Autoxidation Products of the Methanolic Extract of the Leaves of Combretum micranthum Exert Antiviral Activity against Tomato Brown Rugose Fruit Virus (ToBRFV).

Tomato brown rugose fruit virus (ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of C. micranthum, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.

Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid.

While the potato spindle tuber viroid (PSTVd) variant, PSTVd-Dahlia (PSTVd-D or PSTVd-Dwt) induces very mild symptoms in tomato cultivar 'Rutgers', PSTVd-Intermediate (PSTVd-I or PSTVd-Iwt) induces severe symptoms. These two variants differ by nine nucleotides, of which six mutations are located in the terminal left (TL) to the pathogenicity (P) domains. To evaluate the importance of mutations located in the TL to the P domains, ten types of point mutants were created by swapping the nucleotides between the two viroid variants. Bioassay in tomato plants demonstrated that two mutants created on PSTVd-Iwt at positions 42 and 64 resulted in symptom attenuation. Phenotypic and RT-qPCR analysis revealed that mutation at position 42 of PSTVd-Iwt significantly reduced disease severity and accumulation of the viroid, whereas mutation at position 64 showed a significant reduction in stunting when compared to the PSTVd-Iwt infected plant. RT-qPCR analysis on pathogenesis-related protein 1b1 and chalcone synthase genes showed a direct correlation with symptom severity whereas the expansin genes were down-regulated irrespective of the symptom severity. These results indicate that the nucleotides at positions 42 and 64 are in concert with the ones at positions 43, 310, and 311/312, which determines the slower and stable accumulation of PSTVd-D without eliciting excessive host defense responses thus contributing in the attenuation of disease symptom.

Opuntin B, the antiviral protein isolated from prickly pear (Opuntia ficus-indica (L.) Miller) cladode exhibits ribonuclease activity.

An antiviral protein, designated Opuntin B, was purified from Prickly Pear (Opuntia ficus-indica (L.) Miller) Cladode by heat treatment of the extract, protein precipitation by ammonium sulfate treatment followed by ion-exchange chromatography. Assessment of enzymatic activity of the purified protein showed that it degrades total plant genomic RNA, while causing electrophoretic mobility shifting of Cucumber mosaic virus (CMV) RNAs. However, heat-denatured viral RNA became sensitive to degradation upon treatment with antiviral protein. Opuntin B had no DNase activity on native and heat-denatured apricot genomic DNA, and on PCR-amplified coat protein gene of CMV. Using CMV as prey protein and Opuntin B as bait protein, no interaction was found between the antiviral protein and viral coat protein in far western dot blot analysis.

Advances and prospects in biogenic substances against plant virus: A review.

Plant virus diseases, known as 'plant cancer', are the second largest plant diseases after plant fungal diseases, which have caused great damage to agricultural industry. Since now, the most direct and effective method for controlling viruses is chemotherapeutics, except for screening of anti-disease species. As the occurrence and harm of plant diseases intensify, production and consumption of pesticides have increased year by year, and greatly contributed to the fertility of agriculture, but also brought a series of problems, such as the increase of drug resistance of plant pathogens and the excessive pesticide residues. In recent years, biopesticide, as characterized by environmentally safe due to low residual, safe to non-target organism due to better specificity and not as susceptible to produce drug resistance due to diverse work ways, has gained more attention than ever before and exhibited great development potential. Now much progress has been made about researches on new biogenic anti-plant-virus substances. The types of active components include proteins, polysaccharides and small molecules (alkaloids, flavonoids, phenols, essential oils) from plants, proteins and polysaccharides from microorganisms, polysaccharides from algae and oligochitosan from animals. This study summarized the research advance of biogenic anti-plant-virus substances in recent years and put forward their further development in the future.

Tyrosinase inhibitory components from Aloe vera and their antiviral activity.

A new compound, 9-dihydroxyl-2'-O-(Z)-cinnamoyl-7-methoxy-aloesin (1), and eight known compounds (2-9) were isolated from Aloe vera. Their structures were elucidated using 1D/2D nuclear magnetic resonance and mass spectra. Compound 9 exhibited reversible competitive inhibitory activity against the enzyme tyrosinase, with an IC50 value of 9.8 ± 0.9 µM. A molecular simulation revealed that compound 9 interacts via hydrogen bonding with residues His244, Thr261, and Val283 of tyrosinase. Additionally, compounds 3 and 7 were shown by half-leaf assays to exhibit inhibitory activity towards Pepper mild mottle virus.

First discovery of acetone extract from cottonseed oil sludge as a novel antiviral agent against plant viruses.

