Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

A Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)-ELISA, triple antibody sandwich (TAS)-ELISA, antigen coated plate (ACP)-ELISA, and protein-A coated antibody sandwich (PAS)-ELISA. TAS-ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP-ELISA and PAS-ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV-infected plants from asymptomatic plant samples than did TAS-ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS-ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV-indexing of field samples, tissue sampling should be done during the rainy season when most BSV-infected plants express severe symptoms.     

The detection by serological methods of viruses infecting the rose

Homogenates of herbaceous test plants infected with arabis mosaic virus (AMV), prunus necrotic ringspot virus (PNRSV), or strawberry latent ringspot virus (SLRV), and purified virus preparations were used to assess the sensitivities of four serological methods (the enzyme-linked immunosorbent assay - ELISA, immunodiffusion in gels, the latex flocculation assay, and serologically specific electron microscopy -SSEM) for the detection of these viruses. The latex test was up to 250 times more sensitive than gel immunodiffusion, but SSEM and ELISA were respectively up to 1000 and 200 times more sensitive than the latex test. Gel immunodiffusion and latex tests failed to detect any of the viruses in infected roses. Although ELISA reliably detected PNRSV and SLRV when leaves from infected roses were homogenised in a leaf: buffer ratio of 1 g:10 ml, AMV was occasionally undetected. However, when a modified ELISA technique, which reduced non-specific reactions, was used some PNRSV-infected roses were also not detected. Detection by SSEM was c. twice as sensitive as ELISA for all three viruses in rose extracts. The relative advantages of ELISA and SSEM for the detection of plant viruses are discussed.     

Detection of potato leafroll and potato mop-top viruses by immunosorbent electron microscopy

Attachment of virus particles to antiserum-coated electron microscope grids (immunosorbent electron microscopy) provided a test that was at least a thousand times more sensitive than conventional electron microscopy for detecting potato leafroll (PLRV) and potato mop-top (PMTV) viruses. The identity of the attached virus particles was confirmed by exposing them to additional virus antibody, which coated the particles.
PLRV particles (up to 50/μm² of grid area) were detected in extracts of infected potato leaves and tubers, infected Physalis floridana leaves, and single virus-carrying aphids. On average, Myzus persicae yielded 10–30 times more PLRV particles than did Macrosiphum euphorbiae .
PMTV particles (up to 10/μm² of grid area) were detected in extracts of inoculated tobacco leaves, and of infected Arran Pilot potato tubers with symptoms of primary infection. Particles from tobacco leaves were of two predominant lengths, about 125 nm or about 290 nm, and fewer particles of other lengths were found than in previous work, in which partially purified or purified preparations of virus particles were examined, using grids not coated with antiserum.     

Serological studies on cassava latent virus

Particles of cassava latent virus (CLV) were purified by a method that yielded up to 3 mg per 100 g of systemically infected Nicotiana benthamiana leaf. Specific antiserum was prepared and used for enzyme-linked immunosorbent assay (ELISA), which detected purified virus at 5 ng/ml. As estimated by ELISA, CLV antigen reached a greater concentration in leaves of N. benthamiana plants kept at 20–25 °C than in those at 15 °C or 30 °C. CLV was also detected in leaf extracts of naturally infected cassava plants kept at 25 C but its concentration was only 1–7% of that in comparable extracts from N. benthamiana. Staining sections of N. benthamiana leaves with fluorescent antibody indicated that CLV particle antigen accumulates in the nuclei of many phloem cells and of some cells in other tissues. In tests on mosaic-affected cassava plants of Angolan origin, three plants were found in which CLV could not be detected by either ELISA or immunosorbent electron microscopy, or by transmission to indicator plants. This suggests that the mosaic symptoms were caused by a pathogen other than CLV, but no such agent was detected by electron microscopy of leaf extracts. Three kinds of serological test indicated that CLV is related to bean golden mosaic virus. Evidence was also obtained of a distant relationship to beet curly top virus but none was detected to four other geminiviruses.     

