Leaf Disc Assay Research Articles

Effects of Rosmarinus officinalis and Salvia officinalis essential oils on Tetranychus urticae Koch (Acari: Tetranychidae)

Laboratory trials were carried out to characterize Rosmarinus officinal and Salvia officinalis essential oils and to evaluate their pesticidal activities against Tetranychus urticae. Slide-dip and leaf-disk bioassays were employed to study the mortality caused by these plant oils on two-spotted spiders. Different dilutions of both essential oils (0.10–0.25%, v/v) caused acute contact toxicity, although the sage extract showed greater acaricidal activity than rosemary oil. Mortality rates of 95–100% were observed at all the sage oil dosages and when rosemary emulsions contained at least 0.20% of essential oil. In the residual contact experiments (leaf-disk assays), 0.15–0.25% of sage oil or 0.25% of rosemary extracts significantly reduced mite survival. Leaf-disk bioassays were also used to evaluate the effects of oils on T. urticae fecundity. The total number of eggs oviposited decreased as the oil dosage increased and rate reductions were significant when 0.15–0.25% of sage or 0.25% of rosemary extracts were sprayed onto leaf-disks. The effect of oil treatments on the number of emerged larvae was similar to that on egg oviposition. Mite eggs were more susceptible to sage residuals, and significant reductions in larva emergence rates were observed with both sage dosages after 8 days of treatment. Rosemary and sage extracts showed no insecticidal activity against Ceratitis capitata, although sage oil greatly attracted adult flies (see supplementary file).

Localization, concentration, and transmission efficiency of Banana bunchy top virus in four asexual lineages of Pentalonia aphids.

Banana bunchy top virus (BBTV) is the most destructive pathogenic virus of banana plants worldwide. The virus is transmitted in a circulative non-propagative manner by the banana aphid, Pentalonia nigronervosa Coquerel. In this work, we examined the localization, accumulation, and transmission efficiency of BBTV in four laboratory-established lineages of Pentalonia aphids derived from four different host plants: taro (Colocasia esculenta), heliconia (Heliconia spp.), red ginger (Alpinia purpurata), and banana (Musa sp.). Mitochondrial sequencing identified three and one lineages as Pentalonia caladii van der Goot, a recently proposed species, and P. nigronervosa, respectively. Microsatellite analysis separated the aphid lineages into four distinct genotypes. The transmission of BBTV was tested using leaf disk and whole-plant assays, both of which showed that all four lineages are competent vectors of BBTV, although the P. caladii from heliconia transmitted BBTV to the leaf disks at a significantly lower rate than did P. nigronervosa. The concentration of BBTV in dissected guts, haemolymph, and salivary glands was quantified by real-time PCR. The BBTV titer reached similar concentrations in the guts, haemolymph, and salivary glands of aphids from all four lineages tested. Furthermore, immunofluorescence assays showed that BBTV antigens localized to the anterior midguts and the principal salivary glands, demonstrating a similar pattern of translocations across the four lineages. The results reported in this study showed for the first time that P. caladii is a competent vector of BBTV.

Competence of Frankliniella occidentalis and Frankliniella intonsa strains as vectors for Chrysanthemum stem necrosis virus

The vector competence of Frankliniella occidentalis for Chrysanthemum stem necrosis virus (CSNV) was evaluated. Three vector strains with distinct competences for Tomato spotted wilt virus (TSWV) transmission were investigated, including an artificially selected strain (TsH) that has a particularly high competence (>90 %). Newly hatched larvae of F. occidentalis were given an acquisition access period of 5 days on CSNV-infected D. stramonium leaves, and reared to maturity. Their transmission efficiencies were examined using a leaf disk assay using Petunia x hybrida leaves. Following the leaf disk assay, the virus accumulation in the vectors was examined via a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of their bodies. The results showed that the CSNV acquisition and transmission efficiency of the TsH strain did not differ from those of the others, indicating that the competence of F. occidentalis as a vector for CSNV is not related to that for TSWV. The CSNV transmission and acquisition efficiencies of two F. intonsa strains (Hiroshima and Fukuoka) were also evaluated. In Hiroshima strain, 35 % of adults were viruliferous, but only two transmitters (3 %) were observed. In Fukuoka strain, 6 % were viruliferous, and no transmitters were observed. These results indicate that F. intonsa cannot be a major vector for CSNV. The accumulation of CSNV in the adults of F. occidentalis and F. intonsa evaluated using DAS-ELISA showed a significant difference in ELISA values among transmitter, viruliferous non-transmitter, and non-viruliferous individuals. These results clearly demonstrated that only transmitters that accumulated a threshold quantity of virus can transmit CSNV to plants.

