Quantification of Leptosphaeria maculans Growth in Cotyledons of Brassica napus Using ELISA

The result of the Leptosphaeria maculans/Brassica napus interaction is usually assessed by symptom scoring following a cotyledon-inoculation test. However, an early evaluation of the interaction, and reliable quantitaive data of fungal growth inside plant tissues are needed to supplement the visual assessment of the symptoms. For this purpose, we developed a quantitatve double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using rabbit polyclonal antisera directed against soluble mycelial proteins. The specificity of the serum was first assessed by immunoblotting following isoelectric focusing of soluble proteins (Western blot) and by DAS-ELISA. Except for Alternaria brassicae, no cross-reactions were observed with my celial extracts of saprophytes or pathogens of B. napus following DAS-ELISA. Although Tox⁺ and Tox⁰ isolates of L. maculans were unequivocally discriminated by Western blot, they were quantitatively indistinguishable following ELISA, thus enabling us to analyse a wide range of L. maculans isolates in planta. The detection limit of the assay was less than 10 ng of fungal proteins per ml of plant extract. For a given isolate, time-course studies showed that fungal growth in cotyledons was correlated with symptom scoring. In the case of hypersensitive response, only 34% of the plants were ELISA-positive, and these plants never contained more than 10 ng of fungal protein per cotyledon. In contrast, in the cases, of susceptibility, 100% of the plants were ELISA-positive and fungal protein content was higher than 10 μg per cotyledon. Moreover, significant differences in ability to colonize the tissues were observed among Tox⁺ isolates. Finally, using the ELISA quantification, intermediate symptoms could be differentiated as lateresistance responses or susceptibility.     

An Improved Antiserum for Sensitive Serologic Detection of Chickpea chlorotic dwarf virus

A Syrian chickpea isolate of Chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus, family Geminiviridae) was purified and yielded 0.6–0.8 mg of purified virus per kg of infected chickpea tissue. The purified preparations were injected into a rabbit and an antiserum of good quality was obtained and used to evaluate different serological tests for the detection of CpCDV in infected chickpea leaf tissue and extracts. CpCDV was detected in sap dilutions of 1/640 by double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) and dot‐blot ELISA, and in sap dilutions of 1/1280 by direct antigen‐coating (DAC)‐ELISA using CpCDV immunoglobulin G (IgG) at 0.5 μg/ml. The antiserum was also able to detect the capsid protein of CpCDV by Western blot using raw antiserum at a dilution of 1/2000. The CpCDV raw antiserum (third bleeding) produced had a titre of 1/320 000 when determined by tissue‐blot immunoassay (TBIA); whereas, coating ELISA plates with CpCDV IgG at a concentration of 0.004 μg/ml was enough to detect the virus by DAS‐ELISA in a sap dilution of 1/20 using an enzyme conjugate at a dilution of 1/2000.     

The susceptibility and response of inbred and outbred Mimulus guttatus to infection by Cucumber mosaic virus

The deleterious effects of inbreeding have been well documented, but only recently have studies begun to explore the consequences of inbreeding for important ecological interactions. We examined the effects of inbreeding on the interaction between host and pathogen using the mixed-mating Mimulus guttatus (Scrophulariaceae) and Cucumber mosaic virus (CMV). Inbred (self) and outbred M. guttatus from two California populations (M5 and M13) were rub-inoculated with CMV and compared to sham-inoculated controls. Flower production by outbred plants in host population M5 showed little effect of the inoculation treatment, but inoculation reduced flower production of inbred plants by 12%, indicating that inbreeding reduces tolerance to CMV infection. This interaction fell short of significance, however. The effects of inbreeding and CMV inoculation on biomass in M5 varied significantly across the 15 families used in this experiment, indicating genetic variation in the effect of inbreeding on resistance or tolerance to CMV. CMV infection reduced biomass in host population M13, but there were no significant interactions between virus treatment and level of inbreeding for either flower production or biomass. Enzyme linked immunosorbent assay (ELISA) was used to detect CMV in host tissues. In both populations, mean ELISA absorbance values of inoculated plants were nearly identical for self and outcross hosts, indicating equal susceptibility to CMV. In outbred plants of population M5, flower production did not change with increasing ELISA absorbance, but in inbred plants it declined, indicating reduced tolerance to CMV infection. The results from this study suggest that pathogens may become increasingly detrimental as host populations become more inbred.     