A novel acetone extract from cottonseed oil sludge was firstly discovered against plant viruses including Tobacco mosaic virus (TMV), Rice stripe virus (RSV) and Southern rice black streaked dwarf virus (SRBSDV). Gossypol and ß-sitosterol separated from the acetone extract were tested for their effects on anti-TMV and analysed by nuclear magnetic resonance (NMR) assay. In vivo and field trials in different geographic distributions and different host varieties declared that this extract mixture was more efficient than the commercial agent Ningnanmycin with a broad spectrum of anti-plant-viruses activity. No phytotoxic activity was observed in the treated plants and environmental toxicology showed that this new acetone extract was environmentally friendly, indicating that this acetone extract has potential application in the control of plant virus in the future.

Screening anti-southern rice black-streaked dwarf virus drugs based on S7-1 gene expression in rice suspension cells.

Southern rice black-streaked dwarf virus (SRBSDV) is a rice pathogen that had an outbreak in southern China in 2010 and caused significant crop losses. Therefore, screening for effective antiviral drugs against SRBSDV is very important. This study used rice suspension cells infected with SRBSDV by polyethylene glycol-mediated uptake for screening antiviral drugs. SRBSDV P7-1, which is coded by the S7-1 gene, has an intrinsic ability to self-interact to form tubules that play an important role in viral infection. Therefore, relative expression level of the SRBSDV S7-1 gene in infected rice suspension cells was assayed by real-time quantitative polymerase chain reaction to evaluate the antiviral activities of various drugs. Dufulin displayed the highest inhibitory activity against SRBSDV S7-1 expression. In addition, changes in peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) activities were determined in inoculated and noninoculated cells. The results showed that both POD and PPO activities increased upon dufulin treatment. Furthermore, the validity of this approach was confirmed in an in vivo experiment in which dufulin was found to effectively inhibit SRBSDV.

Elevated CO2 spurs reciprocal positive effects between a plant virus and an arbuscular mycorrhizal fungus.

Plants form ubiquitous associations with diverse microbes. These interactions range from parasitism to mutualism, depending partly on resource supplies that are being altered by global change. While many studies have considered the separate effects of pathogens and mutualists on their hosts, few studies have investigated interactions among microbial mutualists and pathogens in the context of global change. Using two wild grass species as model hosts, we grew individual plants under ambient or elevated CO(2), and ambient or increased soil phosphorus (P) supply. Additionally, individuals were grown with or without arbuscular mycorrhizal inoculum, and after 2 wk, plants were inoculated or mock-inoculated with a phloem-restricted virus. Under elevated CO(2), mycorrhizal association increased the titer of virus infections, and virus infection reciprocally increased the colonization of roots by mycorrhizal fungi. Additionally, virus infection decreased plant allocation to root biomass, increased leaf P, and modulated effects of CO(2) and P addition on mycorrhizal root colonization. These results indicate that plant mutualists and pathogens can alter each other's success, and predict that these interactions will respond to increased resource availability and elevated CO(2). Together, our findings highlight the importance of interactions among multiple microorganisms for plant performance under global change.

Thermotherapy, chemotherapy, and meristem culture in banana.

Bananas that provide a staple food to the millions of people are adversely affected by several viruses such as Banana bunchy Top Virus (BBTV), Banana Streak Virus (BSV), and Cucumber Mosaic Virus (CMV). These viruses are known to have a devastating effect on crop production and constraint to the international exchange and conservation of banana germplasm-a cornerstone for breeding new cultivars. The viruses are particularly problematic in vegetative propagated crops, like bananas, because of their transmission in the planting material. Different virus eradication techniques have been developed, such as thermotherapy, chemotherapy, and meristem culture for providing virus-free planting material. Meristem culture proved to be the most effective procedure to eradicate phloem-associated viruses. This method requires isolation of meristematic dome of plant under the aseptic conditions and culture in an appropriate nutrient medium to develop new virus-free plants. Thermotherapy is another widely used virus eradication technique, which is initially carried out on in vivo or in vitro plants and eventually combined with meristem culture technique. The plantlets are initially grown at 28°C day temperature and increase it by 2°C per day until reaches 40°C and the night temperature at 28°C; maintain plants at 40°C for 4 weeks; excise meristem and culture onto the regeneration medium. In chemotherapy technique, antiviral chemical compound Virazole(®) is applied on meristem culture. Combination of these techniques is also applied to improve the eradication rate.

Improved recovery of cryotherapy-treated shoot tips following thermotherapy of in vitro-grown stock shoots of raspberry (Rubus idaeus L.).

