Superoxide generation in extracts from isolated plant cell walls is regulated by fungal signal molecules

ABSTRACT Fractions solubilized with NaCl from cell walls of pea and cowpea plants catalyzed the formation of blue formazan from nitroblue tetrazolium. Because superoxide dismutase decreased formazan production by over 90%, superoxide anion (O(2) ) may participate in the formation of formazan in the solubilized cell wall fractions. The formazan formation in the fractions solubilized from pea and cowpea cell walls was markedly reduced by exclusion of NAD(P)H, manganese ion, or p-coumaric acid from the reaction mixture. The formazan formation was severely inhibited by salicylhydroxamic acid and catalase, but not by imidazole, pyridine, quinacrine, and diphenyleneiodonium. An elicitor preparation from the pea pathogen Mycosphaerella pinodes enhanced the activities of formazan formation nonspecifically in both pea and cowpea fractions. The suppressor preparation from M. pinodes inhibited the activity in the pea fraction in the presence or absence of the elicitor. In the cowpea fraction, however, the suppressor did not inhibit the elicitor-enhanced activity, and the suppressor alone stimulated formazan formation. These results indicated that O(2) generation in the fractions solubilized from pea and cowpea cell walls seems to be catalyzed by cell wall-bound peroxidase(s) and that the plant cell walls alone are able to respond to the elicitor non-specifically and to the suppressor in a species-specific manner, suggesting the plant cell walls may play an important role in determination of plant-fungal pathogen specificity.     

Pea extracellular Cu/Zn-superoxide dismutase responsive to signal molecules from a fungal pathogen

We previously reported that the release of O₂ ⁻ from isolated pea cell walls was enhanced by a 70-kDa glycoprotein elicitor but was suppressed by mucin-type glycopeptide suppressors (supprescins A and B) prepared from pycnospore germination fluid of Mycosphaerella pinodes, causal agent of Mycosphaerella blight of pea. Here, we show that superoxide dismutase (SOD) in the apoplast fluid/cell wall of pea seedlings responds to the fungal elicitor and suppressor molecules. In a pharmacological study and with internal amino acid sequencing, the apoplastic SOD in a pea cultivar Midoriusui was found to be a Cu/Zn type SOD. We cloned a full-length cDNA of the Cu/Zn-SOD and designated it as PsCu/Zn-SOD1. An increase in PsCu/Zn-SOD1 mRNA and the PsCu/Zn-SOD1 protein was induced by treatment with the elicitor more intensively than by wounding. Such induction by the elicitor or wounding, however, was inhibited by the concomitant presence of supprescins. The SOD activity of recombinant PsCu/Zn-SOD1 was regulated directly by these signal molecules in a manner similar to their effect on the SOD activity in the apoplastic fluid and in the cell wall-bound proteins. Based on these findings, we discuss a role for PsCu/Zn-SOD1 in the pea defense response. The nucleotide sequence data of PsCu/Zn-SOD1 reported are available in the DDBJ/EMBL/GenBank databases under accession number AB189165.     

Metabolism of substituted diphenylether herbicides in plants. I. Enzymatic cleavage of fluorodifen in peas (Pisum sativum L.)

A “soluble” glutathione S-transferase that catalyzes the cleavage of the herbicide, 2,4′-dinitro-4-trifluoromethyl diphenylether (fluorodifen), was isolated and partially characterized from epicotyl tissues of pea seedlings. A 32-fold purification of the enzyme was achieved by differential centrifugation, ammonium sulfate precipitation, Sephadex gel filtration, and DEAE-cellulose ion exchange chromatography. The enzyme had a pH optimum of 9.3–9.5 and was specific for reduced glutathione, with an estimated apparent K_m value of 7.4 × 10^?4M. Limited specificity studies with four substituted ¹⁴C-labeled diphenylether compounds indicated that fluorodifen was the only effective substrate, with an estimated apparent K_m value of 1.2 × 10^?5M. Differences and similarities between the pea epicotyl enzyme and other plant and animal glutathione S-transferases were discussed from the standpoint of substrate specificity, pH optima, distribution, stability, and inhibitor studies.     

