for insect resistance genes: The focus of this research has switched from screening large numbers of crude plant extracts to screening more purified fractions of 60 crude extracts that were previously identified in our lab as having a high level of activity against the cowpea weevil. About 6 of 30 extracts (at various levels of purification) still show a high level of activity against the cowpea weevil. About 75% of the crude extracts that were active against the cowpea weevil are also active against the common bean weevil. (B) Trying to understand the population dynamics of insect populations associated with insect resistance: When 3 populations from a lab-selected biotype (cowpea weevil selected against resistant cowpea TVu 2027) of the cowpea weevil were isolated, each of the populations began showing severe inbreeding depression after about 10-15 generations. Individuals from the 3 populations were intercrossed, and it appears that they are more
vigorous than the original wild-type population. (C) Modification and refinement of the cowpea weevil model bioassay system: An in vivo bioassay was developed that can provide a yes / no answer to whether a compound is active against the cowpea weevil at 500ppm using 12-13 ug of test compounds. The answer can be obtained in less than 10 days.
Impacts
Results of this research will eventually provide new genes for resistance to insect pests in the family Bruchidae (and probably other Coleoptera), and provide new methodologies for detecting resistance. Also, this research will contribute to the development of new germplasm release strategies.
Publications
- Murdock, L.L., S.S. Nielsen and R.E. Shade. 2000. Lectins and protease inhibitors as plant defense against insects. ABSTR PAP AM CHEM S 219: U47 Part 1 MAR 26, 2000.
- Koiwa, H., M.P. D'Urzo, K. Zhu-Salzman, J.I. Ibeas, R.E. Shade, L.L. Murdock, R.A. Bressan and P.M. Hasegawa. 2000. An in-gel assay of a recombinant western corn rootworm (Diabrotica virgifera virgifera) cysteine proteinase expressed in yeast. Analytical Biochem. 282: (1) 153-155.
- Koiwa, H., R.E. Shade, K. Zhu-Salzman, M.P. D'Urzo, L.L. Murdock, R. A. Bressan and P.M. Hasegawa. 2000. A plant defensive cystatin (soyacystatin) targets cathepsin L-like Digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera). FEBS LETTERS 471: (1) 67-70.
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Progress 10/01/98 to 09/30/99
Outputs
(I) Prospecting for insect resistance genes: (1) Seeds of Ca. 100 legume accessions (850 total) were collected from the tropics and sub-tropics. Crude seed extracts from ca. 100 legume accessions (650 total) were screened for insecticidal activity using a cowpea weevil (CPW) model bioassay system. Ca. 18 (93 of 650 total) of the 100 crude extracts screened were CPW-active. Ca. 9 of the 18 extracts had at least one active fraction. Plans are to screen seed extracts of 150 new legume accessions, and isolate proteins from the remaining CPW-active fractions during 1999-2000. (2) In some cases, CPW-active crude extracts are screened for activity against other stored grain pests such as the common bean weevil. (3) All CPW-active crude extracts were also screened for activity against the western corn rootworm (L. Murdock). Screening CPW-acitve extracts for western corn rootworm activity will not continue through 1999-2000. (II) Evaluation of gene products using a cowpea
weevil model system: (1) The insecticidal activity of cysteine proteinase inhibitors (alone and in combination with other compounds) are being evaluated for additivity and synergy (S. Nielsen, R. Bressan & L. Murdock). (2) Bioassays of several phage-selected forms of a CPW-active cysteine protease inhibitor are being evaluated to determine whether more potent forms can be evolved (H. Koiwa, M. Hasegawa, R. Bressan & L. Murdock). (III) Gene Deployment Strategies: Cowpea weevil populations from diverse environments may be similar to inbred lines. Heterosis may occur when diverse populations are hybridized (L. Murdock & L. Kitch). (1) We will continue trying to understand the population dynamics of insects where diverse insect populations (within species) interface (biotype X wild-type interactions). A hybrid population may not only be more virulent to a previous source of resistance, but may be more vigorous and difficult insect to deal with in general. (2) We will continue trying to
understand insect and plant variability associated with plant resistance and insect biotypes. (IV) Modification and refinement of the cowpea weevil model bioassay system: (1) Over the past two years, our standard artificial cowpea seed was reduced from 500 mg to 28 mg, greatly reducing the amounts of test compound required, and at the same time, variation in larval feeding rates was reduced. It is now possible rear single larvae in a 15 mg cowpea pellet. These modifications allow us to reliably identify CPW-acitve compounds in vivo (active at 500ppm) using less than 40 ug of a test compound. In conjunction with the Purdue Insect Feeding Monitor, CPW-active compounds can be identified within 10 days of egg hatch. (2) We are now working on a in vivo bioassay that will provide a yes/no answer in 5 days to whether a compound has activity against the cowpea weevil at 1000ppm. (V) Development of the Purdue Insect Feeding Monitor: (1) We continue attempting to acquire more information from
the ultrasonic emissions detected by the feeding monitor. Over the next year, we will continue trying to develop procedures for delineating cowpea weevil larval instars. (Furgason and Murdock).
