Symptomless Tissues Research Articles

Limited asymptomatic colonization of apple tree shoots by Neonectria ditissima following infection of leaf scars and pruning wounds

AbstractEuropean apple canker, caused by Neonectria ditissima, is an important disease of apple (Malus domestica). The fungus may reside in the tree without causing symptoms for up to a few years, thus making canker control difficult. Asymptomatic infections established in the nursery can result in severe canker outbreaks in newly established apple orchards. It has been suggested that N. ditissima might colonize the tree beyond the infection point during the asymptomatic stage. We investigated whether N. ditissima can colonize the internal tissues of apple shoots, both prior to and after visual symptoms. Apple trees were artificially inoculated via pruning wounds and leaf scars; then the pathogen was tracked at the inoculation point and beyond with isolation or real‐time quantitative PCR (qPCR). Before visual symptoms, N. ditissima could be detected in the infected pruning cut or leaf scar, but not at a distance of 10–15 mm from the entry point, or greater. Conversely, after symptom expression, the pathogen could be detected in the symptomless tissue at 10–15 mm from a canker lesion. This study demonstrated that the asymptomatic infection by N. ditissima can be detected using qPCR and that the pathogen does not grow systemically much beyond the initial entry point inside the plant before visual canker symptoms appear.

A multiplex qPCR TaqMan-assay to detect fungal antagonism between Trichoderma atroviride (Hypocreaceae) and Botrytis cinerea (Sclerotiniaceae) in blackberry fruits using a de novo tef1-α- and an IGS-sequence based probes

The aim of this study was to design a Trichoderma atroviride-specific qPCR oligo set, evaluate its specificity, and standardize a methodology that quantifies antagonism against Botrytis cinerea in blackberry fruits (Rubus adenotrichos Schltdl.). Primers and probe were designed based on the nuclear translation elongation factor 1-alpha (tef1-α) of T. atroviride. A commercial IGS-based oligo set was used to quantify B. cinerea. The specificity of the designed oligo set, along with ITS-based oligo sets, was assessed using other Trichoderma species and B. cinerea. Multiplex qPCR assays were performed using DNA from B. cinerea, T. atroviride, and blackberries inoculated with these fungi. Assays with the tef1-α oligo set showed high sensitivity and reproducibility. In inoculated fruits, T. atroviride and B. cinerea were quantified simultaneously, including in symptomless tissues. This work standardized a qPCR methodology that specifically targets a T. atroviride isolate. This newly-designed qPCR oligo set could be useful in future biological control programs.

Antioxidant activity in endophytic fungi isolated from Gynostemma pentaphyllum Makino

Gynostemma pentaphyllum Makino is a well known edible and medicinal plant in oriental countries, its dominant active constituents are mainly Gypenosides(GP). Endophytic fungi with antioxidant activity were screened and identified from gynostemma pentaphyllum, and gynostemma pentaphyllum saponin (GP) was used as host. A total of 10 strains of endophytic fungi were isolated from the root of healthy, living, and symptomless tissues of Gynostemma pentaphyllum Makino. The extract from a strain (G4) showed antioxidant activity and had the same RF value in TLC, because the retention time of real gynostemma pentaphyllum saponin (GP) and its extract was the same as that of natural gynostemma pentaphyllum (GP) in HPLC. So the strain G4 of endophytic fungus appears to produce the same components as its host.

Formation of satellite uredinia as an important trait related to grapevine colonization by Phakopsora meliosmae‐myrianthae

Phakopsora meliosmae‐myrianthae, the causal agent of Asian grapevine leaf rust, significantly reduces the photosynthetic efficiency of grapevine leaves in green symptomless tissues surrounding lesions. This study took a close look at grapevine leaf colonization kinetics by P. meliosmae‐myrianthae and compared it to P. pachyrhizi–soybean and Uromyces appendiculatus–bean colonization. It is already known from the literature that soybean rust, similar to grapevine rust, has a negative effect on leaf photosynthesis greater than would be expected based on visual lesions. However, in contrast to soybean and grapevine rusts, the effect of bean rust on leaf photosynthesis is proportional to the diseased leaf area. Colonization progress was monitored by fungal biomass assessed via histological staining and quantitative polymerase chain reaction (qPCR). Individual lesions of P. meliosmae‐myrianthae on grapevine, P. pachyrhizi on soybean and U. appendiculatus on common bean leaves were evaluated every 3–4 days, and the number of uredinia was counted. Staining showed that mycelial colonization did not extend beyond the lesion border. The number of P. pachyrhizi and P. meliosmae‐myrianthae uredinia within the lesions increased over time (on average 14‐fold), whereas the number of U. appendiculatus uredinia remained the same. These findings were corroborated by qPCR, which revealed a greater increase in fungal biomass for Phakopsora spp. than for U. appendiculatus until 12 days post‐inoculation. The high number of satellite uredinia within lesions might be directly related to the impact of this pathogen in photosynthetic efficiency on symptomless areas of diseased grapevine leaves. This study identified accelerated formation of satellite uredinia as an important feature of grapevine colonization by P. meliosmae‐myrianthae.