Characterization of European Plasmopara viticola isolates with reduced sensitivity to cymoxanil

Cymoxanil has been used for over 30 years to control grape downy mildew (Plasmopara viticola) in European vineyards, prevalently in mixture with other fungicides active on this disease. In the 1990’s cases of P. viticola resistant to cymoxanil were detected using a leaf disc assay. In this study, we establish that the presence of only 1 % of resistant isolates in a P. viticola population will allow the detection of cymoxanil resistance in the leaf disc assay. A poor correlation (R = 0.194) was observed between the leaf disc assay and a whole- plant test for 38 P. viticola field populations collected in 2004. Over 60 % of these populations were characterized as fully sensitive in a whole-plant assay compared to 10 % in the leaf disc assay. Five P. viticola field isolates resistant to cymoxanil reverted to full sensitivity after six to nine transfers to untreated vines, indicating that cymoxanil resistance in P. viticola is unstable. Two European P. viticola populations sensitive to cymoxanil became resistant when transferred 12–14 times on vines treated with cymoxanil. In contrast, two populations originating from the USA and three monozoospore isolates from France retained full sensitivity to the fungicide after 13 cycles on cymoxanil-treated plants. Whole-plant experiments were conducted in the laboratory to compare the efficiency of spray programs to delay the development of cymoxanil resistance. Whereas the continuous use of cymoxanil alone quickly selected for resistance, the mixture of cymoxanil and folpet applied either continuously or in strict or block alternation effectively prevented the development of resistance over 10 generations of the fungus. These results demonstrate that resistance to cymoxanil in P. viticola can be managed with appropriate spray programs.

Preliminary evidence of recovery from Tomato spotted wilt virus infection in Frankliniella occidentalis individuals

AbstractIn this study we analysed the ability of individual thrips to transmit Tomato spotted wilt virus (TSWV) in a population of Frankliniella occidentalis over their lifespan as adults (about 10 days). In three experiments a total of 636 thrips were individually tested for their transmission capacity through leaf disc assays using four inoculation access periods (IAPs). Almost half of the transmitting thrips maintained the capacity to infect leaf discs in each of the four IAPs, confirming the persistent propagative nature of the transmission modality. Nevertheless, a relevant number of thrips (9.25% of transmitter thrips) was able to transmit in the early phases of their adult life (for the first two IAPs), but did not transmit the virus for the remainder of their lifetime. We compared the virus titer of these individuals at the end of the fourth IAP with that of individuals that maintained transmission ability in the four IAPs and showed a statistically significant difference. This difference could be evidence for recovery from TSWV infection in individual thrips.

Preventative and Curative Effects of Several Plant Derived Agents Against Powdery Mildew Disease of Okra

The preventative and curative effects of some plant derived agents based on plant extracts or essential oils were studied at different concentrations against Erysiphe cichoracearum DC. ex Merat, the causal pathogen of okra powdery mildew by the detached leaf-disk and potted plants bioassays. Through detached leaf-disk assay, the highest mean preventative effect (97.74%) was recorded by neem seed oil followed by jojoba oil (89.82%) and extract of Rynoutria sachalinensis (82.77%). Neem seed oil at 1% was the most effective agent followed by jojoba oil and extract of R. sachalinensis at 1.5% and 2%, respectively, where they suppressed E. cichoracearum completely. Potted plants assay revealed that neem seed oil, jojoba oil and extract of R. sachalinensis as well as the fungicide (active ingredient dinocap) showed higher preventative efficacy at all leaf olds treated after 7 and 14 days of inoculation as compared with extracts of henna and garlic. Moreover, the preventative efficacy partly remained apparent after 14 days of inoculation at all leaf olds tested. In field trials through 2010 and 2011 growing seasons, when the first symptoms of powdery mildew appeared naturally, 1.5% jojoba oil, 2% extract of R. sachalinensis and 1% neem seed oil were sprayed individually twice on grown plants to evaluate their efficacy on controlling powdery mildew, growth and yield of okra. Resulted showed that neem seed oil was the most effective agent and highly decreased the disease severity to 29.92%, recorded the highly curative effect (68.15%) and also improved plant growth and pods yield.