Impacts
The development of the biomonitor bioassay has made it possible to screen large numbers of plant extracts for insecticidal proteins in a relatively short time. At least two insecticidal proteins are ready to move into legume seeds once a plant regeneration system becomes available. With a better understanding of insect / plant interactions (evolution of insect biotypes) it will be possible to use newly discovered genes more efficiently.
Publications
- Shade, R. E., L. L. Murdock and L. W. Kitch. 1999. Interactions between cowpea weevil (Coleoptera: Bruchidae) populations and Vigna (Leguminosae) species. J. Econ. Entmol. 92(3): 740-745.
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Progress 10/01/97 to 09/30/98
Outputs
Prospecting for insect resistance genes: (1) Over 750 legume accessions have been collected from the humid and arid tropics and sub-tropics. Seed crude extracts from over 550 legume accessions have been screened for insecticidal activity using a cowpea weevil (CPW) model bioassay system. Ca. 75 of the 550 crude extracts screened were CPW-active. Ca. 55 of the CPW-active crude extracts had at least one active fraction. Three CPW-active proteins have purified.). Our plan is to screen seed extracts from at least 150 more new legume accessions, and isolate proteins from the remaining CPW-active fractions during 1998. (2) All CPW-active crude extracts (and in many cases more purified fractions) are being screened for activity against other stored grain pests such as common bean weevil and lesser grain borer. (3) All CPW-active crude extracts are also being screened for activity against the western corn rootworm (L. Murdock). Evaluation of gene products using a cowpea
weevil model system: (1) The insecticidal activity of cysteine proteinase inhibitors (alone and in combination with other compounds) are being evaluated for additivity and synergy (S. Nielsen, R. Bressan & L. Murdock). (2) Bioassays of several phage-selected forms of a CPW-active cysteine protease inhibitor are being evaluated to determine whether more potent forms can be evolved (H. Koiwa, M. Hasegawa, R. Bressan & L. Murdock). Gene Deployment Strategies: Cowpea weevil populations from diverse environments may be similar to inbred lines. Heterosis may occur when diverse populations are hybridized (L. Murdock & L. Kitch). We will continue to focus on understanding the population dynamics of insects where diverse insect populations (within species) interface (biotype X wild-type interactions). A hybrid population may not only be more virulent to a previous source of resistance, but a more vigorous, more difficult to deal with, insect may result.Modification and refinement of the cowpea
weevil model bioassay system: Over the past two years, our standard artificial cowpea seed was reduced from 500 mg to 28 mg, greatly reducing the amounts of test compound required, and at the same time, variation in larval feeding rates was reduced. It now appears possible to reduce the required amount of test compound to 15 mg/ replication (1 CPW larva completing development consumes ca. 15 mg of seed tissue). These modifications allow us to reliably identify CPW-acitve compounds in vivo (active at 500ppm) using only 35 ug of a test compound. CPW-active compounds can be identified by monitoring (Purdue Insect Feeding Monitor) CPW feeding behavior for 15 min. at from 2-10 days post egg hatch (Murdock). Development of the Purdue Insect Feeding Monitor: We continue attempting to acquire more information from the ultrasonic emissions detected by the feeding monitor. Over the next year, we will attempt to develop procedures for aging cowpea weevil larvae (i. e. delineation of larval
instars) (Furgason and Murdock).
