Investigates the ability of plant extracts from Lens culinaris to protect zucchini from the Zucchini yellow mosaic virus (ZYMV).

Evaluate the impact of extracts from the Lens culinaris plant on a number of physiological and biochemical parameters in squash leaves infected with ZYMV in this work. Compared to the untreated leaves, ZYMV infected leaves showed a range of symptoms, such as severe mosaic, size reduction, stunting, and deformation. Analysis of physiological data revealed that L. culinaris extract lectin therapies and viral infections had an impact on metabolism. Protein, carbohydrate, and pigment levels were all lowered by viral infection. However, phenolic compounds, total protein, total carbohydrates, total amino acids, proline, total chlorophyll and peroxidases levels are considerably elevated with all extract therapies. The other biochemical parameters also displayed a variety of changes. Moreover shoot length, number of leaves and number of flowers was significantly increased compared to viral control in all treatments. The L. culinaris extract treatment increases the plant's ZYMV resistance. This is detectable through reduction of the plants treated with lentil lectin pre and post virus inoculation, reduction in disease severity and viral concentration, and percentage of the infected plants has a virus. All findings demonstrate significant metabolic alterations brought by viral infections or L. culinaris extract treatments, and they also suggest that exogenous extract treatments is essential for activating the body's defences against ZYMV infection.

Overexpression of GmAKT2 potassium channel enhances resistance to soybean mosaic virus.

BACKGROUND: Soybean mosaic virus (SMV) is the most prevalent viral disease in many soybean production areas. Due to a large number of SMV resistant loci and alleles, SMV strains and the rapid evolution in avirulence/effector genes, traditional breeding for SMV resistance is complex. Genetic engineering is an effective alternative method for improving SMV resistance in soybean. Potassium (K+) is the most abundant inorganic solute in plant cells, and is involved in plant responses to abiotic and biotic stresses. Studies have shown that altering the level of K+ status can reduce the spread of the viral diseases. Thus K+ transporters are putative candidates to target for soybean virus resistance. RESULTS: The addition of K+ fertilizer significantly reduced SMV incidence. Analysis of K+ channel gene expression indicated that GmAKT2, the ortholog of Arabidopsis K+ weak channel encoding gene AKT2, was significantly induced by SMV inoculation in the SMV highly-resistant genotype Rsmv1, but not in the susceptible genotype Ssmv1. Transgenic soybean plants overexpressing GmAKT2 were produced and verified by Southern blot and RT-PCR analysis. Analysis of K+ concentrations on different leaves of both the transgenic and the wildtype (Williams 82) plants revealed that overexpression of GmAKT2 significantly increased K+ concentrations in young leaves of plants. In contrast, K+ concentrations in the old leaves of the GmAKT2-Oe plants were significantly lower than those in WT plants. These results indicated that GmAKT2 acted as a K+ transporter and affected the distribution of K+ in soybean plants. Starting from 14 days after inoculation (DAI) of SMV G7, severe mosaic symptoms were observed on the WT leaves. In contrast, the GmAKT2-Oe plants showed no symptom of SMV infection. At 14 and 28 DAI, the amount of SMV RNA in WT plants increased 200- and 260- fold relative to GmAKT2-Oe plants at each time point. Thus, SMV development was significantly retarded in GmAKT2-overexpressing transgenic soybean plants. CONCLUSIONS: Overexpression of GmAKT2 significantly enhanced SMV resistance in transgenic soybean. Thus, alteration of K+ transporter expression is a novel molecular approach for enhancing SMV resistance in soybean.

The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus.

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. One of the plant glutathione S-transferase (GST) genes, NbGSTU4, responds as an upregulated gene in Nicotiana benthamiana post BaMV infection. In order to identify the role of NbGSTU4 in BaMV infection, the expression of NbGSTU4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in N. benthamiana in BaMV inoculation. The results show a significant decrease in BaMV RNA accumulation when the expression level of NbGSTU4 is reduced; whereas the viral RNA accumulation increases when NbGSTU4 is transiently expressed. Furthermore, this study identified that the involvement of NbGSTU4 in viral RNA accumulation occurs by its participation in the viral early replication step. The findings show that the NbGSTU4 protein expressed from Escherichia coli can interact with the 3' untranslated region (UTR) of the BaMV RNA in vitro in the presence of glutathione (GSH). The addition of GSH in the in vitro replication assay shows an enhancement of minus-strand but not plus-strand RNA synthesis. The results suggest that the plant GST protein plays a role in binding viral RNA and delivering GSH to the replication complex to create a reduced condition for BaMV minus-strand RNA synthesis.