Production of transgenic eggplant (Solanum melongena L.) resistant to Colorado Potato Beetle (Leptinotarsa decemlineata Say)

 A modified gene of Bacillus thuringiensis var. Tolworthi (Bt), encoding a coleopteran insect-specific CryIIIB toxin, was transferred via Agrobacterium tumefaciens to the female parent of the eggplant commercial F₁ hybrid ‘Rimina’. One-hundred and fifty eight transgenic plants were regenerated and tested by PCR and NPTII expression assays. The presence of the CryIIIB toxin in leaf extracts was demonstrated in 57 out of 93 transgenic plants tested by DAS-ELISA assay. High Bt-expressing plants contained a 74-kDa protein cross-reacting with serum anti-CryIIIB toxin. Twenty three out of 44 S. melongena plants tested by insect bioassay showed significant insecticidial activity on neonate larvae of Colorado Potato Beetle (CPB). The Bt transgene and the toxic effect on CPB larvae were transmitted to progenies derived by selfing. Thus, transgenic Bt eggplants represent a very effective means of CPB pest control. Received: 25 November 1996/Accepted: 31 January 1997     

Comparison of ELISA and immunoblotting techniques for the detection of cherry mottle leaf virus

Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1:51 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study. Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate-trapped antigen ELISA (PTA-ELISA) and dot-blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS-ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA-ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse-grown plants.     

Detection of Double-Stranded RNA (DSRNA) in Crude Extracts of Virus-Infected Plants by Indirect ELISA

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by indirect ELISA (ELISA-I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)-infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA-I 50 days after inoculation. ELISA values then obtained with the In-Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.     

Toxic and Antifeedant Effects of Different Pesticidal Plant Extracts against Beet Armyworm (<i>Spodoptera exigua</i>)

The beet armyworm (BAW), Spodoptera exigua (Lepidoptera: Noctuidae) is a highly destructive pest of vegetables and field crops. Management of beet armyworm primarily relies on synthetic pesticides, which is threatening the beneficial community and environment. Most importantly, the BAW developed resistance to synthetic pesticides with making it difficult to manage. Therefore, alternative and environment-friendly pest management tactics are urgently required. The use of pesticidal plant extracts provides an effective way for a sustainable pest management program. To evaluate the use of pesticidal plant extracts against BAW, we selected six plant species (Lantana camara, Aloe vera, Azadirachta indica, Cymbopogon citratus, Nicotianatabacum, and Ocimum basilicum) for initial screening experiment. Four out of six plant species such as A. indica, N. tabacum, C. citratus and O. basilicum showed promising mortality of more than 50%. Therefore, we selected these four plant extracts for the subsequent experiments. Through contact bioassay, A. indica showed high mortality 66.63%, followed by the N. tabacum 53.33%, at 10% w/v concentration. Similarly, N. tabacum showed the highest mortality rate, 66% at 10% w/v concentration, followed by the A. indica 46% through feeding bioassay. Furthermore, the feeding deterrence assay showed that C. citratus had a high antifeedant index (−50) followed by A. indica (−39), and N. tabacum (−28). In living plant assay, the N. tabacum extract showed a low mean damage score 3.6 on living cotton plant followed by C. citratus 4.5 and A. indica 5.5. Hence, extracts of three plant species provided promising results against the BAW, which can minimize the use of synthetic chemicals, particularly for small landholding farmers. Further studies are also required to evaluate the effects of these plant extract against BAW on cotton plants under field conditions to optimize the further use.     

Papaya transformed with the Galanthus nivalis GNA gene produces a biologically active lectin with spider mite control activity

Snowdrop (Galanthus nivalis) lectin has previously been shown to have anti-feedant and insecticidal activity towards sap-sucking insects. However, its effectiveness against plant-parasitic mites has not been demonstrated. In this study, the commercial papaya (Carica papaya L.) cultivar Kapoho, which is highly susceptible to mites, was transformed with the snowdrop lectin (G. nivalis agglutin [GNA]) gene. Polymerase chain reaction confirmed the presence of the transgene and six independent transformed lines were selected for expression analysis. Western blot analysis showed that the lines expressed a recombinant protein with a molecular weight similar to that of the native snowdrop lectin. Leaf extracts containing the recombinant GNA protein agglutinated trypsinized rabbit erythrocytes thus, showing the GNA protein to be biologically active. ELISA and indirect measurement from the agglutination assay showed there to be variation in GNA expression among the lines produced. A laboratory bioassay using carmine spider mites (Tetranychus cinnabarinus) suggested improved pest resistance in the transgenic papaya plants. This is the first report that a transgenic plant expressing the GNA gene possesses enhanced resistance to a plant-parasitic mite.     