Impacts
(N/A)
Publications
- Koiwa, H., Shade,R.E., Zhu-Salzman, K., Subramanian,L., Nielsen,S.S., Murdock, L.L., Bressan, R.A. and Hasegawa,P.M. 1998. Phage display selection can differentiate insecticidal activity of soybean cystatins. Proceedings of the National Academy of Science (In Press).
- Shade, R. E., Murdock, L.L. and Kitch, L. 1998. Interactions between cowpea weevil populations and Vigna species. (Submitted to J. Econ. Entomol, AES# 15773).
- Zhu-Salzman, K., Shade, R.E., Koiwa, H. R., Salzman, A., Narasimhan, M., Bressan, R.A., Hasegawa, P.M. and Murdock,L.L. 1998. Carbohydrate-binding and resistance to proteoloysis control insecticidal activity of Griffonia simplicifolia Lectin II (GS-II). Proceedings of the National Academy of Sciences (in press).
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Progress 10/01/96 to 09/30/97
Outputs
Prospecting for insect resistance genes: Over 700 legume accessions have been collected from the humid and arid tropics and sub-tropics. Seed extracts from over 500 legume accessions have been screened fo insecticidal activity using a cowpea weevil model bioassay system. Ca. 60 crude extracts were CPW-active. Ca. 75% of the CPW-active crude extracts had at least one active fraction. Further fractionation and protein purification is in progress. All 60 CPW-active crude extract (and in some cases more purified fractions) will be screened for activity against other stored grain pests such as the common bean weevil and lesser grain borer. CPW-active crude extracts are also being screened for western corn rootworm activity (L. Murdock). Our plan is to screen seed extracts from at least 200 more legume accessions (currently in cold storage) during 1998. (2) Cowpea seed (from plants having been through a tissue culture cycle) that were selected from variety 'TARS 36' for
resistance to the cowpea weevil and azuki bean weevil were intercrossed and self-pollinated. A large sample of the resulting progeny showed no resistance. The remaining progeny will be evaluated in 1998 (R. Bressan & P. Hasegawa).Evaluation of gene products using a cowpea weevil model system: (1) The insecticidal activity of three soybean cysteine proteinase inhibitors was characterized using dose response studies (S. Nielsen, R. Bressan & L. Murdock). (2) Bioassays of several mutant forms of recombinant Griffonia simplicifolia (GS-II gene) proteins showed that insecticidal activity was reduced by site specific mutations (L. Murdock & K. Zhu). Gene Deployment Strategies: It appears that cowpea weevil populations from diverse enviroments behave similarly to inbred lines, and heterosis occurs when populations are hybridized (L. Murdock & L. Kitch). We will continue focus this phase of our research on understanding the population dynamics of insects where diverse insect populations
(within species) interface (biotype X wild-type interactions). Modification and refinement of the cowpea weevil model bioassay system: Our standard artificial cowpea seed was reduced from 500 mg to a compressed pellet of 28 mg, greatly reducing the amounts of test compound required and reducing the sample variation in larval feeding rates. CPW-active compounds can be identified by monitoring (Purdue Insect Feeding Monitor) CPW feeding behavior in the 28 mg pellets for 15 min. at ca. 10 days post egg hatch. These modifications allow us to identify CPW-acitve compounds in vivo (active at 1000ppm) using only 225 ug of a test compound.
Impacts
(N/A)
Publications
- PITTENDRIGH, B.R., HUESING, J. E., SHADE, R. E., and L. L. MURDOCK. 1997. Effects of lectins, CRY1A/CRY1B Bt delta-endotoxin, PAPA,protease and alpha-amylase inhibitors, on the development of the rice weevil, Sitophilus oryzae, using an artificial seed bioassay. Entomologia Experimentalis et Applicata. Vol. 82: 201-211.
- PITTENDRIGH, B.R., HUESING, J. E., SHADE, R. E. and L. L. MURDOCK.1997. Monitoring of rice weevil, Sitop[hilus oryzae, feeding behavior in maize seeds and the occurrence of supernumerary molts in low humidity conditions. Entomologia Experimentalis et Applicata. Vol. 83: 225-231.