The regulation of the SARK promoter activity by hormones and environmental signals

The Senescence Associated Receptor Protein Kinase (PSARK) promoter, fused to isopentenyltransferase (IPT) gene has been shown to promote drought tolerance in crops. We dissected PSARK in order to understand the various elements associated with its activation and suppression. The activity of PSARK was higher in mature and early senescing leaves, and abiotic stress induced its activity in mature leaves. Bioinformatics analysis suggests the interactions of multiple cis-acting elements in the control of PSARK activity. In vitro gel shift assays and yeast one hybrid system revealed interactions of PSARK with transcription factors related to abscisic acid and cytokinin response. Deletion analysis of PSARK, fused to GUS-reporter gene was used to identify specific regions regulating transcription under senescence or during drought stress. Effects of exogenous hormonal treatments were characterized in entire plants and in leaf disk assays, and regions responsive to various hormones were defined. Our results indicate a complex interaction of plant hormones and additional factors modulating PSARK activity under stress resulting in a transient induction of expression.

Patterns of virulence variation in the interaction between Lactuca spp. and lettuce powdery mildew (Golovinomyces cichoracearum)

Abstract Golovinomyces cichoracearum sensu stricto (lettuce powdery mildew) is a common pathogen of the family Asteraceae (Compositae), mainly of species of the tribe Lactuceae. However, information on the natural distribution and virulence variation of G. cichoracearum on wild Lactuca spp. is rather limited. In the Czech Republic, samples of G. cichoracearum were collected in wild populations of prickly lettuce ( Lactuca serriola ) during 2005–2008. These isolates of G. cichoracearum were screened on Lactuca species ( L. sativa, L. serriola, L. sativa × L. serriola, L. saligna and L. virosa ) by using a leaf-disc assay. The main goals were to assess virulence variation in natural populations of this pathogen, determine host-pathogen reaction patterns and search for the race-specificity in interactions between Lactuca species and isolates of G. cichoracearum . On the basis of results from the inoculation studies, a Lactuca spp. differential set was developed for characterization of virulence variation of this pathogen. The experimental work was divided into four sequential steps. In 2005, mainly L. serriola accessions were used for the virulence study. In 2006, the set was enlarged with genotypes of L. sativa , and L. sativa × L. serriola hybrids. In 2007, genotypes of L. sativa and L. serriola previously showing clear differential responses to G. cichoracearum isolates were selected and the set was supplemented with some accessions of L. saligna and L. virosa . Finally, in 2008, the number of differentials was settled on 13 accessions of Lactuca spp. (six cultivars of L. sativa ; one L. sativa × L. serriola hybrid; two accessions of L. serriola , L. saligna and L. virosa ) that showed broad variation in host-pathogen reaction patterns. Overall, the inoculation studies confirmed the existence of many pathogen races, and demonstrated that the interaction between Lactuca spp. and G. cichoracearum is race-specific. In the future, more experimental studies on the pathogenic variability of G. cichoracearum s. str. , as well as application of the genetic and molecular genetic approaches, is essential to understand this host-pathogen interaction in detail. These data may be important for applications in crop breeding and improvement.

Shikimate leaf disc assay for early detection of glyphosate resistance in Conyza canadensis and relative transcript levels of EPSPS and ABC transporter genes

Nol N, Tsikou D, Eid M, Livieratos IC & Giannopolitis CN (2012). Shikimate leaf disc assay for early detection of glyphosate resistance in Conyza canadensis and relative transcript levels of EPSPS and ABC transporter genes. Weed Research52, 233–241.SummaryTwenty‐two biotypes of Conyza canadensis (Canadian fleabane, horseweed) from a conventional orchard in Crete displayed varying degrees of reduced glyphosate susceptibility in standard whole plant assays. A refined shikimate leaf disc assay was developed to precisely determine the resistance levels, permitting early detection of resistance evolution and integrated management of the weed. The 5‐enolpyruvoylshikimate‐3‐phosphate synthase (EPSPS) homologue genes (1 and 2) were sequenced for three different biotypes (one of reduced susceptibility from Crete, one resistant from mainland Greece and one resistant from the USA), and no amino acid substitution of Pro106 was found. Real‐time qRT‐PCR was used to study the expression profiles for EPSPS and the M10 and M11 ABC transporter genes, following glyphosate application. The expression levels of the EPSPS genes were not significantly altered following glyphosate application in any biotype, but both M10 and M11 were found to be highly upregulated in glyphosate‐treated reduced susceptibility or resistant biotypes and not in a susceptible biotype. These results are in accordance with data recently reported by other researchers, supporting a role of the M10 and M11 ABC transporter genes in glyphosate resistance in Conyza canadensis, because of reduced translocation.