H2O2 production by copper amine oxidase, a component of the ecto-apyrase (ATPase)-containing protein complex(es) in the pea cell wall, is regulated by an elicitor and a suppressor from Mycosphaerella pinodes

Blue native PAGE analysis for cell wall proteins from pea epicotyls demonstrated that cell wall-associated ecto-apyrase (ATPase) formed a large protein complex(es) ranging from 450 to 900?kDa; one of the components of the complex was copper amine oxidase (CuAO), which catalyzes the oxidation of amines with the subsequent generation of ammonia and hydrogen peroxide. CuAO activity was coordinately regulated in vitro with ATP-hydrolyzing activity by an elicitor and a suppressor from Mycosphaerella pinodes. Moreover, treatment of cell wall proteins with the suppressor caused the appearance of the apyrase monomer. On the basis of these results, M. pinodes may target the apyrase-containing protein complex(es) of the host to attenuate cell wall-based, extracellular defense(s) including the production of hydrogen peroxide.     

Microscopic, Biochemical and Physiological Assessment of the Effect of Methylene Bisthiocyanate on the Sapstain Fungus Ophiostoma floccosum

In vitro effects of methylene bisthiocyanate (MBT) on hyphal morphology and ultra-structure of Ophiostoma floccosum were examined using differential interference contrast, epifluorescence and transmission electron microscopy (TEM). To understand the mode of action of MBT, experiments were undertaken to measure potassium ion (K⁺) leakage from cells, oxygen consumption, glucose and ATP levels. Differential interference contrast microscopy indicated that MBT caused rapid changes in O. floccosum hyphae resulting in extensive vaculoation and accumulation of granular materials within the cytoplasm. Epifluorescence microscopy provided evidence that MBT treatment causes a loss in the permeability properties of the plasma membrane. TEM showed retraction of the plasma membrane from the cell wall, aggregation of cytoplasmic contents, vesiculation of membranous components, a dramatic increase in vacuolation, and eventually a complete loss in the integrity of organelles. There was a rapid efflux of intracellular K⁺ ions from cells, a substantial loss in K⁺ ions occurring within the first 5 min of MBT treatment. The rate of K⁺ leakage was MBT concentration treatment-time dependent. The study also showed that the effect of lower concentrations of MBT (0.01 and 0.1 mM) on respiratory activity was negligible. However, at the same concentrations, glucose consumption and ATP production were affected. Taken together, these observations suggest that the target site of MBT in O. floccosum alters membrane properties and uncouples oxidative phosphorylation from the respiratory chain.     

Dynamics of conidial adhesion and elicitor uptake in relation to pisatin accumulation in aqueous droplets on the endocarp of pea pods

The adhesion of conidia of Monilinia fructicola to, and uptake of a biotic elicitor (pea-M. fructicola diffusate preparation) and two abiotic elicitors (actinomycin-D and CuCl₂) by endocarp tissue of pea pods has been investigated in relation to the elicitation of pisatin. Conidia were found to rapidly adhere to the endocarp surface and were not readily dislodged by washing. The dynamics of elicitor uptake from the bathing solutions varied with the elicitor treatments. In the case of the biotic elicitor, bioassays of the solutions remaining on the endocarp surface for residual elicitor activity indicated there was a gradual loss of elicitor from the bathing solution. By 10 h, approximately 41% of the original elicitor activity had disappeared from the bathing solution. Direct measurement of actinomycin-D in the bathing solution showed that uptake appeared to begin about 6 h after its application. On the other hand, direct measurement of Cu²⁺ in the bathing solution showed that approximately 60% of the original concentration of Cu²⁺ rapidly disappeared from the bathing solution in the first 30 min.The results highlight the need for sustained contact between plant and fungus or elicitor in the bathing fluid of the infection-droplet or of the elicitor solution for the maximum outcome of the pisatin response.     

A binding protein for fungal signal molecules in the cell wall of Pisum sativum

In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 3.6.1.5.) with ATP-hydrolyzing activity that was regulated by an elicitor and suppressors of defense from pea pathogen Mycosphaerella pinodes. The ATPase-rich fraction was purified from pea cell walls by NaCl solubilization, ammonium sulfate precipitation, and chromatography with an ATP-conjugated agarose column and an anion-exchange column. The specific activity of the final ATPase-rich fraction increased 600-fold over that of the initial NaCl-solubilized fraction. The purified ATPase-rich fraction also had peroxidase activity and generated superoxide, both of which were regulated by the M. pinodes elicitor and suppressor (supprescins). Active staining and Western blot analysis also showed that the ATPase was copurified along with peroxidases. In this fraction, a biotinylated elicitor and the supprescins were bound primarily and specifically to ca. 55-kDa protein (CWP-55) with an N-terminal amino acid sequence of QEEISSYAVVFDA. The cDNA clone of CWP-55 contained five ACR domains, which are conserved in the apyrases (NTPases), and the protein is identical to a pea NTPase cDNA (GenBank accession AB071369). Based on these results, we discuss a role for the plant cell wall in recognizing exogenous signal molecules.     

