Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

Dynamics of Meloidogyne incognita Virulence to Resistance Genes Rk and Rk2 in Cowpea

The virulence index of three Meloidogyne incognita field isolates to the resistance gene Rk in cowpea was 0%, 75%, and 120%, with the index measured as reproduction on resistant plants as a percentage of the reproduction on susceptible plants. Continuous culture of the 75% virulent isolate on susceptible tomato for more than 5 years (about 25 generations) resulted in virulence decline to about 4%. The rate of the decline in virulence was described by exponential decay, indicating the progressive loss of virulence on a susceptible host. The 120% virulent isolate declined to 90% virulence during five generations on susceptible cowpea. Following virulence decline, the two isolates were compared over 5 years in inoculated field microplots both separately and as a mixture on susceptible, gene Rk, and gene Rk² cowpea plants. At infestation of the plots, the two isolates were 1.2% and 92.0% virulent, respectively, to gene Rk and 0.2% and 8.1% virulent, respectively, to gene Rk². Virulence to gene Rk in the two isolates and in mixture increased under 5 years of continuous Rk cowpea plants to 129% to 172% and under Rk² cowpea plants to 113% to 139 % by year 5. Virulence to gene Rk² increased during continuous cropping with Rk cowpea plants to 42% to 47% and with Rk² cowpea plants to 22% to 48% by year 5. Selection of Rk²-virulence was slower in the isolate with low itt-virulence. The virulence to both genes Rk and Rk² in the mixed population was not different from that in the highly virulent isolate by year 5 of all cropping combinations. Selection of Rk²-virulence on plants with Rk, and vice versa, indicated at least partial overlap of gene specificity between Rk and Rk² with respect to selection of nematode virulence. This observation should be considered when resistance is used in cowpea rotations.     

Fitness of Virulent Meloidogyne incognita Isolates on Susceptible and Resistant Cowpea

A study of life-history traits was made to determine factors associated with the fitness of Meloidogyne incognita isolates virulent to resistance gene Rk in cowpea. Egg hatch, root penetration, egg mass production, and fecundity (eggs per egg mass) of avirulent and virulent phenotypes were compared among M. incognita isolates, isofemale lines, and single descent lines over multiple generations on resistant and susceptible cowpea. Variation (P ≤ 0.05) in both hatch and root penetration rates was found among isolates at a given generation. However, this variation was not consistent within nematode lines among generations, and there was no correlation with level of virulence, except for penetration and virulence on resistant cowpea at generation 20. Resistant and susceptible cowpea roots were penetrated at similar levels. Differences in reproductive factors on resistant plants were correlated with levels of virulence expression. In some isofemale lines, single descent lines, and isolates, lower (P ≤ 0.05) rates of egg mass production and fecundity on susceptible cowpea were associated with virulence to Rk, indicating a trade-off between reproductive fitness and virulence. Other virulent nematode lines from the same isolates did not have reduced reproductive ability on susceptible cowpea over 27 generations. Thus, virulent lineages varied in reproductive ability on susceptible cowpea, contributing to adaptation and maintenance of virulence within M. incognita populations under stabilizing selection.     

Sources of Resistance to Cowpea Bacterial Blight Disease in Nigeria

The development of resistant crop varieties depends on the reliability of the method of screening for resistance. One of the techniques widely used in evaluating field resistance is the field screening method. However, most cowpea varieties regarded as having field resistance are often found to be susceptible in farmers’ fields, where the inoculum density can be higher. This study rescreened 96 cowpea varieties, evaluated earlier in breeders’ fields as resistant to Xanthomonas campestris pv. vignicola, in field and greenhouse under high inoculum pressure. There were significant differences (P ≤ 0.05) in the reactions of cowpea varieties to bacterial blight in terms of disease incidence and severity. Results from the field screening showed that there were 69 susceptible, 25 moderately susceptible, and 2 resistant varieties. In artificial inoculation in the greenhouse IT81D‐1228‐14, IT82E‐16, IT93K‐2271‐2‐2, TVu 1235, and TVu 4630, which were moderately susceptible in the field showed a susceptible reaction. Tvu 12349 and Tvu 15549 gave consistent reactions in the field and in the greenhouse and are therefore good sources of stable resistance to bacterial blight pathogen. Stem canker incidence also varied significantly (P ≤ 0.05) among the cowpea varieties. Seventeen varieties did not manifest canker symptoms.     