- NTOUKAM, G., L.W. KITCH, R.E. SHADE and L.L. MURDOCK. 1997. A novel method for conserving cowpea germplasm and breeding stocks using solar disinfestation. J. Stored Prod. Res. Vol. 33: 175-179
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Progress 10/01/95 to 09/30/96
Outputs
Prospecting for insect resistance genes: (1)Seed extracts from >200 legume sp. collected from the humid and arid tropics and sub-tropics have now been screened for insecticidal activity using a cowpea weevil model bioassay system. Ca. 10% of extracts show activity and will be further fractionated and assayed(L. Murdock). (2)Seeds from T2 and T3 cowpea plants (not transformed) are being evaluated for resistance (somaclonal variation) using the azuki bean weevil and cowpea weevil. Number of "resistant" seeds observed is significantly higher than expected by chance (p>.001) (R.Bressan & P. Hasegawa). Evaluation of gene products using a cowpea weevil model system: (1) The insecticidal activity of three soybean cysteine proteinase inhibitors is being characterized(S. Nielsen, R. Bressan & L. Murdock). (2)A comparison of a native and recombinant protein from Griffonia simplicifolia (GS-II) showed the recombinant significantly less active than native(L. Murdock & K. Zhu).
Gene Deployment Strategies: Biology of selected cowpea weevil populations was characterized on cowpea-weevil-resistant Vigna species. There are significant differences between cowpea weevil populations for virulence to resistance,and a significant difference in resistance levels between accessions within a species.
Impacts
(N/A)
Publications
- PITTENDRIGH, B.R., HUESING, J. E., SHADE, R. E., and L. L. MURDOCK. (1995). Artificial seed system for the rice weevil, Sitophilus oryzae (L.). Submitted toEntomologica experimentalis et applicata (Accepted forpublication).
- PITTENDRIGH, B.R., HUESING, J. E., SHADE, R. E. and L. L. MURDOCK. 1995). Supernumerary molts in the rice weevil, Sitophilus oryzae(L.) submitted to Oecologia(Accepted for publication).
- SHADE, R.E., KITCH,L. W., MENTZER, P. E. and L. L. MURDOCK. 1996. Selection of a cowpea weevil biotype virulent to cowpea weevil resistant Landrace TVu 2027. J. Econ. Entomol.89(5): 1325-1331.
- ZHU, K., HUESING, J. E., SHADE, R.E., BRESSAN, R. A., HASEGAWA, P. M. and L. L. MURDOCK. 1996. An Insecticidal GlCNac - specific lectin gene from Griffonia simplicifolia (Leguminoseae). Plant Physiol. 110:195-202.
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Progress 10/01/94 to 09/30/95
Outputs
Prospecting for insect resistance genes: (1)Seed extracts from 100 legume sp. collected in TX, Puerto Rico and Honduras are being screened for activity using a cowpea weevil model bioassay system. Ca. 10% of extracts show activity and will be further fractionated and assayed(L. Murdock). (2)Seeds from T2 and T3 cowpea plants (not transformed) are being evaluated for resistance (somaclonal variation) using the azuki bean weevil. No. of "resistant" seeds observed is significantly higher than expected by chance (p>.001)(R.Bressan & P. Hasegawa). Evaluation of gene products using a cowpea weevil model system: (1) The activity of three soybean cysteine protease inhibitors is being characterized(S. Nielsen, R. Bressan & L. Murdock). (2)A comparison of a native and recombinant protein from Griffonia simplicifolia (GS-II) showed the recombinant significantly less active than native(L. Murdock & K. Zhu). Gene Deployment Strategies: Biology of selected cowpea weevil ecotypes is
being characterized on cowpea-weevil-resistant Vigna species. There are significant differences between cowpea weevil ecotypes for virulence to resistant V. unguiculata, V.oblongifolia, V. luteola, and V. vexillata. Insect variants: A large variant of the cowpea weevil collected in Maroua, Cameroon appeared in a laboratory culture population. This variant is ca. 30% heavier and has a net replacement rate twice that of the wild-type. Size of variant is clearly inherited, but the genetics are not fully understood.