Abundances of thrips on plants in vegetative and flowering stages are related to plant volatiles

Western flower thrips (WFTs), Frankliniella occidentalis Pergrande, and onion thrips (OTs), Thrips tabaci Lindeman, are two cosmopolitan insect pests of agricultural and horticultural plants. Understanding the occurrence and development of thrips on plants is crucial for identifying suitable plants that can be used for developing a “push-pull” strategy against thrips. In this study, the dynamics of WFTs and OTs on plants (Allium fistulosum L., Medicago sativa L., Luffa cylindrica (L.) Roem., Ocimum basilicum L., and Schizonepeta tenuifolia (Benth.) Briq.) were investigated for two consecutive years (2018–2019). Throughout the survey, the abundances of both thrips species were strongly associated with plant species and plant phenology; both thrips species were present at relatively high densities on M. sativa but very low densities on O. basilicum and S. tenuifolia. Populations of both thrips species greatly increased during plant flowering. A Y-tube olfactory test was used to study the effects of plant volatiles in mediating thrips behaviour and showed that volatiles of M. sativa were attractive to both thrips species whether emitted by the plant in the vegetative or flowering stage, while volatiles of O. basilicum and S. tenuifolia were repellent to thrips. Additionally, because of the presence of a high number of floral chemicals, both thrips species exhibited a greater preference for volatiles emitted by plants in the flowering period over those emitted by plants in the vegetative period. Our observations indicate that plant species and flowering status play an important role in the abundance dynamics of thrips and that the volatiles of flowering plants attract thrips more strongly than volatiles emitted by vegetative plants. These findings can facilitate the screening of attractive/unattractive plants for developing push-pull strategies to control thrips.     

Purification and characterisation of the dimeric group 12 allergen from Blomia tropicalis heterologously expressed by Escherichia coli Top10F´

Successful research in the wide-ranging field of allergy is usually achieved by definition not only of physicochemical and immunological properties of natural, but also recombinant allergens. Blomia tropicalis mite is a well-known source for various groups of hypersensitivity-causing proteins. The goal of the present work was to produce, purify and characterise by in silico, biochemical and immunological methods the recombinant group-12 allergen of B. tropicalis. The recombinant Blo t 12 aggregation capacity as well as the affinity to antibodies from BALB/c immunised mice and B. tropicalis-sensitised human donors were investigated through in silico analyses, dynamic light scattering, SDS-PAGE, ELISA and Western blot. The presence of Blo t 12 within B. tropicalis extracts was also determined by ELISA and Western blot. High concentrations of dimeric rBlo t 12 were detected through SDS-PAGE next to other aggregates and the results were confirmed by data from DLS and Western blot. The YITVM peptide was predicted to be the most aggregation-prone region. The IgE-reactivity of rBlo t 12 was not completely abolished by aggregate formation but it was significantly decreased compared to rBlo t 5, or B. tropicalis extracts. Natural Blo t 12 may naturally dimerises, but it was detected in non-delipidified B. tropicalis extracts in low amounts. Given that this allergen may be a specific marker for B. tropicalis allergy, the recombinant Blo t 12 herein obtained is characterised as a mid-tier allergen in Brazilian atopic patients and may be useful for the improvement in precision allergy molecular diagnostic applications.

     

Evaluation of an enzyme-linked immunosorbent assay for detection of Mycosphaerella pinodes in pea seeds

A polyclonal antiserum was raised against soluble mycelial extracts of Mycosphaerella pinodes aiming at pathogen detection in infected pea seeds by ELISA. When tested against the homologous antigen, it allowed the detection of 5 ng fungal soluble protein ml^-1 buffer, by double-antibody sandwich ELISA (DAS-ELISA). Positive reactions were obtained with isolates of M. pinodes of wide geographical origins but also with all tested isolates of Ascochyta pisi and Phoma medicaginis var. pinodella, two closely related pathogens forming with the target organism the Ascochyta complex. Out of the 11 other genera of pea seed-borne fungi tested, only two (Alternaria sp. and Stemphylium sp.) cross-reacted strongly by both antigen-coated plate (ACP-ELISA) and DAS-ELISA. Cross-absorption of the crude antiserum could not lead to a species-specific antiserum; however, a combination of P. medicaginis var. pinodella and Stemphylium sp. antigens resulted in an antiserum preferentially recognising A. pisi and M. pinodes. The cross-absorbed antiserum detected 50 and 500 ng of fungal protein ml^-1 buffer and healthy seed extracts respectively. DAS-ELISA proved suitable for the detection and quantification of M. pinodes in infected pea seeds tested singly.     