Introduction and Expression of Cowpea Trypsin Inhibitor (CpTI) Gene in Transgenic Tobacco

A trypsin/chymotrypsin inhibitor (CpTI) was purified from mature cowpea seeds. A full length cDNA clone encoding the trypsin/chymotrypsin inhibitor protein was isolated from a cDNA library that was constructed using poly(A⁺) RNA from developing seeds. Tobacco protoplasts were transformed with a construct composed of CaMV 35S promoter, NPTII gene, CpTI gene, and NOS terminator, using polyethylene glycol (PEG)-mediated direct gene transfer. The CpTI gene has been integrated into the plant genome, and the expressed CpTI protein from transgenic plants was catalytically active.     

Effect of Co-infection by Fusarium oxysporum and Cowpea Mosaic Virus on the Growth and Colonization of Cowpea Seedlings (Vigna unguiculata [L.] Walp.)

Combined infection of cowpea seedlings (c. v. ‘California Blackeye”) by cowpea mosaic virus (CPMV) and Fusarium oxysporum induced greater losses in leaf area, fresh and dry weights than infection by either pathogen alone. The growth of seedlings infected by F. oxysporum f. sp. tracheiphilum was less than that of comparable seedlings infected by F. oxysporum f. sp. phaseoli. The virus infectivity of extracts of the trifoliate leaves of dual-infected plants was significantly higher than that of comparable extracts from the leaves of plants singly infected with CPMV. The nature of the effects of multiple infection in cowpea is discussed.     

Potato virus-Y multiplication in susceptible tobacco cultivar and transgenic breeding line producing coat protein mRNA

Changes in ribonucleases (RNases) and glucose-6-phosphate dehydrogenase (G6P DH) activities, their content and subcellular localisation were studied in relation to virus multiplication in susceptible (cv. Samsun) or resistant (transgenic breeding line NCTG 83) tobacco plants infected with the potato virus Y^N (necrotic strain of PVY). Activities of RNases and G6P DH from diseased susceptible tobacco plants were markedly increased during the experimental period and significantly correlated with the multiplication curve of the PVY^N. In contrast, the activities of RNases and G6P DH were not changed after PVY inoculation of resistant breeding line NCTG 83 producing the CP mRNA of PVY. Changes in the content and in the subcellular localisation of RNases and G6P DH isozymes were also determined in mesophyll protoplasts isolated from healthy as well as PVY^N infected plants of both cultivars by differential centrifugation of broken protoplasts on day eight post inoculation (the culmination of multiplication curve of PVY and enhanced activity of both enzymes). The chloroplasts fraction from infected protoplasts showed an enhanced content of RNases (192.4% when compared with that from healthy control ones), and of G6P DH (174.4 %). The cytosol fraction from infected protoplasts contained slightly enhanced levels of G6P DH (117.4 %) and considerably enhanced levels of RNases (141.7 %). No significant differences in the activities, contents and subcellular localisation of RNases and/or G6P DH isozymes were observed in the resistant line NCTG 83. This is in accordance with no detectable contents of PVY. This revised version was published online in July 2006 with corrections to the Cover Date.     

Specific binding of a Verticillium dahliae phytotoxin to protoplasts of cotton, Gossypium hirsutum

Summary The occurrence of specific, high-affinity binding sites for a protein-lipopolysaccharide (PLP) phytotoxin purified from culture filtrates of a virulent Vertidllium dahliae isolate has been demonstrated in cotton protoplasts. Binding of the ¹²⁵I-radiolabelled PLP-complex to protoplasts from cotyledon tissue was saturable and with an affinity (K_d = 17.3 nM) comparable with the concentration required for biological activity. A single class of binding site, accessible at the surface of the intact protoplasts, was found and the maximal number of binding sites were estimated as 2.41 × 10^–16 moles per protoplast. The binding affinity to protoplasts proved near identical to that found with purified plasma membrane fractions from roots. When cultivars exhibiting resistance or susceptibility towards the pathogen were compared, no significant differences were found in the affinity of binding, but five times as many binding sites per protoplast and sixteen times as many binding sites per mg membrane protein were found in the resistant cultivar.Abbreviations PLP protein-lipopolysaccharide - k_d dissociation constant - B_max maximal number of binding sites - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Effect of Fungicides on the Viability of Phylophthora cactorum Propagules m the Soil