Impacts
(N/A)
Publications
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Progress 10/01/93 to 09/30/94
Outputs
Bioassay Development:(1) A more rapid, efficient bioassay (the MP (16) system) was developed using the Purdue Insect Feeding Monitor. Compound required for assay was reduced from 35 to 2 mg, and time from 50 to 10 d. The MP(16) system was used determine activity of rGS-II against the CPW(Coop.L. Murdock & K. Zhu).(2) An assay using the azuki bean weevil (ABW), highly sensitive to alph-amylase inhibitor(AAI), was developed for screening potential AAI-transformed cowpea seeds (four man-hours / month / >5,000 seeds)(Coop. L. Murdock). Ca. 7,000 T1 and 1000 T2 seeds have been screened using the ABW (Coop. R. Bressan & M. Hasegawa). Gene discovery for insect resistance: Plant extracts (local and exotic plants) at various levels of purification are moving through our bioassay system. Plant exploration in Texas, focusing on legumes, yielded seed from ca. 30 sp.(Coop. L. Murdock). Gene Deployment Strategies: The biology of 20 CPW ecotypes is being characterized to determine
the diversity within the CPW species. One ecotype from Nigeria is as virulent CPW resistant cowpea 'TVu 2027' as our laboratory-selected biotype was after 40 generations of selection. When varieties carrying the TVu 2027 gene are released within the range of the Nigeria ecotype they will appear susceptible, and when released within the range of the Cameroon ecotype will appear highly resistant. Resistance to insect populations, rather than species will need to be recognized when deploying resistant germplasm (L. Murdock & L Kitch).
Impacts
(N/A)
Publications
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Progress 10/01/92 to 09/30/93
Outputs
Bioassays: (A) Screening Bt's detected 3 with activity against the cowpea weevil(CPW). CryIA/IB caused delayed development and increased mortality at 25 ug/g (Coop. L. Murdock and W. Moar, Auburn U.). (B) The CPW and azuki bean weevil (ABW) were used to identify transgenic garden pea seeds expressing the alpha-amylase inhibitor (AAI-Pv) gene. Seeds with high % AAI-Pv (bioassay) contained high % AAI-Pv in chemical assay. More than half of the seeds were immune to the CPW and ABW (Coop. L. Murdock; M. Chrispeels, U. Cal. San Diego; and T. Higgins, CSIRO, Australia). (C) Screening plant extracts for bruchid-active proteins: Various taxa were screened, with emphasis on legumes. Of several compounds studied, GSA-II is most interesting (Coop. L. Murdock, J. Huesing). Development of Bioassays: Corn rootworm larvae feeding on corn callus survived to instar II (7 d.) before fungal contamination destroyed the callus. Larval wt. was >100 times the wt. of neonate larvae. Wt. gain
and developmental stage may be used to identify transgenic callus (Coop. L. Murdock, R. Bressan and M. Hasegawa). Factors affecting expression of bruchid-resistant TVu 2027: Experiments show that location of seed production field, seed moisture during dry matter accumulation, seed storage moisture, and insect ecotype, affect the expression of resistance. TVu 2027 seed grown and stored under high moisture in S. Nigeria would appear susceptible, while seed grown and stored in low moisture in S. Niger would appear resistant.
Impacts
(N/A)
Publications
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Progress 10/01/91 to 09/30/92
Outputs
(I) Biomonitor : Newest Purdue Insect Feeding Monitor (PIFM) in operation. With PIFM, 1 technician can screen 8000 cowpea (CP) lines/year for cowpea weevil (CPW) resistance. When PIFM and standard bioassay results were compared, the PIFM identified resistance more quickly, and with more information. (Coop. L. Murdock and E. Furgason) (II) Developing Gene Deployment Strategies: (1) CPW Biotype: After 77 generations of selection on CPW-resistant CP var. TVu 2027, we have a biotype virulent to TVu 2027. The biotype is being used to identify new genes for CPW resistance. Three CPW-resistant Vigna sp. are resistant to the biotype, and assumed to have non-TVu 2027 resistance genes. (Coop. L. Kitch and L. Murdock) 2) Cowpea weevil Ecotypes: We have observed a CPW population from Nigeria with virulence equal to our biotype at 40 generations of selection. TVu 2027-derived varieties will appear susceptible if released within Nigerian ecotype range. We are characterizing CPW
ecotypes and using them to screen plant-defense gene products before they are engineered into plants. To date, there has been no differential ecotype response to several gene products. (III) Bioassays (Maruca testulalis X Bacillus thuringiensis): Three Bt proteins showed high activity against the Maruca pod borer larvae. The concentrations of CryIA(b), CryIC and CryIIA required to kill 100% of the larvae were 0.1, 0.6 and 1.0 ug/ml Mt diet respectively. (Coop. L. Murdock and W. Moar, Auburn Univ.).
