Response to selection for grain yield and correlated response for grain size and earliness in cowpea based on early generation testing

Large grain size and earliness to flowering are traits critical to cowpea adoption in the West African savannahs. This study reports on the correlated response to selection in these two traits in the two populations in which selections were made primarily for grain yield potential. Further, the present study reports on the effectiveness of early generation selection for grain yield. Each of the two populations, SARC 2 and SARC 3, was derived from a cross between an adapted parent Marfo-Tuya and an exotic breeding line that has large grain size and early flowering. Replicated yield evaluation of F₅ families showed that grain yield of Marfo-Tuya was not different from those of the highest yielding families in each population. Increases in grain size of individual families over Marfo-Tuya were large and, the response to selection averaged 5.3 and 3.9 g/100 seeds in the SARC 2 and SARC 3 populations, respectively. Response to selection in days to flowering averaged 3 days in SARC 3, whereas response to selection was not observed in SARC 2. Retrospective selection in the F₃ at 40% intensity was efficient in identifying a high proportion of elite families in each population.     

Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation

Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasiteswith a broad host range which includes economically importantcrop species. Cowpea (Vigna unguiculata L. Walp) is an importantfood and fodder legume grown in many regions where root-knotnematodes are a major problem in production fields. Severalsources of resistance to root-knot nematode have been identifiedin cowpea, including the widely used Rk gene. As part of a studyto elucidate the mechanism of Rk-mediated resistance, the histologicalresponse to avirulent M. incognita feeding of a resistant cowpeacultivar CB46 was compared with a susceptible near-isogenicline (in CB46 background). Most root-knot nematode resistancemechanisms in host plants that have been examined induced ahypersensitive response (HR). However, there was no typicalHR in resistant cowpea roots and nematodes were able to developnormal feeding sites similar to those in susceptible roots upto 9–14 d post inoculation (dpi). From 14–21 dpigiant cell deterioration was observed and the female nematodesshowed arrested development and deterioration. Nematodes failedto reach maturity and did not initiate egg laying in resistantroots. These results confirmed that the induction of resistanceis relatively late in this system. Typically in pathogen resistanceHR is closely associated with an oxidative burst (OB) in infectedtissue. The level of reactive oxygen species release in bothcompatible and incompatible reactions during early and latestages of infection was also quantified. Following a basal OBduring early infection in both susceptible and resistant roots,which was also observed in mechanically wounded root tissues,no significant OB was detected up to 14 dpi, a profile consistentwith the histological observations of a delayed resistance response.These results will be useful to design gene expression experimentsto dissect Rk-mediated resistance at the molecular level. Key words: Cowpea, histology, hypersensitive response, Meloidogyne incognita, reactive oxygen species, root-knot nematode, Vigna unguiculata Received 5 November 2007; Revised 22 January 2008 Accepted 23 January 2008     

Cowpea mosaic virus nanoscaffold as signal enhancement for DNA microarrays

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.     

Chemical Composition of the Essential Oil of Nigeria Grown Hoslundia opposita Vahl (Lamiaceae) Dried Leaves and Its Bioactivity against Cowpea Seed Bruchid

Due to several ecological and human hazards of synthetic pesticides in postharvest crop protection, there is the need to search for eco‐friendly alternatives. In this study, chemical composition and insecticidal activities of essential oil (EO) obtained from Hoslundia opposita dried leaves were evaluated against cowpea seed bruchid. Eight constituents, predominated by oxygenated monoterpenes (78.86%), were identified using Gas Chromatography (GC)/MS. The constituents were 1,8‐cineole ( 1 ; 61.15%), followed by α‐terpineol ( 2 ; 16.81%), β‐phellandrene ( 3 ; 13.24%), β‐farnesene (4; 3.55%), α‐pinene ( 5 ; 1.89%), Germacrene D ( 6 ; 1.83%), cis‐sabinene hydrate ( 7 ; 0.90%) and caryophyllene ( 8 ; 0.63%). In fumigation bioassay, at 6 h after exposure (HAE), 0.78 ml EO/l air caused 35.33% mortality which was significantly lower than 60.90% and 63.6% observed at 3.15 and 6.25 ml/l air, respectively. Mortality reached 90.0% at 24 HAE regardless of the applied concentration. Lethal time for 50% of the bruchids (LT₅₀) at concentration of 0.78 ml/l air (6.89 h) was higher than the LT₅₀ at 3.15 and 6.25 ml/l air (4.72 and 4.44 h, respectively). H. opposita EO reduced Callosobruchus maculatus oviposition, while progeny emergence observed in EO‐treated seeds (2.42 – 25.73) was significantly (P < 0.05) lower than 51.56 observed in control. The results confirm H. opposita EO's potentials for control of cowpea bruchids.     

A wounding-induced PPO from cowpea (Vigna unguiculata) seedlings

Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH(4))(2)SO(4) precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (K(m)=9.86mM, V(max)=24.66 EU [DeltaAmin(-1)]) and catechol (K(m)=3.44mM, V(max)=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.     