Benzothiadiazole (BTH) induces resistance to Pepper golden mosaic virus (PepGMV) in pepper (Capsicum annuum L.).

Pepper is an economically important crop in many countries around the world but it is susceptible to many diseases. In Mexico, diseases caused by bipartite begomoviruses have emerged as important problems in pepper. Several control strategies have been explored wiht little success; most of them are based on the avoidance of virus transmission and the breeding for resistance. Abiotic inducers can act at various points in the signaling pathways involved in disease resistance, providing long-lasting, wide-spectrum resistance. Benzothiadiazole (BTH) shares the property of activating the systemic acquired resistance pathway downstream from the SA signaling. In this work, resistance to PepGMV infection was induced in pepper plants by activating the SA pathway using BTH treatment. The resistance was characterized by evaluating symptom appearance, virus accumulation and viral movement. Our results showed that BTH could be an attractive alternative to induce geminivirus resistance in pepper plants without a significant damage of the fruit quality and productivity.

Benzothiadiazole (BTH) induces resistance to Pepper golden mosaic virus (PepGMV) in pepper (Capsicum annuum L.)

Pepper is an economically important crop in many countries around the world but it is susceptible to many diseases. In Mexico, diseases caused by bipartite begomoviruses have emerged as important problems in pepper. Several control strategies have been explored wiht little success; most of them are based on the avoidance of virus transmission and the breeding for resistance. Abiotic inducers can act at various points in the signaling pathways involved in disease resistance, providing long-lasting, wide-spectrum resistance. Benzothiadiazole (BTH) shares the property of activating the systemic acquired resistance pathway downstream from the SA signaling. In this work, resistance to PepGMV infection was induced in pepper plants by activating the SA pathway using BTH treatment. The resistance was characterized by evaluating symptom appearance, virus accumulation and viral movement. Our results showed that BTH could be an attractive alternative to induce geminivirus resistance in pepper plants without a significant damage of the fruit quality and productivity.

Construction of effective inverted repeat silencing constructs using sodium bisulfite treatment coupled with strand-specific PCR.

RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of inverted repeat (IR) constructs during cloning and plant transformation. Although such constructs have been shown to be stabilized with introns and efficiently induce RNA silencing, we found that the Pdk intron did not stabilize South African cassava mosaic virus (SACMV) silencing constructs. Therefore, we developed a method for producing long SACMV IR constructs through bisulfite-induced base pair mismatches on the sense arm prior to IR assembly. Expression of SACMV BC1 mismatched IR constructs in the model test plant Nicotiana benthamiana resulted in a reduction in viral BC1 transcript levels, hence viral replication, upon SACMV infection. Mismatched SACMV AC1 IR constructs induced PTGS more efficiently in a N. benthamiana callus system than nonmismatched IR constructs. Our novel method for IR construct generation should be applicable to many sequences where the generation of these constructs has proven difficult in the past.

A new seco-neolignan glycoside from the root bark of Ailanthus altissima.

A new seco-neolignan glycoside, seco-dehydrodiconiferyl alcohol-4-O-ß-D-glucopyranoside, together with eight known compounds, were obtained from the EtOH extract of the root bark of Ailanthus altissima. Their structures were elucidated based on the spectroscopic data. Three neolignan glycosides including 7,9,9'-trihydroxy-3,3',5'-trimethoxy-8-O-4'-neolignan-4-O-ß-D-glucopyranoside, sonchifolignan B and citrusin B exhibited moderate in vitro inhibitory effect on tobacco mosaic virus replication with IC50 values 0.30, 0.35 and 0.26 mmol L⁻¹, respectively.

The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis.

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.

An increasing the resistance of field tomato to Pepino mosaic virus.