Vein-clearing followed by downward rolling and necrosis of leaves and severe stunting of groundnut (Arachis hypogaea) plants were caused by cowpea mild mottle virus (CMMV). The virus was readily transmitted by mechanical sap inoculations to groundnut and to 10 plant species belonging to Leguminosae, Chenopodiaceae and Solanaceae. Chenopodium quinoa and Beta vulgaris were good diagnostic hosts. Diseased sap remained infective at 10^–3 but not 10^–4, when stored 8 to 9 days at 25 °C; for 10min at 75 °C but not 80°C. In limited tests, virus was not seed-transmitted m groundnut or soybean. Virus was transmitted by Bemisia tabaci but not by Aphis craccivora or Myzus persicae. An antiserum for CMMV was produced and virus was serologically related to CMMV reported on cowpea and groundnut crinkle virus (GCV) from West Africa. Employing carbon diffraction grating replica as a standard the modal length of virus particles to be 610 nm. Infected cells contained large number of virus particles associated with endoplasmic reticulum.     

Release of Antifungal Phenolic Compounds from Cucumber Mosaic Virus-Infected and Noninfected Cowpea Protoplasts

Fungitoxic phenolic compounds were released from cucumber mosaic virus (CMV)-infected and noninfected cowpea protoplasts. These compounds were presumed by thin layer chromatography as similar compounds released into the leaf ambient fluids when CMV-infected cowpea leaves were incubated in water. Larger amounts of the compounds were released from CMV-infected cowpea protoplasts than from noninfected protoplasts.     

Expression of genes for resistance to potato virus Y in potato plants and protoplasts

Plants of several potato clones with major gene resistance to potato virus Y (PVY) developed necrotic local lesions and systemic necrosis after manual inoculation with common (PVY_o) or veinal necrosis (PVY_N) strains of the virus. The clones reacted similarly, although their resistance genes are thought to be derived from four different wild species of Solarium. Mesophyll protoplasts from each clone became infected when inoculated with RNA of PVY_o by the polyethylene glycol method. The proportion of protoplasts infected, assessed by staining with fluorescent antibody to virus particles, was similar to that of protoplasts of susceptible potato cultivars. In contrast, plants of potato cultivars Corine and Pirola, which possess gene Ry from S. stoloniferum, developed few or no symptoms when manually inoculated or grafted with PVY_o. Moreover, only very few protoplasts of these cultivars produced virus particle antigen after inoculation with PVY_o RNA. The extreme resistance to PVY of cvs Corine and Pirola was therefore expressed by inoculated protoplasts whereas the resistance of the necrotic-reacting potato clones was not.     

A Serious Disease of Groundnut Caused by Cowpea Mild Mottle Virus in the Sudan

A serious disease of groundnut (Arachis hypogaea L.) characterized by stunting of plants, downward rolling, mottling, general chlorosis and reduced size of the leaflets was observed in the Sudan. Surveys conducted from 1992 to 1994 showed that this disease was restricted to irrigated groundnut crops grown between the two Niles. The virus had slightly flexuous filamentous particles (626 nm long) and was transmitted by whiteflies. It was identified serologically as cowpea mild mottle virus (CPMMV). This appears to be the first record of natural occurrence of CPMMV on groundnut in the Sudan and the first evidence that it causes a disease of major economic importance.     

Blockage of stylet tips as the mechanism of resistance to virus transmission by Aphis gossypii in melon lines bearing the Vat gene

Aphis gossypii is the main virus vector in muskmelon crops. The melon gene Vat confers resistance to non‐persistent virus transmission by this aphid. The mechanism of this resistance is not well understood, but no relationship has been detected between resistance and the probing behaviour of aphids on resistant plants. Results presented here suggest that temporary blockage of aphid stylet tips preventing virus particle release may explain the resistance conferred by Vat gene. We performed experiments in which viruliferous aphids were allowed to probe different sequences of resistant (Vat‐bearing) and/or susceptible melon plants. The results demonstrated that A. gossypii inoculates Cucumber mosaic virus (CMV) efficiently in susceptible plants having previously probed resistant plants, showing that the resistance mechanism is reversible. Furthermore, the infection rate obtained for susceptible plants was the same (25%) regardless of whether the transmitting aphid had come directly from the CMV source or had subsequently probed on resistant plants. This result suggests that virus is not lost from stylet to plant during probing of resistant plants, supporting the temporary blockage hypothesis. We also found that the ability of Myzus persicae to transmit CMV is noticeably reduced after probing on resistant plants, providing evidence that this aphid species also responds to the presence of the Vat gene. Finally, we also found that in probes immediately after virus acquisition M. persicae inoculates resistant plants with CMV more efficiently than susceptible plants, perhaps because the Vat gene product induces increased salivation by this aphid.     