Sensitivity of Erysiphe necator to Demethylation Inhibitor Fungicides in Virginia.

Grape powdery mildew (Erysiphe necator) isolates were collected from 2005 to 2007 from vineyards mostly in Virginia but also some in Maryland, North Carolina, and Pennsylvania. Using a leaf disc assay, the isolates were tested against five demethylation inhibitor (DMI) fungicides. Most isolates exhibited reduced sensitivity to the five DMIs when compared with a sensitive group (n = 12) and compared with unexposed populations reported from other areas. The median resistance factor (RF) was highest for tebuconazole (RF = 399) and myclobutanil (RF = 378), followed by triflumizole (RF = 70), triadimefon (RF = 62), and fenarimol (RF = 44). The sensitive group used as the basis for comparison appears to have been more sensitive than unexposed isolates in New York and California. Our finding that the greatest resistance shift occurred with tebuconazole and myclobutanil contrasts with earlier reports from New York and California, where the greatest resistance shift was observed with triadimefon or triadimenol. Sensitivities to all five DMI fungicides were strongly correlated (pairwise r values of 0.70 to 0.87) but our data suggest that some may retain greater utility than others.

Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.

Resistance gradient of black pod disease in cocoa and selection by leaf disk assay

To characterize the gradient of resistance to black pod disease experimentally, the leaf disk assay was applied to 217 cocoa genotypes. The parameters time interval, number of leaves evaluated per genotype and distribution of experimental treatments were evaluated. Significant differences between genotypes were found, resulting in the grouping in five homogenous groups (p < 0.01). In the assessments with the leaf disk test, five and seven days after inoculation, the F value was highest after seven days, clearly separating the resistant from susceptible genotypes. There was no difference between the clones behavior and the use of more than one box to group the genotypes (p = 0.56) and the use of different leaves of the same genotype (p = 0.08). The resistance gradient observed indicates variability enough for mapping and cocoa breeding in the population.

The Susceptibility of Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) to Spinosad

The susceptibility of Leptinotarsa decemlineata (Say) adults to spinosad was determined by leaf disk and glass jar techniques under laboratory conditions. The mortalities of the treated adults were recorded 72 h post treatments. LC50s values in leaf disk and glass jar methods for adults were 98.50 and 108.34 mg a.i/L, respectively. A significant linear relationship between LC50 values of individual population across test methods was detected (r = 0.93, P < 0.05). Both bioassay techniques were suitable for monitoring resistance to spinosad in Colorado potato beetle adult populations. Glass jar technique, however, exhibited less variability in LC50 estimates and showed a higher degree of sensitivity than the leaf disk method. Filter paper and leaf disk techniques for larvae were two bioassay methods used to determine spinosad susceptibility in L. decemlineata population. Both bioassay techniques exhibited a different level of susceptibility of the larvae to spinosad; however, the confidence interval values from filter paper method were narrow than the leaf disk assay technique. A significant direct relationship between LC50 values of individual population across test methods was observed (r = 0.96, P < 0.05). The glass jar and filter paper testing methods are simple and sensitive test techniques for measuring susceptibility of Colorado potato beetle adults and larvae to spinosad, respectively. The efficacy of spinosad can be conserved for a long period of time if it is judiciously rotated with other suitable insecticides in a spray program and the number of application is restricted.

Silencing of Aphid Genes by dsRNA Feeding from Plants

BackgroundRNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.Methodology/Principal FindingsIn this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.Conclusions/SignificanceSimilar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.