Development of three fusarium crown rot causal agents and systemic translocation of deoxynivalenol following stem base infection of soft wheat

Fusarium pseudograminearum, F. culmorum and F. graminearum are the most important fusarium crown rot (FCR) causal agents. They have the common ability to biosynthesize deoxynivalenol (DON). To elucidate the behaviour of each of the three species, a comparative study was carried out to investigate symptom progression, fungal systemic growth and translocation of DON following stem base inoculation of soft wheat. FCR symptoms were mainly localized in the inoculated area, which extended up to the second node for all inoculated species. Only the most aggressive strains caused symptoms up to the third node. Real‐time quantitative PCR showed that fungal colonization reached the third node for all the tested species, but a low percentage of plants showed colonization above the third node following inoculation with the most aggressive strains. Fungal growth was detected in symptomless tissues but none of the three species was able to colonize as far as the head tissues. However, even if the pathogens were not detected in the heads, DON was detected in head tissues of the plants inoculated with the most aggressive strains. These results demonstrate that F. pseudograminearum, F. culmorum and F. graminearum, under the same experimental conditions, follow a similar pattern of symptom progression, fungal colonization and DON translocation after stem base infection. Differences in the extent of symptoms, fungal colonization and mycotoxin distribution were mainly attributable to strain aggressiveness. These findings provide comparative information on the events following infection of the stem base of wheat by three of the most important FCR casual agents.

A spatial temporal analysis of the Fusarium graminearum transcriptome during symptomless and symptomatic wheat infection.

SummaryFusarium head blight of wheat is one of the most serious and hazardous crop diseases worldwide. Here, a transcriptomic investigation of Fusarium graminearum reveals a new model for symptomless and symptomatic wheat infection. The predicted metabolic state and secretome of F. graminearum were distinct within symptomless and symptomatic wheat tissues. Transcripts for genes involved in the biosynthesis of the mycotoxin, deoxynivalenol, plus other characterized and putative secondary metabolite clusters increased in abundance in symptomless tissue. Transcripts encoding for genes of distinct groups of putative secreted effectors increased within either symptomless or symptomatic tissue. Numerous pathogenicity‐associated gene transcripts and transcripts representing PHI‐base mutations that impacted on virulence increased in symptomless tissue. In contrast, hydrolytic carbohydrate‐active enzyme (CAZyme) and lipase gene transcripts exhibited a different pattern of expression, resulting in elevated transcript abundance during the development of disease symptoms. Genome‐wide comparisons with existing datasets confirmed that, within the wheat floral tissue, at a single time point, different phases of infection co‐exist, which are spatially separated and reminiscent of both early and late infection. This study provides novel insights into the combined spatial temporal coordination of functionally characterized and hypothesized virulence strategies.

Efficacy of dimethyl disulfide (DMDS) for the control of chrysanthemum Verticillium wilt in Italy

Chrysanthemum Verticillium wilt caused by V. dahliae is one of the most damaging soil-borne diseases affecting chrysanthemum production in Italy and in Europe. After the definitive phase-out of methyl bromide and the non-inclusion or limitation of other fumigants a great deal of research to identify alternative treatments for controlling soil-borne pathogens has been conducted. One of the possible alternatives is the use of the new fumigant dimethyl disulfide (DMDS), which is also found in minor amounts in nature as a compound that plays a role in the global sulfur cycle. Two greenhouse trials were carried out over two years (2013–2014), to evaluate the efficacy of DMDS (600 kg ha−1) on Verticillium wilt development and chrysanthemum stems yield in naturally infected soil. Treatment with DMDS significantly reduced the incidence of Verticillium wilt compared to the untreated control and results were similar to those obtained with a standard chemical fumigation (Chloropicrin or Metam sodium). Furthermore, stem yields obtained with DMDS and standard chemical fumigations were significantly higher than untreated control. This study shows that V. dahliae was not only isolated from diseased tissues, but also from symptomless tissues in residual chrysanthemum stems. Results suggest the importance of removing chrysanthemum residues infected with V. dahliae, for managing Verticillium wilt and the effectiveness of DMDS fumigation as well as standard chemical fumigations in decreasing V. dahliae residual stems colonization.