Localization and responsiveness of a cowpea apyrase VsNTPase1 to phytopathogenic microorganisms

Apyrases (NTPases) are associated with both compatible and incompatible interactions between plants and microorganisms. Previously we reported that the ATPase activities of cell-wall-bound apyrases of several leguminous plants, such as pea, cowpea, soybean, and kidney bean, were enhanced by a glycoprotein elicitor and were inhibited in a species-specific manner by mucin-type glycopeptide suppressors secreted from a pea pathogenic fungus, Mycosphaerella pinodes. In this study, we isolated two apyrase genes, VsNTPase1 and VsNTPase2, from a cDNA library of Vigna sinensis Endl. cv. Sanjakusasage. Based on phylogenetic analysis, VsNTPase1 may belong to a group that responds to environmental stimuli. In a transient assay using DNA bombardment, a fusion protein of green fluorescent protein (GFP) and the N-terminal putative signal sequence of VsNTPase1 was distributed in the nucleus, cytoplasm (cytoskeletal structure), and cell wall. On the other hand, a fusion protein of GFP and the N-terminal putative VsNTPase2-signal sequence was localized in the cytoplasm, especially in small particles (perhaps mitochondria). A recombinant VsNTPase1 expressed in Spodoptera frugiperda 21 cells responded directly to signal molecules from several phytopathogenic microorganisms. Here, we discuss the role of apyrases in recognizing and responding to exogenous signals. The nucleotide sequences of VsNTPase1 and VsNTPase2 in this article have been submitted to DDBJ as accession numbers AB196769 and AB196770, respectively.     

Characterization of herbicidal injury by acifluorfen-methyl in excised cucumber (Cucumis sativus L.) cotyledons

Initial signs of herbicidal injury by several diphenyl ether herbicides were monitored by following the efflux of ⁸⁶Rb⁺ from treated cucumber (Cucumis sativis L.) cotyledons after exposure to light (600 μE m^?2 sec^?1; measured as PAR, i.e., photosynthetically active radiation between 400 and 700 nm). This very sensitive, rapid, and quantitative bioassay proved quite useful in (a) a structure-activity correlations study of the diphenyl ether compounds investigated and (b) an examination of herbicidal characteristics. The following diphenyl ether herbicides were analyzed: acifluorfen, sodium 5-[2-chloro-4-(trifluormethyl)phenoxy]-2-nitrobenzoate; acifluorfen-methyl (MC-10108), methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate; bifenox, methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate; nitrofen, 2,4-dichlorophenyl p-nitrophenyl ether; nitrofluorfen, 2-chloro-1-(4-nitrophenoxy)-4-(trifluoromethyl)benzene; oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene; MC-7783, potassium 5-(2,4-dichlorophenoxy)-2-nitrobenzoate; and MC-10982, ethyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate. Of the compounds investigated, acifluorfen-methyl (AFM) had the greatest degree of herbicidal activity. Cucumber cotyledons placed in high light (600 μE m^?2 sec^?1; PAR) with 10 nM AFM showed a significant increase in the efflux of ⁸⁶Rb⁺ within 2 to 4 hr. Light was required for herbicidal activity by AFM, and when treated cotyledons were returned to darkness, no further damage to the tissue occurred. By decreasing the quantity of light, the effect of the compound was delayed, although the magnitudes of the responses at the different intensities (600, 300, 150, and 75 μE m^?2 sec^?1; PAR) were nearly equal. By increasing the length of time of dark pretreatment with 1 μM AFM, ⁸⁶Rb⁺ efflux could be detected as early as 10 to 15 min after exposure to light (600 μE m^?2 sec^?1; PAR). Following light activation of AFM there was a simultaneous efflux of ⁸⁶Rb⁺, ³⁶Cl^?, ⁴⁵Ca²⁺, 3-O-methyl-[¹⁴C]glucose, and [¹⁴C]methylamine⁺. These data suggest the initial response to the herbicidal activity of AFM is expressed as a general increase in membrane permeability.     

