Prospects of engineered forms of resistance against tomato spotted wilt virus

Tomato spotted with virus (TSWV) is deviant from most other plant viruses in having (mainly) a negative-strand RNA genome and a lipid envelope, and in being propagatively transmitted by thrips. Due to increasing resistance of thrips to insecticides as well as to a relative shortage of natural resistance genes suitable for breeding, genetically engineered forms of resistance to TSWV infections are urgently needed. This review will discuss the recently approved strategy of N gene-mediated protection, which has been shown to hold even against thrips-mediated virus inoculation, as well as a number of potential strategies that are likely to be developed in the near future.     

Changes in ultrastructure and protein metabolism of tobacco plants infected by tomato spotted wilt virus

As the result of electron microscope investigation of ultra-thin sections of the tissues infected by tomato spotted wilt virus it was shown that ultrastructural changes in the cells depend on the virus virulence. The isolate with low virulence induces mostly virus-specific changes (virus particles and virus inclusion bodies); the isolate with high virulence besides the virus-specific changes causes essential non-specific violation of cell organelle structure that could be the consequence of pathological action of the virus. It was determined that severe virus infection results in the decrease of general content of the proteins in the leaves. At the same time it induces formation of at least three pathogenesis-associated proteins (PR-proteins) and two antiviral factors of the types AVF (6) and IVR (7) active towards tobacco mosaic virus.     

Monoclonal antibodies against tomato spotted wilt virus: Characterisation and application*

Mouse hybridoma cells, secreting monoclonal antibodies (MCA) against tomato spotted wilt virus, were produced and screened for virus specificity by an indirect triple antibody ELISA, using a rabbit polyclonal antiserum for antigen trapping. A Bulgarian virus isolate from tobacco was used for immunisation of mice and rabbits. One fusion eventually led to 10 stable hybridoma cell lines, all of which produced antibodies of IgG-type though of different subgroups. Since none of the MCAs reacted with TSWV structural proteins after electrophoresis and transfer to nitrocellulose, other methods were chosen to examine their protein specificity. Purified viral cores and detergent-solubilised envelope proteins were used as antigens for ELISA, or, alternatively, glycosylated viral envelope proteins were trapped onto microtitre plates coated with lectins in order to detect MCAs specific for them. Both methods, independently, led to the identification of two MCAs that were specific for envelope proteins of TSWV. Only these two antibodies reacted with intact TSWV particles when examined by immunogold labelling in the electron microscope. The reaction of all MCAs with 11 different TSWV isolates eventually led to the selection of one core- and one envelope-specific antibody for routine use. Core-specific MCAs revealed serological differences between isolates belonging to the common serotype (= lettuce serotype), but did not react with the serotype TSWV-I. When comparing different ELISA procedures, broadest reactivity and highest sensitivity with different isolates were obtained in an indirect test procedure, using goat anti-mouse antibody conjugates.     

Infection ofArabidopsis thaliana ecotype columbia by tomato spotted wilt virus

Mechanical inoculation ofArabidopsis thaliana ecotype Columbia with tomato spotted wilt virus led to viral replication and spread as determined by dot blot and ELISA analysis. Severe symptoms were observed three to four weeks post-inoculation. Early symptoms were manifested as chlorotic spots on uninoculated leaves. Later in the infection process, some plants showed complete chlorosis and wilting prior to bolting. Bolts that were developed by infected plants were chlorotic and deformed. These preliminary results suggest thatA. thaliana could become a model system for the genetic analysis of host factors required for the replication of viruses in the family Bunyaviridae, which includes viruses that cause important diseases of both plants and animals.     

Use of Pseudomonas fluorescens-based formulations for management of tomato spotted wilt virus (TSWV) and enhanced yield in tomato

Strains of Pseudomonas fluorescens were investigated for biocontrol efficacy against tomato spotted wilt virus (TSWV) in tomato both alone and in mixtures. P. fluorescens strains applied to seed, soil and foliage or as a seedling dip significantly reduced TSWV, with a concomitant increase in growth promotion in both the glasshouse and field. Two native strains (CoP-1 and CoT-1) and one foreign strain (CHAO) reduced TSWV. In P. fluorescens-treated tomato plants, increased activity of polyphenol oxidase, β-1,3-glucanase and chitinase was observed, and induction of chitinase was confirmed by western blot analysis. Induction of new protein (18 kDa) detected by SDS-PAGE in P. fluorescens-treated tomato plants was not found in healthy and P. fluorescens-untreated virus inoculated control plants. Indirect ELISA clearly showed a reduction in viral antigen concentration in P. fluorescens-treated tomato plants corresponding to reduced disease ratings. All the P. fluorescens-treated tomato plants also showed enhanced growth and yield compared to control plants. Hence, plant growth promoting rhizobacteria (PGPR) could play a major role in reducing TSWV and increasing yield in tomato plants.     