Rapid and sensitive detection of Lily symptomless virus by reverse transcription loop-mediated isothermal amplification

Lily symptomless virus (LSV) is one of the most prevalent viruses that infect lily plants worldwide. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of LSV, using two primer pairs that specifically amplified the conserved sequence of LSV coat protein. The optimum reaction conditions were as follows: 4mM MgCl2 and 0.8M betaine with incubation at 64°C for 30min. The limit of detection of LSV from infected lily leaves was 10-fold higher for RT-LAMP than for conventional RT-PCR. Moreover, RT-LAMP detected LSV in not only symptomatic, but also in symptomless tissues of infected plants. These findings indicate that our RT-LAMP method for LSV can serve as a low-cost, simple, and rapid alternative to conventional detection assays.

Powerful qPCR assays for the early detection of latent invaders: interdisciplinary approaches in clinical cancer research and plant pathology.

Latent invaders represent the first step of disease before symptoms occur in the host. Based on recent findings, tumors are considered to be ecosystems in which cancer cells act as invasive species that interact with the native host cell species. Analogously, in plants latent fungal pathogens coevolve within symptomless host tissues. For these reasons, similar detection approaches can be used for an early diagnosis of the invasion process in both plants and humans to prevent or reduce the spread of the disease. Molecular tools based on the evaluation of nucleic acids have been developed for the specific, rapid, and early detection of human diseases. During the last decades, these techniques to assess and quantify the proliferation of latent invaders in host cells have been transferred from the medical field to different areas of scientific research, such as plant pathology. An improvement in molecular biology protocols (especially referring to qPCR assays) specifically designed and optimized for detection in host plants is therefore advisable. This work is a cross-disciplinary review discussing the use of a methodological approach that is employed within both medical and plant sciences. It provides an overview of the principal qPCR tools for the detection of latent invaders, focusing on comparisons between clinical cancer research and plant pathology, and recent advances in the early detection of latent invaders to improve prevention and control strategies.

Fasciation in Crassula argentea: molecular identification of phytoplasmas and associated antioxidative capacity

The present study reports on phytoplasma induced fasciation in Crassula argintea (Crassulaceae). DNA was extracted from symptomless and fasciated tissues and amplified by nested PCR using universal primers P1/P7 followed by R16F2n/R16R2 produced amplicons of 1.2 Kb. The nucleotide sequence analyses of the amplicons indicated that fasciated plants were infected by phytoplasma. Phylogenetic analysis placed the Crassula fasciation phytoplasmas in 16SrII-D group. Histochemical staining for reactive oxygen species indicated that phytoplasma infected (PI) tissues possess significantly higher levels of hydrogen peroxide (H2O2) rather than superoxide (O2 ·-) as compared with symptomless tissues. PI tissues were also associated with a significant increase in antioxidant enzyme activities (catalase, peroxidase, polyphenol oxidase, and glutathione reductase) and electrolyte leakage as compared with symptomless tissues.

Molecular identification of phytoplasmas in fasciated cacti and succulent species and associated hormonal perturbation

Fasciation, a frequent phenomenon in Cactaceae, has been attributed to various causes. The present study reports on phytoplasma-induced fasciation in Euphorbia coerulescens (Euphorbiaceae), Orbea gigantea (Asclepiadaceae), Opuntia cylindrica (Cactaceae), and Senecio stapeliiformis (Asteraceae). DNA was extracted from symptomless and fasciated tissues and amplified by nested PCR using universal primers P1/P7 followed by R16F2n/R16R2 produced amplicons of 1.2 Kb. The nucleotide sequence analyses of the amplicons indicated that fasciated plants were infected by phytoplasma. Phylogenetic analysis placed the cacti fasciation phytoplasmas in 16SrII group. The hormonal content of symptomless and fasciated tissues including indole-3-acetic acid (IAA), kinetin (Kin), N6-benzyladenine (BA), abscisic acid (ABA), and gibberellic acid (GA3) was quantified by high performance liquid chromatography (HPLC). The results indicated that fasciation in O. gigantea was correlated with the accumulation of Kin and IAA increasing five and two times, respectively, as compared to symptomless tissue. However, there was no consistent pattern of hormones in other fasciated species (E. coerulescens, O. cylindrica, and S. stapeliiformis), suggesting that different plant species might have different mechanism to develop fasciation associated with phytoplasma infection.