Impacts
(N/A)
Publications
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Progress 10/01/90 to 09/30/91
Outputs
Biomonitor (Ultrasonic insect feeding monitor): A protocol developed for identifying Vigna seeds resistant to the cowpea weevil (CPW) is also suitable for identifying Vigna seeds resistant to the azuki bean weevil (Coop. L.L. Murdock and A. Bahagiawati) Development of biomonitor techniques for characterizing CPW responses to single-gene plant products (lectins, proteinase inhibitors and alpha-amylase inhibitors) continues. (Coop. L.L. Murdock and K. Zhu) A new, more sensitive, more user-friendly biomonitor with greater capacity was completed and is operating. (Coop. L.L. Murdock, E.S. Furgason, P.E. Mentzer, and L. Sudlow) Biological Assay (CPW X Vigna: Artificial seed assays have identified new, potent, single-gene products (lectin and alpha-amylase inhibitor) having a detrimental effect on CPW larvae. Assays using artificial and intact seeds have detected significant differences in CPW ecotype responses to single-gene products and resistant Vigna seed. Ecotypes
(insect differentials) will be used to help characterize resistance. (Coop. L.L. Murdock, S.S. Nielsen, J.E. Huesing and J. Parker) Biological Assay (Granary Weevil X Maize): An artificial seed assay for the adult GW is completed, but the larval assay is not. When oviposition can be controlled on artificial maize seeds, we will begin evaluating candidate compounds (those effective against the CPW) for their affects on GW biology. (Coop. L.L.
Impacts
(N/A)
Publications
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Progress 10/01/89 to 09/30/90
Outputs
Biomonitor (an ultrasonic device for detecting and quatitatively measuring insect feeding within fibrous substrates): A seed screening protocol was developed for the biomonitor to rapidly identifying cowpea cowpea weevil (CPW) resistance cowpea seeds. One set of 30, 10-s. samples was sufficient to identify resistant TVu 2027 seeds. Larval feeding rates were 50% lower on resistant than on susceptible seeds, while the coefficient of variation on resistant seeds was double that on susceptible seeds. Using this same protocol to screen representative accessions of three known sources of resistance, all resistant lines were identified. Biomonitor techniques are being developed for characterizing larval feeding behavior and larval biology when larvae are red compounds having an adverse affect on the insect's biology. When CPW larvae fed on artificial seeds containing wheat germ aglutinin (a lectin) and E-64 (a cysteine proteinase inhibitor), the biomonitor results showed
a characteristic response for each compound (Coop. L.L. Murdock). Identifying Sources of Resistance: accessions of several wild cowpea species were screened for CPW resistance. Results showed that some accessions of: 1) V. vexillata were immune, 2) V. oblongifolia were highly resistant, and 3) V. luteola were moderately resistant (Coop. L.W. Kitch and L.L. Murdock).
Impacts
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Progress 10/01/88 to 09/30/89
Outputs
Biomonitor: An ultrasonic device (pat. no. 4,809,554) that detects and measures feeding rates of insects feeding within fibrous substrates, such as seeds was developed (Co-inventors: E. S. Furgason and L. L. Murdock). Biomonitor studies show that: (1) insect feeding is detected when they bite or tear their food substrate; (2) the number of signals generated by cowpea weevil (CPW) larvae/unit time is proportional to the number of insects present; (3) many different insect species can be detected; and (4) resistant and susceptible cowpea seeds can be identified by monitoring seeds for 10 min. These results suggest that the biomonitor has many potential uses: (a) detection of hidden insect infestations (IPM); (b) screening for insect resistance; (c) insect life history studies; and (d) measuring the impact of factors (temperature, fumigants, and food chemicals) on insect feeding. Solar Heater: A simple solar heater which can generate sufficient heat to kill CPW and
common bean weevil infestations in their host seeds has been developed. Temperatures of 60C for 1 hr, necessary for killing all stages of the CPW can be achieved using the solar heater. The solar heater has potential for managing stored grain pests, especially in developing countries where small quantities of grain are handled (Coop: L.L. Murdock). Biological Assays: Assays of potential plant defense chemicals suggest that certain plant lectins have a detrimental affect on cowpea weevil developmental rate and mortality (Coop: L. L. Murdock and J.E.