A new interspecific pear cultivar Yutaka: highly resistant to the two major diseases scab and black spot on Asian pears

Phytoplasma suspected symptoms of little leaf, flat stem, witches’ broom and leaf yellowing were recorded on the four legume species, cowpea (Vigna unguiculata (L.) Walp.), pigeon pea (Cajanus cajan (L.) Millsp.), lentil (Lens culinaris Medikus) and mung bean (Vigna radiata (L.) Wilczek) in the states of Delhi, Uttar Pradesh (UP) and Kerala from 2014 to 2016. DNA specific fragments of approximately 1.3 kb were amplified from symptomatic samples of cowpea, pigeon pea, lentil and mung bean in nested PCR assays by using two sets of universal phytoplasma nested specific primers P1/P7 followed by 3Far/3Rev. No DNA amplifications were observed in any of the non-symptomatic legume samples with same primer pairs. Pair wise sequence comparison, phylogeny and virtual RFLP analysis of 16S rDNA sequences of the four legume species confirmed the association of four different groups and subgroups of phytoplasmas in the present study. The mung bean witches’ broom at Delhi was identified to be associated with strain related to ‘Ca. P. aurantifolia’ (16SrII-D), pigeon pea little leaf at Faizabad, UP with strain related to ‘Ca. P. phoenicium’ (16SrIX-C), lentil witches’ broom at Faizabad, UP with ‘Ca. P. trifolii’ (16SrVI-D) and cow pea flat stem disease at Kerala with ‘Ca. P. cynodontis’ (16SrXIV-A). Association of ‘Ca. P. cynodontis' (16SrXIV-A) infecting cowpea, ‘Ca. P. trifolii’ (16SrVI-D) in lentil and phytoplasmas strain related to ‘Ca. P. phoenicium’ (16SrIX-C) infecting pigeon pea are the new reports to the world.     

柳枝稷种植年限对土壤盐分离子的影响

为探明能源植物柳枝稷对盐碱土的改良效果,于2014—2015年,在宁夏大学西大滩盐碱地改良试验站田间条件下,设置1、3、5 a3个不同种植年限柳枝稷处理(T1、T3、T5),以附近未种植柳枝稷的盐碱荒地作为对照(CK),研究了不同种植年限柳枝稷对盐碱土盐分离子的影响。结果表明,随着种植年限增加,柳枝稷对土壤盐分离子的作用效果增强,土壤中的Na~+、SO_4~(2-)、Cl~-含量随种植年限增加而降低,在柳枝稷生育末期T5处理与CK相比,Na~+、SO_4~(2-)、Cl~-含量分别下降了52.64%、40.37%和63.68%;土壤中的Ca~(2+)、Mg~(2+)含量与柳枝稷种植年限呈正相关关系,在柳枝稷生育末期T5处理Ca~(2+)、Mg~(2+)含量分别比CK增加了2.22%、48.08%。本试验条件下,柳枝稷种植5年后对土壤Na~+、SO_4~(2-)和Cl~-等盐分离子的降低作用最为明显。     

Studies on the systemicity of [14C]metalaxyl in cowpea (vigna unguiculata (L.) walp.)

[¹⁴C]metalaxyl applied to seeds, roots and leaves of cowpea was readily taken up by all plant parts and a major fraction of it was retained within the treated leaves and seeds but not in the roots. The fungicide exhibited ambimobility inside the plant. When applied to the middle of leaflets, metalaxyl mobilised towards proximal and distal parts of the leaflet. Intraleaflet movement seemed to result from translocation through veins and diffusion through cell walls and intercellular spaces. Accumulation of metalaxyl at tips and margins of primary leaves followed application to roots. Metalaxyl was readily extracted from most plant parts except cotyledons and only 0.8% of the fungicide applied as a seed treatment was recovered from 50-day-old plants.     

A pea NTPase, PsAPY1, recognizes signal molecules from microorganisms

Apyrases (E.C.3.6.1.5; NTP-NDPases) are distributed in the cytosol, nuclei, cytoskeleton, and on the surface of plant cells. Some may play an important role in signal transduction from exogenous stimuli. We previously found a protein of ca. 55-kDa (CWP-55) in an ATPase-rich fraction from the pea cell wall bound to the elicitor and supprescins (suppressors of defense) from pea pathogen Mycosphaerella pinodes. We cloned the cDNA of CWP-55 that coincided with PsAPY1, one of two NTPase clones in a pea cDNA library. An analysis with a green fluorescent protein fusion protein indicated that PsAPY1 was distributed in the cell wall, nucleus, and cytoplasm. The recombinant PsAPY1 expressed in Escherichia coli had ATP-hydrolyzing activity responsive not only to the elicitor and supprescins from the pea pathogen but also to other elicitors such as a bacterial harpin, a yeast extract, and a synthetic glycopeptide. Biotinylated fungal signal molecules were bound to the recombinant PsAPY1 specifically. Resonant mirror detection confirmed such binding characteristics of PsAPY1. Based on these results, we discuss the role of cell-wall-bound NTPases in recognizing and responding to microorganisms on the cell wall surface.     