Use of Reverse Transcription Loop‐Mediated Isothermal Amplification for the Detection of Zucchini yellow mosaic virus

AbstractA Reverse Transcription Loop‐Mediated Isothermal Amplification (RT‐LAMP) assay was employed to develop a simple and efficient system for the detection of Zucchini yellow mosaic virus (ZYMV) in squash and melon plants. The RT‐LAMP assay took 30 min under isothermal condition at 64°C by employing a set of four primers targeting ZYMV. The sensitivity of RT‐LAMP was 10‐fold greater than that of the RT‐PCR assay in the detection of ZYMV in infected tissues of squash and melon. No reaction was detected from the tissues of healthy plants by either RT‐LAMP or RT‐PCR assay. The RT‐LAMP product of the tested samples can be visualized by staining directly in the tube with SYBR Green I dye. The sensitivity of SYBR Green I staining method is similar to that analyzed by gel electrophoresis. Field‐grown squash and melon plants were tested using RT‐PCR and RT‐LAMP. Both RT‐LAMP and PCR could detect ZYMV in symptomatic or symptomless tissues of infected plants. However, the RT‐LAMP assay is superior to RT‐PCR because it is rapid, simple, and highly sensitive; therefore, RT‐LAMP is a useful and practical method for detection of ZYMV in cucurbits.

A Nested PCR Assay for Detecting Valsa mali var. mali in Different Tissues of Apple Trees.

A nested polymerase chain reaction (PCR) assay for detecting Valsa mali var. mali, the causal agent of apple tree Valsa canker, was developed. One pair of genus-specific primers was designed based on the ribosomal DNA internal transcribed spacer conservative sequence of the Valsa genus and one pair of species-specific primers was designed based on the specific sequence of V. mali var. mali. The specificity of the genus-specific and species-specific primers was evaluated against 10 V. mali var. mali isolates, 10 V. mali var. pyri isolates, 4 isolates from closely related Valsa spp., and 8 isolates from fungal species that are commonly isolated from naturally infected apple bark tissue. A distinct band of 348 bp in length was detected in all V. mali var. mali isolates but not in other tested species and the V. mali var. pyri variety. The sensitivity of this assay was evaluated by serial dilutions of DNA extracted from V. mali var. mali pure cultures and apple bark tissues with or without visible symptoms. The results showed that the assay was able to detect as little as 100 fg of DNA in mycelial samples and apple bark tissues with visible symptoms, whereas the lowest detectable concentration was 10 pg of DNA in symptomless apple bark tissues. The efficiency of the nested PCR assay was compared with that of fungal isolation assays. All symptomless and symptomatic samples from which the pathogen was successfully isolated yielded a PCR product of the expected size. The detection rate of nested PCR for symptomless samples was 64.7%, which was much higher than the detection rate of 20.6% by fungal isolation. The PCR analysis of different symptomless tissues showed that the incidence of V. mali var. mali was different in different tissues of apple trees. The average incidence of V. mali var. mali was 89% in terminal buds, 71% in internodes, and 48% in bud scale scars. Moreover, the incidence of V. mali var. mali in nonsymptomatic tissues was higher in orchards where more trees were infected. Taken together, the assay developed in this study can be used for rapid and reliable detection of V. mali var. mali in tissues of apple trees with or without symptoms and also for monitoring the presence of the pathogen at an early stage of disease development.

Spatiotemporal distribution of flavescence dorée phytoplasma in grapevine

In this study quantitative real‐time PCR was used to follow the seasonal changes of flavescence dorée phytoplasma (FDp) titre in grapevines of cv. Modra frankinja (syn. Blaufränkisch) and cv. Refošk (syn. Refosco'd'Istria) from two vineyards located in climatically different vine‐growing regions of Slovenia. Besides its known presence in the leaf veins, FDp was also detected in flowers, berry tissues and tendrils. In plants with high concentrations of FDp in tissues with symptoms, phytoplasma was also detected in symptomless tissues. A trend of decreasing FDp titre in all examined symptomless tissues from June to July and an increasing one throughout the growing season in tissues with symptoms was recorded. Accordingly, FDp was present in detectable amounts in flowers, petioles and veins of almost all infected plants in the late spring, and was detected in all examined tissue types in summer, with the highest titre in berries in August. The study showed that in the absence of plant health measurements an FDp infection may spread exponentially by a factor of 40 per year.