Midgut infection by tomato spotted wilt virus and vector incompetence of Frankliniella tritici

Abstract:  The mechanism leading to vector competence of thrips species to transmit tomato spotted wilt virus (TSWV) is not well characterized. We investigated the interaction of TSWV and the non-vector species Frankliniella tritici . A monoclonal antibody to the non-structural protein (NSs) of the TSWV was used to detect TSWV replication within the thrips by immunofluorescence microscopy and enzyme-linked immonosorbent assay (ELISA). TSWV was acquired by F. tritici , replicated and moved within the alimentary canal of F. tritici similar to a known vector of TSWV, Frankliniella occidentalis . However, virus was not found in the salivary glands of F. tritici , which is a prerequisite to virus transmission. Thus, movement to the salivary glands may determine vector incompetence of F. tritici .     

Thrips tabaci and tomato spotted wilt virus: inheritance of vector competence

Populations of onion thrips, Thrips tabaci Lindeman (Thysanoptera: Thripidae), were shown to differ significantly in their ability to transmit an isolate of tomato spotted wilt virus (Tospovirus: Bunyaviridae) (TSWV) collected from potato [Solanum tuberosum L. (Solanaceae)]. To gain an understanding of the basis for this variation, we generated reciprocal crosses between an efficient and an inefficient transmitting population. The resulting F₁ progeny and progeny from the parental populations were tested for their ability to transmit TSWV. Our results indicate that the ability to transmit TSWV efficiently by T. tabaci is inherited as a recessive trait.     

接种番茄斑萎病毒番茄植株对西花蓟马生物学特性的影响

西花蓟马Frankliniella occidentalis(Pergande)是我国的一种重要入侵害虫。本文研究了西花蓟马在番茄3种处理(健康CK、机械接种番茄斑萎病毒MI、机械损伤MD)叶片上的生长发育、存活及种群增长。结果表明:健康、机械接种番茄斑萎病毒、机械损伤叶片上的发育历期依次为12.68、12.99和11.79d。雌雄成虫寿命和雌虫繁殖能力在各处理叶片上差异不显著(P>0.05)。健康、机械接种番茄斑萎病毒、机械损伤叶片上的内禀增长率依次为0.1362、0.1526和0.1292d-1。本研究表明,接种番茄斑萎病毒的番茄叶片未缩短西花蓟马发育历期,也不能延长寿命及提高产卵量,不能明显加速种群数量增长。这意味着番茄斑萎病毒对西花蓟马在番茄叶片上的生物学特性未能产生明显的有利作用。     

Developmentally regulated Arabidopsis thaliana susceptibility to tomato spotted wilt virus infection

Tomato spotted wilt virus (TSWV) is one of the most devastating plant viruses and often causes severe crop losses worldwide. Generally, mature plants become more resistant to pathogens, known as adult plant resistance. In this study, we demonstrated a new phenomenon involving developmentally regulated susceptibility of Arabidopsis thaliana to TSWV. We found that Arabidopsis plants become more susceptible to TSWV as plants mature. Most young 3-week-old Arabidopsis were not infected by TSWV. Infection of TSWV in 4-, 5-, and 6-week-old Arabidopsis increased from 9%, 21%, and 25%, respectively, to 100% in 7- to 8-week-old Arabidopsis plants. Different isolates of TSWV and different tospoviruses show a low rate of infection in young Arabidopsis but a high rate in mature plants. When Arabidopsis dcl2/3/4 or rdr1/2/6 mutant plants were inoculated with TSWV, similar results as observed for the wild-type Arabidopsis plants were obtained. A cell-to-cell movement assay showed that the intercellular movement efficiency of TSWV NSm:GFP fusion was significantly higher in 8-week-old Arabidopsis leaves compared with 4-week-old Arabidopsis leaves. Moreover, the expression levels of pectin methylesterase and β-1,3-glucanase, which play critical roles in macromolecule cell-to-cell trafficking, were significantly up-regulated in 8-week-old Arabidopsis leaves compared with 4-week-old Arabidopsis leaves during TSWV infection. To date, this mature plant susceptibility to pathogen infections has rarely been investigated. Thus, the findings presented here should advance our knowledge on the developmentally regulated mature host susceptibility to plant virus infection.     

西花蓟马传播番茄斑萎病毒研究进展

西花蓟马Frankliniella occidentalis是世界性重要检疫性害虫之一,不仅直接取食危害作物而且传播病毒,从而造成极为严重的经济损失.由于西花蓟马在我国具有广泛的适生范围,随其入侵我国并随之传播的番茄斑萎病毒(Tomato spotted wilt virus)已在我国不同地域发现,对经济作物已形成严重...     