Serological detection of ryegrass mosaic virus and ryegrass seed-borne virus

Ryegrass mosaic virus (RMV) was reliably detected in both perennial (S24) and Italian (S22) ryegrass, by enzyme-linked immunosorbent assay (ELISA) when plants had been infected for 8 wk. ELISA detected more infections in field-grown perennial ryegrass cv. Premo than either visual assessment or electron microscopy. However, with plants of Italian ryegrass cultivars only recently infected with RMV, positive reactions were more difficult to separate from the reactions of RMV-free plants, which varied considerably with cultivar, some giving high absorbance values. Immunosorbent electron microscopy showed that the RMV antiserum also contained antibodies to ryegrass seed-borne virus (RGSV), suggesting that these high values were caused by RGSV infection in the material tested.     

An overview of arbuscular mycorrhizal fungi–nematode interactions

Plant parasitic nematodes and arbuscular mycorrhizal fungi (AMF) share plant roots as a resource for food and space. The interest in AMF-nematode interactions lies in the possibility of enhanced resistance or tolerance of AMF-infected plants to nematodes, and the potential value of this for control of crop pests. Data collated from previous studies revealed a great diversity of AMF-nematode responses and we seek to generalise from these by evaluating and discussing interactions involving three groups of nematodes distinguished by their mode of parasitism: (i) ectoparasites; (ii) sedentary endoparasites; and (iii) migratory endoparasites. Based on proximity in tissue, we expected that the interactions between endoparasites and AMF would be stronger, i.e. more reciprocal effects of endoparasitic nematodes on AMF, than those between ectoparasites and AMF. Contrary to this hypothesis, we found that, relative to AMF-free plants, AMF-infected plants were damaged more by ectoparasites than by endoparasites. Of the sedentary endoparasites, numbers of root-knot nematodes were reduced more by mycorrhizal infection than were those of cyst nematodes. The reduction in nematode damage by AMF was not different for root-knot or cyst nematode infested plants. Migratory endoparasitic nematodes were the only group whose numbers were greater on AMF-infected plants. However, the experiments involving migratory nematodes were characterised by relatively high levels of AMF infection and little nematode damage compared to the other feeding types. The outcomes of the AMF-nematode interactions are determined by many factors during the interactions between organisms and their physical, physiological and temporal environments. Assessing effects by recording plant sizes and total nematode or AMF populations at the end of experiments gives very little information on the mechanisms of the interactions. It is time to stop doing studies of black boxes and time to start observing processes, directly by using microscopy and indirectly by application of molecular genetics.     

Utilization of noxious weeds for the management of plant-parasitic nematodes infesting some vegetable crops: Einsatz von schädlichen unkräutern zur bekämpfung von pflanzenparasitären nematoden auf verschiedenen gëmusekulturen

Significant reduction was observed in the population of plant-parasitic nematodes, Meloidogyne incognita, Rotylenchulus reniformis and Tylenchorhynchus brassicae infesting eggplant and cauliflower when given root-dip treatment in the leaf extracts of Argemone maxicana and Solanum xanthocarpum at different concentrations and dip durations. The root-knot development and larval penetration of second stage juveniles of M. incognita were also inhibited, may be due to bare-root dip treatment in leaf extracts of both the weed plants. Leaf extracts of S. xanthocarpum caused more inhibition in root-knot development, nematode multiplication of reniform and stunt nematodes than that of A. maxicana. Plant growth improvement was noted which seems to be due to dip treatment and reduction in the population of parasitic nematodes. The efficacy of root-dip treatment with respect to improvement in plant growth of eggplant and cauliflower and reduction in root-knot development and nematode population, increased with increasing the concentration of leaf extracts and dip durations.     

Narcissus late season yellows potyvirus: symptoms, properties and serological detection

Narcissus late season yellows virus (NLSYV) was obtained, apparently unmixed with other viruses, in selected naturally infected narcissus plants. The virus was not transmitted by manual inoculation to any of the 12 herbaceous species tested but was transmitted by Myzus persicae to virus-tested narcissus plants, which then developed symptoms characteristic of late season yellows. Typically, symptoms develop late in the growing season but their expression is unreliable. NLSYV particles, which measured c. 750 ± 12 nm, were purified from fresh leaf extracts of narcissus in 0·3 m sodium citrate containing 10 mm sodium EDTA and 10 mm sodium DIECA by differential centrifugation followed by clarification with diethyl ether, gel filtration and isopycnic banding in caesium sulphate. Using antibodies from antisera prepared to virus particles, NLSYV was detected by ELISA in 14 of the 18 cultivars examined and nine seemed totally infected. Detection of the virus by immunosorbent electron microscopy was as sensitive as by ELISA but neither test detected all infections. The virus was detected by ELISA only in samples collected after flowering and capsid antigen concentration was greatest in the distal region of leaves. The reliability of detection depended on the cultivar. Immuno-electron microscopy tests confirmed the occurrence of a distinct potyvirus, presumably narcissus degeneration, in narcissus cv. Grand Soleil d'Or, failed to detect any virus other than NLSYV in narcissus plants affected by white streak disease, and showed that NLSYV is related to three of five other potyviruses tested.     