Physiological and Biochemical Aspects of Symbiotic Nitrogen Fixation in Cowpea (Vigna unguiculata (L.) Walp. var. Tuy) Plants Infected by Cowpea Mosaic Virus

Three-day-old cowpea seedlings were inoculated with the severestrain of Cowpea Mosaic Virus (CpMV) and 24 h later with Bradyrhizobiumsp. cowpea, strain I-125, when virus translocation to rootsstill had not taken place. Plants were harvested at 22, 30,45 and 59 d after germination. Active virus replication wasassociated with increased protein content, detected in the leavesof 22-d-old plants. CpMV infection reduced the total leaf area,dry weight of shoots and the chlorophyll content of the firsttrifoliolate leaf at all experimental times. Low sugar contentwas recorded in leaves of 22- and 30-d-old CpMV-infected plantsand in nodules and roots of 30- and 45-d-old CpMV-infected plants.Up to 45 d, the nodule mass of CpMV-infected plants was lowerthan in controls, but reached control values at the 4th harvest.In CpMV-infected nodules, massive agglomeration of virus particles,crystalline virus inclusions and the proliferation of the endoplasmicreticulum were observed only in those cells containing bacteroids.In 30- and 45-d-old plants, CpMV infection decreased the contentof ureides in nodules, roots, and petioles. Virus infectiondid not alter the -amino-N content of roots and nodules butinduced a transient 74% reduction in the level of -amino-N inpetioles of 45-d-old plants. At the 1st and 2nd harvests theactivity of uricase (EC 1.7.3.3 [EC] ) in the nodules and of pyruvatekinase (EC 2.7.1.40 [EC] ) in the nodules and leaves were decreasedseverely by virus infection. CpMV did not hinder the allantoinase(EC 3.5.2.5 [EC] ) activity in the leaves but caused a 9% transitorydecrease in the activity of this enzyme in nodules of 45-d-oldplants. Measurements of NAD-malic enzyme (EC 1.1.1.38 [EC] ) in nodulesalso showed the non-effect of CpMV on this enzyme, except fora temporary 16% reduction at the 2nd harvest. As compared tocontrols, the relative abundance of ureides in 30-d-old CpMV-infectedplants indicated a 15%, 10%, and 51% reduction in the nodules,roots, and petioles, respectively. Results indicate that atthe time of the 4th harvest the symbiotic process, measuredin terms of ureide content and enzymatic activities, was functioningat a near normal level despite nodule infection by CpMV. Key words: Cowpea Mosaic Virus, nitrogen fixation, cowpea, enzymes, ultrastructure     

Ultrastructure and anatomy of nematode-induced syncytia in roots of susceptible and resistant sugar beet

Summary Using susceptible and resistant sugar beet lines, comparative analyses of root histology and ultrastructure were made during invasion by nematodes and the induction and formation of specific feeding structures (syncytia).The resistant line carried the resistance geneHs1^pro–1.Nematodes were able to invade and induce functional syncytia in roots of resistant and susceptible lines. However, syncytia in resistant roots were smaller and less hypertrophied. The vacuolar system of syncytia in susceptible plants contained many small vacuoles. In resistant plants vacuoles were larger but less numerous. Smooth endoplasmic reticulum prevailed in syncytial protoplasts of susceptible plants, whereas almost only rough endoplasmic reticulum occurred in syncytia in resistant plants. The most conspicuous and hitherto undescribed trait of syncytia in resistant roots was the initial appearance of loose, and later compact, aggregations of the endomembrane system which composed most of the endoplasmicreticulum system of syncytia at later stages. Syncytia in resistant plants usually degraded before the nematodes reached their adult stage. The appearance of membrane aggregations and the other resistance-specific features are discussed in relation to their possible effects on syncytium function and role in nematode resistance.Abreviations DAI days after inoculation - ER endoplasmic reticulum - ISC initial syncytial cell - J2 second-stage juvenile - MA membrane aggregations - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

Cowpea mosaic virus chimeric particles bearing the ectodomain of matrix protein 2 (M2E) of the influenza a virus: Production and characterization