A resistance degree of three cultivars: 'Atol', 'Betalux' and 'Promyk' of Lycopersicon esculentum Mill. to the pathogen was investigated. The most susceptible to Pepino mosaic virus (PepMV) turned out to be the 'Betalux' cultivar, therefore it was chosen for further studies. For this cultivar, under the transmission electron microscope, a few particles of the virus were observed in leaves, and many virions arranged in the form of the bundle--in fruits. An influence of two inducers was examined--benzothiadiazole (BTH) dissolved in the water and chitosan dissolved in the acetic acid as well as the influence of milk with the addition of the acetic acid on the degree of the disease development. Different testing methods were applied: measuring the percentage of leaf surface covered by necroses and yellow spots as well as the percentage of infected area on fruits, detecting the presence of 3.3'-diaminobenzidine peroxidase, hydrogen peroxide and lignin in leaves with colourful histochemical reactions. It was stated that the 0.5 mM solution of BTH induced the highest increase in the systemic acquired resistance (SAR)--mainly in fruits, which were least infected by the virus. The upper leaves, growing above the BTH treatment zone, were also highly protected. The milk with the acetic acid had no influence on improving the condition of younger leaves (growing above the level of sprayed leaves). However, it contributed in a high degree, though a little bit lower than in case of BTH, to the protection of the tomato fruits. Natural resistance of the 'Promyk' cultivar is correlated with the higher level of hydrogen peroxide, the higher activity of the peroxidase and the greater accumulation of the lignin than in case of the susceptible cultivar--'Betalux'. BTH induced defensive reactions at susceptible cultivar of the tomato, which correlated with an increase in the level of the above mentioned indicators of plants resistance, especially of hydrogen peroxide.

[Diseases and its control on Dendrobium].

The diseases on the Dendrobium plants and their occurrence and damage as well as control methods have been investigated and studied. 11 kinds of fungi, 4 kinds of bacteria, 3 kinds of virus and one root-knot nematode were recorded. To control them effectively, cultural methods such as selecting cultivable field, keeping the field sanitation, enhancing cultural operations are mostly recommended. The application of chemical germicides is also stressed.

The effect of an Egyptian isolate of Streptomyces afghanensis on some plant viruses.

An Egyptian isolate of Streptomyces afghanensis was examined for the production of antiviral metabolites. Concentrated broth exudates of this microorganism were tested against tobacco mosaic virus (TMV), tomato mosaic virus (ToMV) and potato virus X (PVX) infecting Nicotiana tabacum L. cv. White Burley. Both concentrated metabolites and their acetone extract inhibited local lesion development of the tested viruses. In all cases, maximum antiviral effect was observed 2 hr after infection. The ultrafiltrate of broth culture reduced the number of local PVX lesions produced on the challenged half leaves in the case of later applications.

Inhibition of proteolytic processing of the polyproteins of cowpea mosaic virus by hemin.

Cleavages of the polyproteins synthesized from cowpea mosaic virus (CPMV) B RNA and M RNA in rabbit reticulocyte lysates are inhibited by hemin. Cleavage of the CPMV B RNA-encoded 200K polyprotein and of the M RNA-encoded 60K intermediary precursor protein were most sensitive to hemin inhibition, while cleavages of other precursor proteins were less sensitive. A significant observation was that at a hemin concentration of 25 microM, but not at higher concentrations, the 60K protein was cleaved to yield the two viral capsid proteins. This cleavage reaction has not been observed in vitro previously.

Antiviral activity of tiazofurine against barley stripe mosaic virus.

Tiazofurine (2-beta-D-ribofuranosylthiazole-4-carboxamide) was found to inhibit replication of barley stripe mosaic virus (BSMV) in barley and wheat plants. Treatment with this nucleoside analogue delayed and inhibited symptom development and suppressed virus multiplication. The most effective concentration applied twice as a foliar spray 3 h and one day after inoculation, was 10(-3) M. Decreased virus multiplication was obtained without marked phytotoxicity. Three weeks after treatment the antiviral effect declined.

Pressure-induced dissociation of brome mosaic virus.

Brome mosaic virus reversibly dissociates into subunits in the pressure range of 600 x 10(5) to 1600 x 10(5) Pa, as demonstrated by studies of the spectral shift of intrinsic fluorescence, of filtration chromatography and of electron microscopy of samples fixed under pressure. Smaller shell particles (T = 1) were detected as intermediates in the dissociation pathway. Dissociation was facilitated by decreasing the concentration, as expected for a multimolecular reaction. The estimated change in volume upon dissociation into 90 dimer particles was -2960 ml/mol. Large increases in the intrinsic fluorescence intensity and in the binding of bis(8-anilinonaphthalene-1-sulfonate) occurred at pressures higher than 1400 x 10(5) Pa. The pressure-dependence profile of the different spectral properties shifted to lower pressures when 5 mM-MgCl2 was included in the buffer or when the pH was raised from 5.5 to 5.9. When the pressure was progressively increased above 1400 x 10(5) Pa, a value that led to 75% dissociation, the capsid subunits lost the ability to reassociate into regular shells and only amorphous aggregates were formed after decompression, as evidenced by both electron microscopy and gel filtration chromatography. The formation of these random aggregates of brome mosaic virus can be explained by a conformational drift of the separated subunits, similar in nature to that found in simpler oligomeric proteins.