Plant Sources of Chinese Herbal Remedies: Effects on Pratylenchus vulnus and Meloidogyne javanica

More than 500 plant species, used alone or in combination, are documented in Chinese traditional medicine to have activity against helminth and micro-invertebrate pests of humans. We subjected 153 candidate medicines or their plant sources to multilevel screening for effectiveness against plant-parasitic nematodes. For extracts effective in preliminary screens, we determined time-course and concentration-response relationships. Seventy-three of the aqueous extracts of medicines or their plant sources killed either Meloidogyne javanica juveniles or Pratylenchus vulnus (mixed stages), or both, within a 24-hour exposure period. Of 64 remedies reported as antihelminthics, 36 were effective; of 21 classi- fied as purgatives, 13 killed the nematodes; of 29 indicated as generally effective against pests, 13 killed the nematodes. Sources of extracts effective against one or both species of plant-parasitic nematodes are either the whole plant or vegetative, storage or reproductive components of the plants. Effective plants include both annuals and perennials, range from grasses and herbs to woody trees, and represent 46 plant families.     

Electron Microscopical and Serological Detection of Virus-like Particles Associated with Lettuce Big Vein Disease

Very labile rod-shaped particles measuring 324 and 152 nm × 18 nm were isolated only from lettuce plants affected with lettuce big vein (LBV) but not from healthy ones. An antiserum to these particles was prepared which enabled us to diagnose LBV-affected plants, using immunosorbent electron microscopy (ISEM) and enzyme-linked immunosorbent assay (ELISA). Clearly positive reactions were obtained in ISEM and ELISA even when symptoms of LBV-affected plants raised in soil from various locations were indistinct. Higher ELISA values were obtained with extracts from leaves than with those from roots. In ISEM high numbers of particles were trapped from extracts of LBV-affected plants with antiserum to tobacco stunt virus.     

The Feeding Behavior of Adult Root-knot Nematodes (Meloidogyne incognita) in Rose Balsam and Tomato

Meloidogyne incognita is a parasitic root-knot nematode that causes considerable yield loss in a wide range of plants. In this study we documented the movement of adult female nematodes for more than 2 hr in micro-slices of infected tomato (Solanum lycopersicum) and rose balsam (Impatiens balsamina) plants using light and video microscopy. Stylet thrusting was followed by short pumping actions of the esophagus, dorsal esophageal gland ampulla, and metacorpal bulb. Regular thrusting was normally accompanied by head turning and always preceded continuous stylet thrusting aimed at a single point (for 20 to 90 sec). Females often held the stylet in a protruded position, while pulsating the metacorpus bulb, for about 30 sec. Subsequently, the stylet was paused in a retracted position for 5 to 40 sec. This sequence of behavior took 290 to 380 sec to complete. The procedure developed in this study provides a useful cytological technique to investigate the interaction between root-knot nematodes and the giant cells formed by infected plants. Scanning electron microscopy revealed that the head of the adult nematode was located in the narrow intercellular spaces among several giant cells. The anterior part of the head of the adult was folded like a concertina, whereas that of the second-stage juvenile was not. The labial disc and medial lips of second-stage juveniles seemed expanded and sturdy, whereas those of the adult were star-shaped, appeared to be contracted, and softer. These morphological differences in the heads of adult and second-stage juveniles are discussed with respect to their movement.     