Survey of faba bean (Vicia faba L.) for viruses in Morocco

A total of 52 faba-bean (Vicia faba L.) fields, located in the main growing areas in Morocco were surveyed for viruses. From 240 samples with symptoms suggestive of virus infection, the following viruses were detected using electron microscopy, serology, and biological indexing: Alfalfa mosaic virus (AMV), bean yellow mosaic virus (BYMV), broad bean mottle virus (BBMV), broad bean stain virus (BBSV), broad bean true mosaic virus (BBTMV), pea earlybrowning virus (PEBV), pea enation mosaic virus (PEMV), pea seed-borne mosaic virus (PSbMV), and a complex of luteoviruses including bean leafroll virus (BLRV). This is the first report of the occurrence of BBTMV, PEMV, PSbMV, and the luteoviruses (including BLRV) of faba bean in Morcco. The luteoviruses and BBMV were found to be the most prevalent. They were detected in 56 and 50%, respectively, of the surveyed fields; while AMV, BBSV, and PEBV were found in single fields only. The remaining viruses were less prevalent, and were detected in a range of 4 to 15% of the fields surveyed. The incidences per field of the prevalent viruses varied and ranged from 1 to 33% for BBMV and up to 20% in the case of luteoviruses. BBMV was found confined to the central and northern parts of the country, BBTMV and PEMV mainly occurred in the central area, while the luteoviruses and BYMV were spread over the faba-bean growing regions of the country.     

Effects of dimethazone (FMC 57020) on chloroplast development: 1. Ultrastructural effects in cowpea (Vigna unguiculata L.) primary leaves

The effects of dimethazone [FMC 57020; 2-(2-chlorophenyl)methyl-4,4-dimethyl-3-isoxalidinone] on the growth and ultrastructure of cowpea (Vigna unguiculata L.) were examined. Seeds were imbibed in 0.5 mM dimethazone for 1 day and grown for 4 to 5 subsequent days in darkness without the herbicide. The herbicide stunted etiolated hypocotyl growth and retarded greening under 150 μmol · m⁻² · sec⁻¹ white light. No effects of dimethazone on the in vivo absorption spectrum of the etiolated primary leaf was detected. The herbicide caused some reduction and disorganization of prothylakoids in etiplasts. After 3 hr of white light chlorophyll accumulation was greatly reduced in treated leaves and ultrastructural development of the chloroplasts of herbicide-treated tissues appeared to be retarded. Pronounced thylakoid disruption was noticed in some cells after 12 hr, was more common after 24 hr, and was found in all cells by 72 hr. Maximally affected plastids lacked thylakoids, had irregular envelopes, and contained numerous vesicles.     

Differential response of plant cell membranes to some vinyl organophosphorus insecticides

The response of plant cell membranes to vinyl organophosphorus insecticides was studied by determining the release of intracellular materials as a measure of membrane permeability and the uptake of [1-¹⁴C]-α-aminoisobutyric acid as a measure of active transport. A pretreatment with chlorfenvinphos (2-chloro-1-(2,4-dichlorophenyl)-vinyl diethyl phosphate) at 0.4 mM or higher concentrations increased the leakage of cell contents from the tissues of pea, corn, and beet, but two other vinyl organophosphorus insecticides, tetrachlorvinphos (2-chloro-1-(2,4,5-trichlorophenyl)-vinyl diethyl phosphate) and phosphamidon (2-chloro-2-diethyl carbamoyl-1-methyl vinyl dimethyl phosphate), had no effect. Simultaneous addition of phospholipids, β-sitosterol, or Ca²⁺ inhibited in varying degrees the chlorfenvinphos-induced permeability, suggesting that the leakage of cell contents might be due to alteration in membrane structure.Chlorfenvinphos or tetrachlorvinphos at 0.1 mM or higher concentrations inhibited the uptake of α-aminoisobutyric acid. The degree of inhibition varied with different plant species. The inhibition was competitive and was not prevented by phospholipids. However, Ca²⁺ and other divalent cations were stimulatory to the uptake of α-aminoisobutyric acid, either in the presence or absence of chlorfenvinphos. Chlorfenvinphos also inhibited plant growth in tobacco callus and pea stem assays.From the differences in effective concentration, structural requirement, and interaction with phospholipids, it is suggested that chlorfenvinphos affected the membrane permeability and active transport by different mechanisms. These effects probably underlie its inhibitory action on plant growth.     