Identification of AFLP markers linked to resistance of cowpea (Vigna unguiculata L.) to parasitism by Striga gesnerioides

AFLP and bulked segregant analysis were used to identify molecular markers linked to resistance of cowpea [Vigna ungiculata (L.) Walp.] to parasitism by Striga gesnerioides (Willd.) Vatke. Segregation analysis of F₂ progeny from a cross of Tvx3236, a Striga-susceptible line, with IT82D-849, a resistant cultivar, showed that resistance to S. gesnerioides race 1 from Burkina Faso was controlled by a single dominant gene, designated Rsg2–1. Three AFLP markers were identified that are tightly linked to Rsg2–1: E-AAC/M-CAA₃₀₀ (2.6 cM), E-ACT/M-CAA₅₂₄ (0.9 cM), and E-ACA/M-CAT_140/150 (0.9 cM), which appears to be codominant. Segregation analysis of a different F₂ population resulting from a cross of the Striga-susceptible line IT84S-2246–4 with Tvu 14676, a S. gesnerioides race 3 resistant line, showed that resistance to S. gesnerioides race 3 was also controlled by a single dominant gene, designated Rsg4–3. Six AFLP markers linked to Rsg4–3 were identified: E-ACA/M-CAG₁₂₀ (10.1 cM), E-AGC/M-CAT₈₀ (4.1 cM), E-ACA/M-CAT₁₅₀ (2.7 cM), E-AGC/M-CAT₁₅₀ (3.6 cM), E-AAC/M-CAA₃₀₀ (3.6 cM), and E-AGC/M-CAT₇₀ (5.1 cM). Segregation analysis of the E-AAC/M-CAA₃₀₀ and E-ACA/M-CAG₁₂₀ markers in recombinant inbred lines derived from IT84S-2049×524B determined that both are located within linkage group 1 of the cowpea genetic map. The identification of AFLP markers linked to Striga resistance provides a stepping stone for a marker-assisted selection program and the eventual cloning and characterization of the gene(s) encoding resistance to this noxious parasitic weed. Received: 24 April 2000 / Accepted: 21 August 2000     

Pseudo-atomic models of swollen CCMV from cryo-electron microscopy data

The capsid of cowpea chlorotic mottle virus (CCMV) can reversibly switch between two forms that are contingent on the charge of acidic residues that are clustered at the quasi-threefold axes of the T=3 icosahedral particle. The quaternary structure conformations are dependent on divalent metal ions and pH and were previously analyzed by crystallography in the native, compact form, and by cryo-electron microscopy in the compact and swollen forms (Speir et al., 1995). In this report we use the atomic models of the three structurally unique viral subunits determined by crystallography for a detailed interpretation of the 28-A-resolution electron density of the swollen form and the production of a pseudo-atomic model of this particle. The model of the quaternary structure conforms with high fidelity to conventional geometric constraints, quasi-equivalence, intersubunit association energies, and the electron density. It was derived by conserving the pentamers and hexamers of subunits whose associated electron densities are strikingly similar in the two forms of the particles. Treating these as rigid units in the modeling implies that the particle flexibility is accommodated primarily by changes in dimer interactions, an observation that is consistent with the flexible C-terminal polypeptide extensions that stabilize this contact in the crystal structure. Because the hexamers and pentamers were incrementally translated and rotated in a screw motion, with energy minimization at each of 28 steps, a path for the expansion is also implied.     

Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.     

Acetyl substitution patterns of amylose and amylopectin populations in cowpea starch modified with acetic anhydride and vinyl acetate

To study the effect of reagent type on the distribution pattern of acetyl groups in acetylated cowpea starch, amylose and amylopectin populations were isolated from the starch granules after modification to a low degree of substitution (DS < 0.1) with acetic anhydride and vinyl acetate, respectively. Slowly reacting reagent vinyl acetate resulted in higher DS values for the amylopectin populations when compared to the rapidly reacting reagent acetic anhydride. The two reagents had similar effects on the acetylation level of amylose, suggesting that the amorphous regions of granules were easily accessible for both reagents. The acetyl substitution patterns were analyzed by enzymatic degradation followed by characterization of the obtained fragments using chromatographic and mass spectrometric techniques. The distributions of acetyl groups along the amylose and amylopectin chains were more clustered for modification with vinyl acetate as compared with modification with acetic anhydride. Between the two acetylation types, pronounced differences in the acetyl substitution patterns were observed for the large fragments obtained after -amylase digestion; only slight differences were exhibited for the small fragments obtained by exhaustive enzymatic digestion of amylose and amylopectin populations.     

Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo

Protein cage nanoparticles have the potential to serve as multifunctional cell targeted, imaging and therapeutic platforms for broad applications in medicine. However, before they find applications in medicine, their biocompatibility in vivo needs to be demonstrated. We provide here baseline biodistribution information of two different spherical protein cage nanoplatforms, the 28 nm viral Cowpea chlorotic mottle virus (CCMV) and the 12 nm heat shock protein (Hsp) cage. In naive and immunized mice both nanoplatforms show similar broad distribution and movement throughout most tissues and organs, rapid excretion, the absence of long-term persistence within mice tissue and organs, and no overt toxicity after a single injection. These results suggest that protein cage based nanoparticles may serve as safe, biocompatible, nanoplatforms for applications in medicine.