Antifungal activity of extracts from five Egyptian wild medicinal plants against late blight disease of tomato

This study was carried out to evaluate the antifungal potential of water and ethanol extracts from aerial parts of five wild medicinal plants collected from Sinai Peninsula, Egypt, and the extracts were tested in vitro and in vivo against Phytophthora infestans, the causal agent of late blight disease of tomato. The five wild medicinal plants used for the study were Asclepias sinaica, Farsetia aegyptia, Hypericum sinaicum, Phagnalon sinaicum and Salvia aegyptiaca. Ethanol extracts were more effective on the pathogen than water extracts at all concentrations used. Water and ethanol extracts of all plants tested reduced mycelial growth and inhibited spore germination of the pathogen with varying degrees. Water and ethanolic extracts also reduced the disease infection with pathogen comparing with control in detached leaves technique. In all experiments, extracts of A. sinaica and F. aegyptia were most effective on the pathogen than other plant extracts. In plot experiment, the water and ethanolic extracts of A. sinaica and F. aegyptia gave the most reduction of late blight disease severity comparing with control. Also, data indicated that after the application of plant extracts, there was an increase in fruit yield of tomato corresponding with the reduction of disease severity . Scanning electron microscopy revealed a negative alteration of pathogen hyphae treated with A. sinaica extract at 20%. This concentration of the same plant extract also reflected in dramatic changes in the cyto-morphology of pathogen hyphae as observed by transmission electron microscopy. These changes resulted in an increase in vacuolisation and lipid contents with consequent reduction of cytoplasm with alteration of cell wall and plasmalemma. The overall results suggested that the use of these Egyptian wild medicinal plant extracts was promising, effective and environment-friendly management measure against Phytophthora blight of tomato and thus, may be used in the production of organically grown vegetables.     

A comparison of serological techniques for detecting and identifying honeybee viruses

The sensitivity and specificity of conventional Ouchterlony gel-diffusion, immuno-osmoelectrophoresis (IO), immune serum electron microscopy (ISEM), “decoration,” radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) tests for detecting black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), and sacbrood virus (SBV) particles in extracts of diseased honeybees were compared. A “slow” ISEM method detected virus particles in extracts of individuals or groups of individuals diluted to 10^?3 and 10^?4, respectively, whereas the IO method and a “fast” ISEM method using protein A were one-tenth as sensitive, and Ouchterlony gel-diffusion tests were only one-thousandth as sensitive. Using the antibody “decoration” technique, mixtures of serologically unrelated virus particles could be resolved. RIA and ELISA were found to be one thousand times more sensitive than ISEM in detecting the particles of BQCV, CBPV, KBV, and SBV; however, nonspecific reactions occurred when using RIA with very dilute particle suspensions, and this made dilution endpoints difficult to assess, but this did not occur when using the ELISA method. There was little difference in the effectiveness of rabbit or hen antisera in the tests, except when protein A was used as it does not combine with hen antibodies.     

The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles.

Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.     

Surface Coat of Meloidogyne incognita

The nematode surface coat is defined as an extracuticular component on the outermost layer of the nematode body wall, visualized only by electron microscopy. Surface coat proteins of Meloidogyne incognita race 3 infective juveniles were characterized by electrophoresis and Western blotting of extracts from radioiodine and biotin-labeled nematodes. Extraction of labeled nematodes with cetyltrimethylammonium bromide yielded a principal protein band larger than 250 kDa and, with water soluble biotin, several faint bands ranging from 31 kDa to 179 kDa. The pattern of labeling was similar for both labeling methods. Western blots of unlabeled proteins were probed with a panel of biotin-lectin conjugates, but only Concanavalin A bound to the principal band. Nematodes labeled with radioiodine and biotin released ¹²⁵I and biotin-labeled molecules into water after 20 hours incubation, indicating that surface coat proteins may be loosely attached to the nematode. Antiserum to the partially purified principal protein bound to the surface of live nematodes and to several proteins on Western blots. Differential patterns of antibody labeling were obtained on immuno-blots of extracts from M. incognita race 1, 2, and 3; Meloidogyne hapla race 2; and Meloidogyne arenaria cytological race B.     

Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora captures nematodes using adhesive polymers present on special hyphae (traps) which form a three-dimensional network. To understand further the adhesion mechanisms, A. oligospora surface polymers were visualized by transmission electron microscopy and characterized by chemical methods. Both traps and hyphae were surrounded by a fibrillar layer of extracellular polymers which stained with ruthenium red. The polymer layer was resistant to most of the chemicals and enzymes tested. However, part of the layer was removed by sonication in a Tris-buffer or by extraction in a chaotropic salt solution (LiCl), and the structure of the polymers was modified by treatment with Pronase E. Chemical analysis showed that the crude extracts of surface polymers removed by sonication or LiCl solution contained neutral sugars, uronic acids and proteins. Gel chromatography of the extracts revealed that the major carbohydrate-containing polymer(s) had a molecular mass of at least 100 kDa, containing neutral sugars (75% by weight, including glucose, mannose and galactose), uronic acids (6%) and proteins (19%). There was more polymer in mycelium containing trap-bearing cells than in vegetative hyphae. SDS-PAGE of the extracted polymers showed that the trap-forming cells contained at least one protein, with a molecular mass of approx. 32 kDa, not present on vegetative hyphae. Examining the capture of nematodes by traps of A. oligospora in which the layer of surface polymers was modified, or removed by chemical or enzymic treatments, showed that both proteins and carbohydrate surface polymers were involved in the adhesion process.     

Possible utilization of weeds for the management of plant parasitic nematodes infesting some vegetable crops

Leaf extracts of noxious weeds such as Solanum xanthocarpum and Argemone maxicana were used as bare-root dip treatment for the management of three important plant-parasitic nematodes, Meloidogyne incognita, Rotylenchulus reniformis and Tylenchorhynchus brassicae infesting tomato (Lycopersicon esculantum ) and chilli (Capsicum annuum) plants. Significant reduction was observed in the root-knot development caused by M. incognita, multiplication of nematode populations of R. reniformis and T. brassicae on both the test plants. Larval penetration of second stage juveniles of M. incognita was also inhibited at various concentrations of leaf extracts and dip durations. Leaf extract of S. xanthocarpum caused relatively more inhibition in root-knot development in case of root-knot nematode, nematode multiplication of reniform and stunt nematodes than that of A. maxicana. Because of dip treatment in leaf extracts of Argemone maxicana and Solanum xanthocarpum, the plants show better growth and at the same time the populations of nematodes such as M. incognita, R. reniformis and T. brassicae significantly decreased, which naturally improved plant growth. The efficacy of root-dip treatment with respect to improvement in plant weight and reduction in root-knot development and nematode populations, increased with increasing the concentration of leaf extracts and dip durations.     

The transmission of European wheat striate mosaic virus by Javesella pellucida (Fabr.) injected with extracts of plants and plant-hoppers

Virus-free individuals of the plant-hopper Javesella pellucida (Fabr.) infected plants with European wheat striate mosaic virus (EWSMV) after being injected at 5° C. with extracts of either plants or hoppers, but extracts of hoppers provided a better inoculum. Hoppers were unable to infect plants until at least 8 days at 20–25° C. after they were injected, and nymphs fed on infected plants similarly required 8 days before they gave infective extracts. Few hoppers survived more than a week after injection with untreated extracts of hoppers or with material sedimented from them by centrifuging the extracts at 8000g, but 60–70% survived injection with purer virus preparations. Injection of the virus seemed harmless, because as many hoppers survived CO₂ anaesthesis + injection, whether or not they later infected plants, as survived anaesthesis without injection. Attempts to determine the properties of the virus in vitro gave inconsistent results, but virus from hoppers was still infective after 10 min. at 30° C, 36 hr. at 5° C, precipitation at pH 4.0, storage for several months at -15° C, or at a dilution equivalent to 0.0014 g. hopper/ml. The best extraction medium contained 0.2 M-Na₂HPO₄+ ascorbic acid + 0.01 M-DIECA at pH 7.0–7.3. In sucrose density-gradients, EWSMV sedimented more slowly than tobacco mosaic virus. No specific particle with which infectivity could be correlated was seen by electron microscopy.     

Little Leaf: A Disease of Sweet Potato in Papua New Guinea Probably Caused by Mycoplasma-Like Organisms

A disease of sweet potato which causes dwarfing and shoot proliferation is reported from the Central Province of Papua New Guinea. The disease primarily effects yield by reducing tuber number and yield reductions of more than 50 % have been recorded. Mycoplasma like organisms have been detected in the phloem cells of diseased plants using electron microscopy and may be the cause of the disease. Early stage infections can be detected using fluorescent light microscopy and this may prove an appropriate method for rapid diagnosis of the disease. Treatment of diseased plants with tetracycline results in temporary symptom remission.