Oxidising activity in root extracts from plants inoculated with virus or buffer that interferes with ELISA when using the substrate 3,3', 5,5'-tetramethylbenzidine

In indirect ELISA using protein A-horseradish peroxidase (HRP) as enzyme conjugate and 3,3′, 5,5′-tetramethylbenzidine (TMB) as substrate, extracts of roots of all cucumber, Chenopodium quinoa and Petunia hybrida plants previously inoculated with virus or buffer produced A₄₅₀ values up to seven-fold greater than those for comparable shoots or for extracts of roots from undisturbed, uninoculated plants, irrespective of the virus antiserum used for detection. This effect was also produced in tests in which no HRP conjugate was used, indicating that root extracts from virus-infected or physically injured plants, but not healthy uninjured plants, contain high levels of a factor able to oxidise TMB. The HRP conjugate/TMB substrate version of ELISA is therefore not reliable for detecting viruses in root extracts of herbaceous plants. In contrast, non-specific reactions were not obtained with root extracts, and viruses were reliably detected, when protein A-alkaline phosphatase was used as conjugate and p-nitrophenyl phosphate as substrate.     

Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

Characterisation and serology of virus-like particles associated with faba bean necrotic yellows

A disease showing chlorosis, leaf rolling and stunting in Vicia faba and other legumes was observed in West Asia and North Africa during 1987–1988. The putative causal agent could not be transmitted mechanically, but could be transmitted by aphids, most efficiently by Acyrthosiphon pisum, in the persistent manner. Further studies revealed isometric virus-like particles (VLPs) closely associated with the disease, although their infectivity could not be demonstrated by membrane feeding. These particles, measuring c. 18 nm in diameter and containing a capsid protein of about 22 kDa and ssDNA of about 1 kb, are hereafter designated faba bean necrotic yellows virus (FBNYV). A high proportion of circular nucleic acid molecules of about 0.9 kb were visualised by electron microscopy. Hybridisation analysis of cloned viral DNA suggests that the circular genome is larger than 1 kb and consists of several components of similar size. An antiserum produced against FBNYV was used in ELISA, immunoelectron microscopy (IEM) and Western blot experiments for virus detection in aphids and field samples and for serological comparison with other viruses. Weak heterologous reactions between FBNYV and subterranean clover stunt virus (SCSV) were detected in IEM, but could not be confirmed in ELISA or Western blots. No serological relationship to banana bunchy top virus (BBTV) was detected. Using a direct tissue blot immunoassay (TBIA), FBNYV was detected in vascular tissue of infected faba bean leaves and stems.     

Production of a heat-labile enterotoxin B subunit-porcine epidemic diarrhea virus-neutralizing epitope fusion protein in transgenic lettuce (Lactuca sativa)

Plant-based vaccines have been produced in transgenic plants including tobacco, potatoes, corn, and rice. However, these plants are not suitable for administration without cooking. To overcome this obstacle, a fusion gene encoding the synthetic enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit genetically fused with a synthetic neutralizing epitope of porcine epidemic diarrhea virus (sLTB-sCOE) was introduced into lettuce cells (Lactuca sativa) by Agrobacterium-mediated transformation methods. The integration and expression of the sLTB-sCOE fusion gene was confirmed in transgenic lettuce by genomic DNA PCR amplification and Northern blot analysis, respectively. Synthesis and assembly of the LTB-COE fusion protein into oligomeric structures with pentamer size were observed in transgenic plant extracts by Western blot analysis with anti-LTB or anti-COE antibodies. The binding of plantproduced LTB-COE to intestinal epithelial cell membrane glycolipid receptors was confirmed by G_M1-ganglioside enzyme-linked immunosorbent assay (G_M1-ELISA). Based on the ELISA results, LTB-COE fusion protein made up about 0.026∼0.048% of the total soluble protein in the transgenic lettuce leaf tissues. The synthesis and assembly of LTB-COE monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of using uncooked edible plant-based vaccines for mucosal immunization.