Raspberry bushy dwarf virus (RBDV) can be efficiently eradicated from raspberry plants (Rubus idaeus) by a procedure combining thermotherapy and cryotherapy. However, the bottleneck of this procedure is that, following thermotherapy, cryopreserved shoot tips become chlorotic during regrowth and eventually die after several subcultures. In addition, survival of heat-treated stock shoots and recovery of cryopreserved shoot tips following thermotherapy are low. The present study focused towards improving regrowth of cryopreserved raspberry shoot tips following thermotherapy. Results showed that preconditioning stock shoots with salicylic acid (SA; 0.01-0.1 mM) markedly increased survival of stock shoots after 4 weeks of thermotherapy. Regrowth of cryopreserved shoot tips following thermotherapy was also significantly enhanced when SA (0.05-0.1 mM) was used for preconditioning stock shoots. Addition of either Fe-ethylenediaminetetracetic acid (Fe-EDTA, 50 mg per L) or Fe-ethylenediaminedi(o)hydroxyphenylacetic acid (Fe-EDDHA, 50 mg per L) to post-culture medium strongly promoted regrowth and totally prevented chlorosis of shoots regenerated from cryopreserved shoot tips following thermotherapy. Using the parameters optimized in the present study, about 80 percent survival of heat-treated stock shoots and about 33 percent regrowth of cryopreserved shoot tips following thermotherapy were obtained. Morphology of plants regenerated from cryopreserved shoot tips following thermotherapy was identical to that of control plants, based on observations of leaf shape and size, internode length and plant height. Optimization of the thermotherapy procedure followed by cryotherapy will facilitate the wider application of this technique to eliminate viruses which can invade meristems.

Potide-G derived from potato (Solanum tuberosum L.) is active against potato virus YO (PVYO) infection.

A PVYO virus-resistant potato (Solanum tuberosum L. cv. Golden Valley) was identified, and further, from its tubers, a small (5.57 kDa) antiviral peptide potide-G was isolated. Application of potide-G on virus susceptible potato (cv. Winter valley) expressed robust resistance to PVYO infection and showed no virus infected morphology. We found that PVYO infection spreads up completely within 3 days post inoculation (dpi) in susceptible cultivar. PVYO was more accumulated toward the basal leaves, when infection occurred longer. Combined results of morphology of PVYO infection, ELISA, RT-PCR, and real-time PCR showed the resistance to the PVYO infection depends on the expression of Ry gene. Indeed, the real-time PCR result showed that the Ry gene up-regulated to 3 times higher in PVYO infected cv. Golden valley. Golden crude protein was found to be active against PVYO infection in the in vivo test. In addition, application of potide-G in a virus susceptible potato potently reduced the viral infection actively with 50 times lower concentration than that of the Golden protein. Further identification of a host-specific resistant gene in a plant and the peptide derived from it offers new opportunities for the development of novel bio-pesticides against plant virus.

[Researches and applications on pesticides from Chinese medicine plant origin].

The research progress on Chinese medicine plant resources with pesticide activities, the active components and their reaction mechanism as well as the application and prospect were reviewed in this paper. Some proposals on the exploitation of traditional Chinese medicine plant origin pesticide were given. It is suggested to found compounds with pesticide activities from heat clearing and toxic clearing medicinal plants.

Experimental and natural weed host-virus relations.

Weeds, as alternative hosts of plant viruses and nutrient plants of virus vectors play important role in virus ecology and epidemiology. The aim of our study was to discover new weed-virus relations. Therefore some weed species were mechanically inoculated with 28 viruses (strains or isolates) maintained in our glasshouse. Different weed species with and without visible symptoms were collected from agro-, water ecosystems and wastelands of Hungary between 1997 and 2003. Virus infections were evaluated by biotests, DAS ELISA serological methods, electronmicroscopy and immunosorbent electronmicroscopy (ISEM). Under glasshouse conditions Ambrosia artemisifolia was considered as a virophob species, showing resistance to all viruses listed above. A series of new artificial (Chenopodium album--SoMV (LH+SH)*, AMV (LH+SH); C. berlandieri--PVY(NTN) (LH), AMV (LH+SH), CMV (LH), SoMV (LH+SH), ObPV (LH+SH), ZYMV-10 (LH): C. ugandae--ObPV (LH), SoMV (L); C. glaucum--ObPV (LH), SoMV (L); Echinocystis lobata--PVX (L), ZYMV (LH+SH); Solanum nigrum--MYFV (LH+SH), PVY(N) (L), PVY(NTN) (LH+SH), SoMV (LH), TMV (SH), CMV (SH); S. dulcamara--CMV-U/246 (SH), PVY(NTN) (LH), SoMV-H (L), TMV-O (L); S. luteum--PVY(N) (SH), PVY(NTN) (LH+L), TMV(SH).) and natural (Asclepias syriaca--TMV, AMV, TSWV; Alisma plantago-aquatica--PVY, SoMV; Ambrosia artemisiifolia--CMV; Chenopodium album--CMV, PVS, PLRV; C. hybridum--CMV; Cirsium canum--CMV, PVM; Carex vulpina--CMV; Comium maculatum--PVY; Datura stramonium--PVA, PVX, PVS, PVM, CMV, TMV; Lysimachia vulgaris--ArMV, BNYVV, CMV, TMV; Lythrum salicaria--ArMV; Malva neglecta--CMV; Mercurialis annua--SoMV; Solanum nigrum--CMV, PVY, PVY(N); Solidago gigantea--CMV, RpRSV, BNYVV; Stenactis annua--PVM, PVA) weed--virus relations were detected. The epidemiological role of perennial hosts (A. syriaca, A. planlago aquatica, C. canurm, L. vulgaris, L. salicaria, S. gigantea) is especially high, because they can serve as infection sources as well as overwintering hosts of different plant viruses.