Transmission of tomato spotted wilt virus by Frankliniella occidentalism median acquisition and inoculation access period

To quantify the transmission of tomato spotted wilt virus (TSWV) by Frankliniella occidentalis, the median acquisition access period (AAP₅₀) and median inoculation access period (IAP₅₀) were determined. These parameters were established using transmission rates obtained after AAPs and in IAPs which both ranged from 5 to 2560 min. An AAP₅₀ of 106 min was found when larvae acquired virus from TSWV-infected Impatiens plants. IAP₅₀s of 58 or 137 min, respectively, were calculated when petunia or Datura stramonium leaf disks were used to test the inoculation efficiency of viruliferous thrips. The virus could successfully be acquired or inoculated in periods of 5 min. Transmission reached an optimum after an AAP of 21.3 h (AAP_opt) and in an IAP of 42.7 h (IAP_opt). These results show that TSWV can be acquired and transmitted efficiently by F. occidentalis in short feeding periods.     

Partial purification and characterization of 2, 4-diacetylphloroglucinol producing Pseudomonas fluorescens VSMKU3054 against bacterial wilt disease of tomato

We find out the antimicrobial potential of partially purified 2,4-diacetylphloroglucinol (DAPG) against Ralstonia solanacearum and fungal plant pathogens isolated from tomato rhizobacterium Pseudomonas fluorescens VSMKU3054. The present study is mainly focused on the control of wilt disease of tomato by our isolate VSMKU3054 and DAPG. The cell free culture filtrate of P. fluorescens VSMKU3054 was significantly arrested the growth of R. solanacearum and fungal pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum compared to control. The existence of DAPG from the crude metabolites of P. fluorescens VSMKU3054 was confirmed on TLC with Rf value 0.34, which is coincide with that of authentic phloroglucinol. The partially purified DAPG exhibited much higher activity against R. solanacearum at 30 µg/ml than the fungal plant pathogens compared to control. The antimicrobial partially purified compound was identified as DAPG by UV, FT-IR and GC–MS analysis. The percentage of live cells of R. solanacearum when supplemented with DAPG at 30 µg/ml, significantly controlled the living nature of R. solanacearum up to 68% compared to tetracycline and universal control observed under high content screening analysis. The selected isolate P. fluorescens VSMKU3054 and DAPG significantly controlled wilt disease of tomato up to 59.5% and 42.12% on 3rd and 7th days compared to positive and negative control by detached leaf assay. Further, in silico analysis revealed that high interaction of DAPG encoding protease with lectin which is associated with R. solanacearum. Based on our findings, we confirmed that P. fluorescens VSMKU3054 and DAPG could be used a potential bio inoculants for the management of bacterial wilt disease of tomato.     

Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus

In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.     

Tactics for management of thrips (Thysanoptera: Thripidae) and tomato spotted wilt virus in tomato

Four studies were conducted in Georgia during spring 1999, 2000, 2001, and 2002 to evaluate various management tactics for reducing thrips and thrips-vectored tomato spotted wilt virus (TSWV) in tomato and their interactions relative to fruit yield. Populations of thrips vectors of TSWV, Frankliniella occidentalis (Pergande) and Frankliniella fusca (Hinds), were determined using flower and sticky trap samples. The management practices evaluated were host plant resistance, insecticide treatments, and silver or metallic reflective mulch. Averaged over all tests, the TSWV-resistant tomato 'BHN444' on silver mulch treatment had the largest effect in terms of reducing thrips and spotted wilt and increasing marketable yield. Of the insecticide treatments tested, the imidacloprid soil treatment followed by early applications of a thrips-effective foliar insecticide treatment provided significant increase in yield over other treatments. Tomato yield was negatively correlated with the number of F. fusca and percentage of TSWV incidence. F. occidentalis per blossom was positively correlated with percentage of TSWV incidence, but not with yield. No significant interactions were observed between cultivar reflective mulch main plot treatments and insecticide subplot treatments; thus, treatment seemed to be additive in reducing the economic impact of thrips-vectored TSWV. Control tactics that manage thrips early in the growing season significantly increased tomato yield in years when the incidence of TSWV was high (>17%).     

Visual representation by atomic force microscopy (AFM) of tomato spotted wilt virus ribonucleoproteins.

Atomic force microscopy (AFM) allows the observation of biological material without fixation procedures. Here we present AFM images of ribonucleoproteins (nucleocapsids) derived from a plant infecting RNA virus (tomato spotted wilt virus, TSWV), which have been recorded in contact mode. The nucleocapsids, prepared from systemically infected leaves of tobacco, were spreaded on a glass surface and dried in air, and appeared as regularly formed rings, resembling the proposed pseudocircular and panhandle structure of encapsidated genomic RNA. Average values between 1300 and 2200 nm of nucleocapsid lengths could be related to dimensions estimated by electron microscopy, thereby validating a filamentous configuration of the TSWV ribonucleoproteins. However, to our knowledge regular, ring-like forms of ribonucleoproteins have not been obtained by electron microscopy, which rather showed an amorphous structure of the virus particles. Hence, the AFM approach provides a starting point for further detailed studies on TSWV ribonucleoprotein complexes.