Phytophthora cinnamomi visible necrotic lesion-colonisation relationships in native flora

Relationships were examined between visible necrotic lesion and colonisation following stem wound inoculated of native plant species of the South-West Botanical Province of Western Australia with Phytophthora cinnamomi. Relationships were determined over a wide range of biotic and abiotic environments of plant taxa, host susceptibility, isolate, phosphite fungicide spray, native community and glasshouse environments, seasons and soil types. Consistent significant relationships were found between necrotic lesions and colonisation over the different environments. Visible necrotic lesions were either in-phase with or greater than colonisation in 55 % of the environments analysed. ANCOVA interactions of total visible necrotic lesion with treatments were not significant for 10 out of 14 interactions, indicating homogeneity of slopes in the majority of cases. Rather than having distinct hemibiotrophic stages, P. cinnamomi switches between necrotic lesions and colonisation of symptomless tissue depending on environmental conditions. Average necrotic lesion/taxa in stems following wound inoculation was significantly positively correlated with mortality following soil inoculation. Determination of visible necrotic lesion was 10 times quicker and 30 times cheaper than colonisation, giving considerable savings for large experiments. Quantification of necrotic lesion-colonisation relationships aids determination of mechanisms of pathogen invasion and host resistance.

Colonization of soft wheat following infection of the stem base by Fusarium culmorum and translocation of deoxynivalenol to the head

Following inoculation of the base of soft wheat seedlings with Fusarium culmorum, disease symptoms typical of crown rot developed at the stem base and extended up to the third node by plant maturity. Fungus was isolated from all tissues exhibiting symptoms but not from symptomless tissues. Histopathological analysis revealed that the fungus was present mainly in the parenchymatic cells of the stem base and colonized the tissues via apoplastic and symplastic pathways. Host response in advance of pathogen colonization was observed. At maturity, plants were divided into sections from the inoculated area to the head. Heads were also separated into grain, rachis and chaff components. Colonization by the fungus was assessed by isolation from surface‐sterilized segments and quantified by real‐time PCR. Disease symptoms and the fungus were detected up to the third node, while deoxynivalenol (DON) was present in all stem segments and heads. Within the head, the DON concentration was higher in the rachis than in the chaff and grain components. These results demonstrate that F. culmorum can extensively colonize stem tissues but not reach the head by the time of plant maturity. In contrast, DON was detected in tissues beyond those colonized by the fungus, translocating to the head where, although accumulating mainly in the rachis, significant quantities accumulated in the grain. These findings indicate that there is a potential threat of contamination of grain with DON where severe crown rot is present in a crop.

Regulation switching of Epichloë typhina within elongating perennial ryegrass leaves

Epichloë and Neotyphodium spp. are clavicipitaceous fungi that form symbiotic endophytic associations with temperate grasses. The growth of these endophytes is strictly intercellular and tightly regulated, being synchronized with that of the host grass. The exception to this synchronized form of growth is the profuse growth of hyphae during development of stromata on reproductive tillers, thereby preventing emergence of the inflorescence. Here we report the occurrence of stromata on leaves of vegetative tillers of perennial ryegrass infected with E. typhina. Stroma formation was confined to leaf blades, being present as a distinct zone within an otherwise symptomless leaf. In some leaf blades two or more stromata zones were present, separated by symptomless tissue. Growth of hyphae in the symptomless zones was seldom branched, orientated parallel with the longitudinal leaf axis, and synchronised with plant growth. In contrast, hyphal growth in the stroma zones was unrestricted and highly branched. Thus in stable zones along a single leaf blade the interaction between E. typhina and its host grass alternated between synchronised, symptomless growth and a pathogenic state with unrestricted hyphal growth. These results demonstrate that the development of an inflorescence is not a prerequisite for hyphal growth leading to stroma formation and provide an ideal experimental system for investigating further how these fungi are able to switch from synchronized, symptomless growth to unrestricted stromal expression.