Impacts
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Progress 10/01/87 to 09/30/88
Outputs
Development of a cowpea weevil (CPW) biotype virulent to mature seed-resistant cowpea variety TVu 2027 (Coop.: L. L. Murdock and L. W. Kitch). After 40 generations (4 years) of severe selection pressure (laboratory) on a wild CPW population from Niger, West Africa, with CPW-resistant variety TVu 2027, a biotype (CPW-B) has evolved; suggesting a potential for field selection of a biotype. The CPW-B is also virulent to resistant cowpea varieties KNW and KNS; suggesting all three varieties have the same genes for resistance (findings support earlier genetic studies). Wild Vigna sp., previously identified as resistant to the CPW, are also resistant to the CPW-B, and are considered new sources of CPW resistance. (2) Assessing the interaction between the cowpea weevil and proteinase inhibitors (Coop: L. L. Murdock and S. S. Nielsen). The detrimental effects of a thiol proteinase inhibitor were reversed by supplementing the CPW's diet with free amino acids; suggesting that
the proteinase inhibitor was inhibiting the insect's digestive enzymes. (3) An ultrasonic device for monitoring insects concealed within plant tissues (Patent to be issued: Co-inventor with L. L Murdock and E. S (Furgason). This ultrasonic device has been demonstrated to detect the feeding of several species of stored grain insects within seeds of different plant species.
Impacts
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Progress 10/01/86 to 09/30/87
Outputs
Introgressing tepary bean resistance to the bean weevil, Acanthoscelides obtectus, into common beans (Coop.: R. C. Pratt.) The bioassay of (Phaseolus vulgaris X P. acutifolius) showed some F(4)BC(2) individuals with testal resistance and some with cotyledon resistance to the bean weevil. Studies now in progress will identify those individual genotypes with both testal and cotyledon resistance. Bases of resistance in TVu 2027 to the cowpea weevil, Callosobruchus maculatus, (Coop.: L. L. Murdock). When the physical structure of TVu 2027 was destroyed by milling, resistance to C. maculatus vanished. Also, when TVu 2027 seeds were softened by increasing seed moisture, resistance to the cowpea weevil was significantly reduced. These results suggest that nature of resistance is biophysical, such as hardness or toughness of seed tissue. Assessing the vulnerability of the cowpea weevil and bean weevil to protease inhibitors (Coop.: L. L. Murdock and S. S. Nielsen). The
within-seed developmental time, mortality, and weight gain of the bean weevil and cowpea weevil were adversely affected when they were fed artificial seeds containing a thiol protease inhibitor (E-64) and certain serine protease inhibitors. When bean weevil larvae were fed both a serine and thiol protease inhibitor, the effects were synergistic.
Impacts
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Progress 08/01/85 to 07/30/86
Outputs
A precision bioassay system has been developed which can be utilized for characterizing the biology of the cowpea weevil and bean weevil, and for conventional screening of cowpea and common bean seeds for insect resistance. The bioassy system has been used to identify a new source of resistance in tepary beans to the bean weevil, and to identify highly resistant cowpea seeds within a population of seeds from the resistant cowpea variety TVu2027. An artificial seed system has been developed that, based on the responses of the cowpea weevil and bean weevil (mortality, fecundity, rate of development, adult longevity and adult weight), mimics a cowpea seed and common bean seed. The artificial seed system has been used to discover compounds which have an adverse effect on the biology of the cowpea weevil and bean weevil, and establish dosage X insect-response curves for these compounds. The artificial seed system has also been used to partially identify the nature of the
resistance in resistant cowpea variety TVu2027.
Impacts
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