Herbicide and inhibitor effects on cell leakage in suspension cultures of tobacco (Nicotiana tabacum)

Effects of selected herbicides and respiratory inhibitors on leakage from tobacco (Nicotiana tabacum) cell suspension cultures were studied. Leakage was monitored by quantitation of fluorescein dye released from preloaded cells and by measuring conductivity changes in the suspension medium. The herbicides ioxynil, Barban, 2,4,5-T, MCPB, and PCP (10^?6 to 10^?4M) caused leakage of fluorescein dye and electrolytes within 2 hr of exposure to the herbicides. Potassium cyanide and 2,4-DNP caused appreciable leakage at the same concentrations. Similar responses were induced by anaerobiosis. Atrazine, metolachlor, paraquat, and nitrofen did not induce leakage when tested at concentrations of 10^?6 to 10^?4M.     

Growth of seedling Achillea millefolium L. (yarrow) in association with pea (Pisum sativum L.)

When Achillea millefolium L. (yarrow) seedlings were grown in the field in association with a pea (Pisum sativum L.) crop vegetative growth of yarrow was significantly reduced by 6 weeks after emergence. Flowering was totally suppressed while the pure stand of yarrow developed flower clusters at 13 weeks after seedling emergence. Rhizome development occurred at 8 weeks after seedling emergence in the pure stand, but not until 15 weeks when grown with pea. The early suppression of seedling yarrow in a glasshouse experiment was associated with root interference, although by 5 weeks shoot interference by pea plants was important in reducing yarrow growth. The greatest suppression of yarrow occurred when both roots and shoots of the two species were allowed to interfere. Yarrow had low aggressivity against pea when grown in various combinations in a replacement series experiment in the glasshouse.     

Detection and identification of seed-borne viruses from cowpea (Vigna unguiculata (L.) Walp.) germplasm

Seedlings from 182 cowpea ( Vigna unguiculata ) pre-introductions/germplasm accessions from 12 countries were tested under greenhouse conditions for six seed-borne viruses. Twenty-one (13.3%) accessions from eight countries were found to be seed-infected with one of the three following viruses: blackeye cowpea mosaic (BlCMV) and cowpea aphid-borne mosaic (CABMV) potyviruses, and cucumber mosaic cucumovirus (CMV). Natural seed transmission incidence of 0–6.9%, 0–13.3%, and 0–2.0% were determined for BlCMV, CABMV and CMV respectively.   Another set of 2930 cowpea germplasm accessions, mostly from Botswana and Senegal (Africa), were examined under field conditions for detection and identification of seed-borne viruses. Only CABMV was detected in this material. Most of the lines were free from other viruses reported in cowpea seed. Eight isolates of BlCMV and 28 isolates of CABMV were derived from cowpea pre-introductions/germplasm accessions evaluated under greenhouse and field conditions.     

Insecticide resistance management of permethrin and acephate against the cowpea curculio,Chalcodermus aeneus Boheman (Coleoptera: Curculionidae), a pest of the southern pea,Vigna unguiculata (L.) Walp.

To evaluate strategies for management of potential resistance of the cowpea curculio, Chalcodermus aeneus Boheman, to sprayer‐applied permethrin and acephate, five management regimes were evaluated on early, midseason and late sequential plantings of southern peas, Vigna unguiculata (L.) Walp. during 1991–1993. Management regimes were: (1) consecutive permethrin (0.11 kg a.i./ha early‐midseason‐late); (2) consecutive acephate (1.1 kg a.i.ha early‐midseason‐late); (3) consecutive mixture (0.055kg a.i./ha permethrin and 0.55kg a.i./ha acephate early‐midseason‐late); (4) alternation (0.11 kg a.i./ha permethrin early and 1.1 kg a.i./ha acephate midseason and late plantings); and (5) an untreated check early, midseason and late. The effect of management regime on the toxicities (LC₅₀) of permethrin and acephate was determined by topical application to next generation adult cowpea curculios reared from infested southern pea pods harvested from untreated plants in the management fields. In the field, infestation varied more by planting date than by management regime. Toxicity of permethrin to adults selected in the consecutive mixture regime was significantly lower in 1991 and 1992 late season and in 1993 midseason than in early season for the respective years. There was a seasonal decrease in the laboratory toxicity of acephate to adults with all management regimes.