[Study on the technique of Rehmannia glutinosa virus-free].

The stem tip from germ-free stem segment of Rehmannia glutinosa cultured in test tube can be induced into virus-free seedling. The experiment showed that the proper disinfectant for stem segments of Rehmannia glutinosa was 0.05% HgCl2. The seedings from stem segments grew better with MS in the concentration of agar 0.7% and pH7. The stem tips could be directly induced to seedlings by using MS + 6-BA(0.05 mg/L). The MS media for seedlings virus-free culture are 1/4 macro-elements + 1/2 micro-elements and using edible sugar instead of sucrose, so the cost of media could be decreased to 48.7%.

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction.

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.

Principles and background for the construction of transgenic plants displaying multiple virus resistance.

We investigated the possibility of reconstructing the 2'-5' oligoadenylate (2-5A) pathway into the plant kingdom to achieve multiple virus resistance. Differently phosphorylated 2-5A trimers and tetramers inhibited TMV RNA translation in cell-free systems. In wheat germ extracts the most potent inhibitors were nonphosphorylated forms of 2-5A. Triphosphorylated forms of 2-5A were deposphorylated and hydrolysed in plant extracts. Since we could not detect homologous DNA to mammalian 2-5A synthetase cDNA in tobacco or potato, we cloned rat 2-5A synthetase cDNA and transformed it by the Agrobacterium-mediated mechanism into tobacco and potato. Transformed tobacco plants were resistant to PVS infection and propagation of PVX was reduced. In transgenic potatoes tolerance to PVX and, in one transgenic clone, also to PVY was observed.

Mutagenicity assay with Salmonella typhimurium revealing biotransformation of antiphytoviral substances by cell-free plant extract.

The mutagenic activity of four antiphytoviral substances was tested in reversion mutagenicity assays with a set of histidine auxotrophic strains of Salmonella typhimurium by means of the preincubation method. A possible metabolic activation of the substances by cell free fractions from maize seedlings (S-14-fraction) and for comparison from mouse liver (S-9 mix) was examined. None of the guanidine, phenyl urea and thiadiazole compounds exerted mutagenic activity in the bacterial strains in experiments without metabolic activation. Cyanoguanidine and N-phenyl-N-carboxyphenylurea became mutagenic for Salmonella strain TA98 after metabolic activation by the S-14 plant fraction. Both substances were not mutagenic in the presence of S-9 mix made from mouse liver. The promutagen cyclophosphamide proved highly mutagenic in experiments with S-14 mediated plant metabolic activation. This kind of bacterial mutagenicity assay is valuable in investigations of potential agrochemicals, as the examples have shown.

On the inhibition of plant virus multiplication by ribavirin.

The inhibition of the replication of potato virus X (PVX), belladonna mottle virus, tobacco mosaic virus, potato virus Y (PVY), and tobacco necrosis virus by ribavirin and pyrazofurin is described with emphasis on the inhibition of PVX by ribavirin. Ribavirin inhibits an early step of PVX replication. The inhibition is reversed to different degrees by all ribo- and deoxyribonucleosides, most strongly by thymidine. In tobacco leaves, nucleosides compete with ribavirin for phosphorylation to monophosphate by a nucleoside phosphotransferase. However, the final and main phosphorylation product of ribavirin is triphosphate. It is suggested that ribavirin triphosphate is the antiviral form and that it acts by inhibiting the capping of viral RNAs. This mode of action cannot be applied to the inhibition of PVY, the RNA of which is probably covalently linked to a protein at the 5'-terminus.

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.