Antiviral effects of dihydroxypropyladenine [RS)-DHPA) and bromovinyldeoxyuridine (BVDU) on plant viruses.

Two established antiviral agents, dihydroxypropyladenine (RS)-DHPA and bromovinyldeoxyuridine (BVDU) were evaluated for their inhibitory effects on 4 plant viruses: Tobacco mosaic virus, potato virus X (PVX), eggplant mosaic virus (EMV) and a potyvirus isolated from Solanum palinacanthum (Poty-Sp). Using the leaf disc incubation test, BVDU proved virtually inactive while (RS)-DHPA efficiently inhibited EMV and Poty-Sp when applied at concentrations as low as 5 mg/l. TMV was less susceptible to the chemical while PVX could be inhibited at drug concentrations of 100 mg/l. To achieve a similar inhibitory effect in the leaf spray test, concentrations up to 250 and 500 mg/l were required. Using these tests no phytotoxicity was observed with (RS)-DHPA at any of the concentrations used.

A new 'sense' RNA approach to block viral RNA replication in vitro.

The use of "antisense" RNA is being widely considered to block specific steps in viral infection. We propose here a new "sense" RNA approach to block viral RNA replication in vitro and possibly in vivo. In the turnip yellow mosaic virus (TYMV) system, the recognition site of the viral replicase (RNA-dependent RNA polymerase) is assumed to be located within the 3' end of the RNA genome. Small "sense" RNAs have been obtained by in vitro transcription of the corresponding cloned cDNAs. Replication of TYMV RNA in vitro is shown here to be blocked only by those RNAs that contain the 3' terminal region of the genome.

Oligonucleotide directed mutagenesis of cauliflower mosaic virus DNA using a repair-resistant nucleoside analogue: identification of an agnogene initiation codon.

Mutation of the initiation codon of the dispensible open reading frame, ORF VII, of cauliflower mosaic virus (CaMV) delayed the appearance of disease symptoms, but the mutants reverted with high frequency. This suggests a role of this start codon in viral expression. Oligonucleotide-directed mutagenesis, utilizing a novel, repair-resistant deoxyguanosine analogue, 2'-deoxy-7-deazainosine (dDI), highly improved the yield of mutants.

Human leukocyte interferon does not inhibit alfalfa mosaic virus in protoplasts or tobacco tissue.

Enzyme-linked immunosorbent assays and local lesion infectivity assays showed that recombinant leukocyte interferons, rIFN-alpha A and rIFN-alpha D, did not reproducibly affect the accumulation of alfalfa mosaic virus in tobacco leaf discs or in tobacco or alfalfa protoplasts when applied within 1 hr after inoculation with virus.

Effect of tiazofurin on tomato plants infected with tomato spotted wilt virus.

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) was examined for its activity against tomato-spotted wilt virus (TSWV) in tomato plants. Solutions containing 50, 100, 200, 400 and 800 mg/l of the drug were sprayed onto the leaves. The results showed that 100 and 200 mg/l were the most efficient concentrations to suppress TSWV infection, thereby delaying the appearance of systemic symptoms. The drug was more effective in controlling TSWV infection when applied after than before virus inoculation. The results suggest that tiazofurin can be used as an efficient antiviral drug in the treatment of TSWV-infected tomato plants.

Specificity of the actinomycin-D-sensitive function of some RNA plant viruses.

The actinomycin-D(AMD)-sensitive step of replication of three plant viruses - cowpea chlorotic mottle virus (CCMV), cowpea mosaic virus, and the cowpea strain of tobacco mosaic virus - in cowpea was specific for each virus. Different combinations of the viruses were examined in which leaves were inoculated with one virus and incubated for 48 h until multiplication was almost resistant to AMD. At that time, the same leaves were inoculated with a different virus and the ability of AMD to inhibit multiplication of the second virus was determined at intervals afterwards. In all combinations tested, the first virus had no effect on the sensitivity of the second virus to AMD. Also, resistance to AMD, which appeared to spread from cell to cell in advance of the virus in mechanically inoculated leaves, did not spread appreciably faster than CCMV into systemically infected upper leaves.