Occurrence and development of Colletotrichum acutatum on symptomless blueberry bushes

The anthracnose fungus Colletotrichum acutatum was detected in symptomless blueberry bushes (Vaccinium spp.) in a Japanese blueberry field. Naturally diseased bushes and their apparently healthy neighbours were selected, and C. acutatum was isolated from the symptomless tissues of each bush from February 2000 to January 2002. Analysis of the diseased bushes during the dormant period revealed that the fungus was able to survive on symptomless tissues, such as shoot bark and bud scales. Furthermore, C. acutatum was consistently isolated from symptomless leaves and shoots of several surrounding symptomless bushes. Arbitrarily primed PCR (ap-PCR) analyses of the fungal isolates obtained from the diseased and symptomless bushes revealed that most C. acutatum isolates were genotypically identical, regardless of their origins. Inoculation tests using leaves of various blueberry cultivars suggested that the presence or absence of symptoms on each bush can not always be explained by differences in cultivar susceptibility, and other factors may be associated with the appearance of symptoms.

Study of endophytic fungal community from different parts of Aegle marmelos Correae (Rutaceae) from Varanasi (India)

Endophytic fungi were isolated from healthy, living, and symptomless tissues of inner bark, leaf, and roots of Aegle marmelos, a well-known medicinal plant, growing in different parts of India including Varanasi. A total of 79 isolates of endophytic fungi were isolated, representing 21 genera, adopting a standard isolation protocol. Members of the deuteromycotina were more prevalent than ascomycotina and others. The result was quite encouraging in terms of maximum isolates recovery from hyphomycetes (78.5%) followed by ascomycetes (8.9%) and coelomycetes (7.6%) respectively, which corroborates previous studies in same area. However, 5.1% isolates remained unidentified and were classified under Mycelia Sterilia. No isolate was obtained from either basidiomycotina or from zygomycotina. Fusarium spp. had maximum colonization frequency (8.00%) in this plant. The other dominant endophytic genera were Aspergillus spp., Alternaria sp., Drechslera sp., Rhizoctonia sp., Curvularia sp., Nigrospora sp., and Stenella sp. Only two ascomycetous members Chaetomium globosum and Emericella sp. (perfect state of Aspergillus sp.) were obtained from the bark sample. These results indicated that distribution of endophytic fungi within the A. marmelos is not even. Bark harbors more endophytic fungi than leaf and root.

First report of Tomato spotted wilt virus (Tospovirus, Bunyaviridae) infecting Euphorbia eritrea and Asclepias curassavica in Liguria, Italy

The known natural hosts of tospoviruses, in particular Tomato spotted wilt virus (TSWV), are steadily increasing worldwide. The Liguria region of north-west Italy is important for the cultivation of vegetable and ornamental crops, both in open fields and under protection. TSWV and Impatiens necrotic spot virus (INSV) were first reported in Liguria in 1989, when many infected crops were found to be severely affected (Vaira et al ., 1993). The virus vector, Frankliniella occidentalis , is present in Liguria on protected crops during the winter and on both protected and field-grown crops during the summer. In 2000, approximately 20% of a batch of around 10 000 glasshouse-grown pot plants of Euphorbia eritrea showed chlorotic and necrotic spots in the stem, starting from the attachment point of true leaves. TSWV, but not INSV, was detected by both ELISA and immunochromatographic lateral flow assay in tissues with symptoms but not in symptomless tissues of the same plants. Only tospovirus particles were detected in tissues with symptoms by electron microscopy. The virus was transmitted mechanically to common herbaceous test plant species, but not to healthy E. eritrea plants. Thus it seems possible that viruliferous thrips fed on, and infected, true leaves and then moved only partly into the stem. Such infected plants were unmarketable. In 2002, a few potted plants of Asclepias curassavica in a glasshouse showed malformation and some necrosis of apical leaves. Again TSWV, but not INSV, was detected by ELISA and immunochromatographic lateral flow assay, and was transmitted to several herbaceous hosts of TSWV. Electron microscopy of negatively stained sap of affected plants revealed only the presence of tospovirus particles. After removal of necrotic tissues, some plants continued to develop malformation and sometimes necrosis on leaves and stems. The Asclepiadaceae and Euphorbiaceae include several host of TSWV (Campbell et al ., 2003; Peters, 2003), but this is the first report of TSWV infection in Euphorbia eritrea and Asclepias curassavica.