The epitope presentation system for the ectodomain of the M2 protein (M2e) of the influenza A virus was constructed on the basis of the cowpea mosaic virus (CPMV) for expression in the plant Vigna unguiculata. CPMV is widely used as a vector to produce immunogenic chimeric virus particles (CVPs) bearing epitopes of various infectious human and animal pathogens. To produce chimeric CPMV particles in plants, two binary vectors were constructed to bear a modified gene coding for the CPMV S-coat protein with insertions of M2e epitopes of human influenza and bird influenza viruses. Antigenic and immunogenic properties of CVPs were investigated in mice immunization experiments. CVPs were shown to induce anti-M2e IgG production and to partly protect mice against a challenge with low doses of the influenza virus. However, low infectivity and immunogenicity of chimeric CPMV particles indicate that the plant virus-based systems for M2e epitope presentation requires further optimization in order to use plants as a possible source of flu vaccines.     

Spatial and Temporal Spread of Cassava Mosaic Virus Disease in Cassava Grown Alone and when Intercropped with Maize and/or Cowpea

The spread of cassava mosaic disease (CMD) and populations of the whitefly vector (Bemisia tabaci) were recorded in cassava when grown alone and when intercropped with maize and/or cowpea. The trials were planted under conditions of high inoculum pressure in 1995 and 1996 at a site in the lowland rainforest zone of southern Cameroon. In the 1995 experiment, the maize and cowpea intercrops reduced the final incidence of CMD in the cassava cvs. Dschang White and Dschang Violet, but not in the more resistant cv. Improved. In the 1996 experiment with cv. Dschang Violet, the maize and cowpea intercrops grown alone or together decreased adult whitefly populations on cassava by 50% and CMD incidence by 20%. The monomolecular population growth model generally provided the best fit for disease progress. Areas under the disease progress curves (AUDPCs) and incidences expressed as multiple infection units were significantly (P<0.05) less for cassava intercropped with maize and/or cowpea than in cassava alone; times to 50% CMD incidence were significantly (P<0.05) longer in all intercrop systems. In 1995 the basic infection rates (r) were similarly low (0.010 per month) in the moderately resistant cv. Dschang Violet intercropped with maize and in all treatments in the more resistant cv. Improved. By contrast, rates were significantly higherfor cv. Dschang Violet alone or with cowpea and in all treatments for the less resistant cv. Dschang White (0.030–0.060). In 1996, r values in cassava grown alone (0.077) were significantly larger (P<0.05) than in the other cropping systems (0.042–0.052). There were no significant differences in the symptom severity in the different cropping systems. Disease foci were isodametric and more compact in plots containing cowpea than in other cropping systems.     

Responses of three genetically different stocks of the honeybee to a virus from bees with hairless-black syndrome

Test samples, each of 50 honeybees (Apis mellifera), from three different stocks (hairless-black resistant, hairless-black susceptible, and commercial) were fed over a 3-day period a total dose per bee of 4 × 10⁹, 4 × 10⁷, 4 × 10⁶, 4 × 10⁴, or 0 virus particles in sucrose syrup. Mortality differences due to dose and stock variables were observed. By the last day of the experiment, mortality in control groups of all three stocks averaged less than 4%, whereas samples of susceptible, commercial, and resistant stocks receiving 4 × 10⁹ virus particles per bee averaged 94, 60, and 40%, respectively. The responses to other doses reflected the same trends.     

Screening for Barley yellow dwarf virus-Resistant Barley Genotypes by Assessment of Virus Content in Inoculated Seedlings

The content of Barley yellow dwarf virus (BYDV) in roots and leaves of barley seedling plants differing in their level of resistance was assessed by quantitative ELISA 1–42 days after inoculation with the strain of BYDV (PAV). High virus accumulation in roots and low concentration in leaves was characteristic of the period 9–15 days after inoculation. In leaves, the differences in virus content between resistant and susceptible genotypes became significant after 15 days and resistance to virus accumulation was better expressed 30–39 days after inoculation. Roots of resistant materials exhibited evident retardation of virus accumulation and the greatest difference in virus content between resistant and susceptible plants was detected 9 days after inoculation. By these criteria, the selected winter and spring barley cultivars and lines (in total 44 materials) fell in to five groups according to field reactions and the presence or absence of the Yd2 resistance gene. There were highly significant and positive relations between ELISA values and 5‐year field data on symptomatic reactions and grain‐yield reductions due to infection. Using the described method, resistant and moderately resistant genotypes (both Yd2 and non‐Yd2) were significantly differentiated from susceptible genotypes. The possible use of this method in screening for BYDV resistance is discussed.     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.