Bottom-up effects mediated by an organic soil amendment on the cabbage aphid pests Myzus persicae and Brevicoryne brassicae

Earthworm-produced compost or vermicompost has been shown to increase resistance of plants to a variety of insect pests, but it is still unclear whether this resistance is dose dependent and whether the mechanisms responsible are the same for insect species with differing feeding habits and preferences. Therefore, we tested the effects of plants grown in various vermicompost concentrations (0, 20, 40, and 60%) on the preference and performance of generalist, Myzus persicae L., and specialist, Brevicoryne brassicae L. (both Hemiptera: Aphididae), aphid pests. Preference was evaluated with leaf disk (apterous) and whole plant (alate) choice assays. After 24 h of feeding, there was no significant negative effect on the feeding preference noted for apterae of either species of any of the treatments tested. To the contrary, apterae B. brassicae showed a significant preference for vermicompost treatments over control leaf disks. Alate M. persicae preferred alighting on control plants over vermicompost-grown plants, but B. brassicae showed no preference toward any of the treatments tested. Both aphid species deposited significantly more nymphs on control plants than on those grown in 20% vermicompost. Furthermore, plants grown in soil amended with 20% vermicompost significantly suppressed mass accumulation, as well as numbers of adults and nymphs of both aphid species compared to controls. These data clearly show that vermicompost soil amendments can significantly influence pest aphid preference and performance on plants and that these effects are not dose dependent, but rather species and morph dependent.

Sensitivity to fungicides in the isolates of Phytophthora infestans (Mont.) de Bary in the Czech Republic from 2003 to 2008

In the growing seasons from 2003 to 2008, 547 isolates of Phytophthora infestans from five regions in the Czech Republic were collected and examined for their sensitivity to the active ingredients (metalaxyl, dimethomorph and propamocarb-HCl) of frequently used fungicides. The response of the isolates to each of these substances was examined using the in vitro amended-agar method; in 352 of these isolates, the sensitivity to metalaxyl was also assessed by the floating leaf-disc assay. The majority of the isolates were sensitive (89.8%) to metalaxyl. Resistant isolates were found only in two of the sample years (2003 and 2008); they represented 58% of the samples in 2003 and only 29% in 2008. Four isolates from 2004 were found to be intermediate for their level of resistance. All the isolates that were tested were sensitive to dimethomorph and propamocarb-HCl; these particular substances completely suppressed mycelial growth at 1 &amp;micro;g a.i. per ml.

A novel recombinant tomato-infecting begomovirus capable of transcomplementing heterologous DNA-B components

The genome of a tomato-infecting begomovirus from Ranchi, India, was cloned, sequenced and analysed. The viral genome shared 88.3% sequence identity with an isolate belonging to the species Tobacco curly shoot virus (TbCSV), and this virus should therefore be considered a member of a new species, tentatively named Tomato leaf curl Ranchi virus (ToLCRnV). The DNA-β molecule, which had 74.5% sequence identity with tomato leaf curl Bangladesh betasatellite (ToLCBDB), is named tomato leaf curl Ranchi betasatellite (ToLCRnB). Phylogenetic analysis revealed that ToLCRnV is related to tomato leaf curl Bangladesh virus (ToLCBDV), tobacco curly shoot virus (TbCSV) and tomato leaf curl Gujarat virus (ToLCGV). An infectivity study with ToLCRnV established the monopartite nature of the viral genome, whereas inoculation with ToLCRnB resulted in increased symptom severity. ToLCRnV could transreplicate DNA-B of tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl New Delhi virus (ToLCNDV), both in N. benthamiana and tomato, although DNA-B accumulation of was less than with the wild-type combinations. ToLCRnB could be efficiently replicated by DNA-A of both ToLCNDV and ToLCGV. A leaf disk assay suggests that DNA-A could transreplicate the homologous DNA-B and DNA-β more efficiently than the heterologous one.

Detection of peroxynitrite accumulation in Arabidopsis thaliana during the hypersensitive defense response

Nitric oxide (NO) is synthesized in plants in response to stress, and its role in signaling is well-documented. In contrast, very little is known about the physiological role of its derivate peroxynitrite (ONOO −), which forms when NO reacts with O 2 - and induces protein modification by tyrosine nitration. Infection with an avirulent pathogen triggers the simultaneous production of NO and reactive oxygen species, as well as an increase in tyrosine nitration, so peroxynitrite could be physiologically relevant during this process. To gain insight into the role of peroxynitrite in plants, we measured its accumulation during the hypersensitive response in Arabidopsis thaliana using the specific peroxynitrite-sensitive fluorescent dye HKGreen-2 in a leaf disc assay. The avirulent pathogen Pseudomonas syringae pv. tomato, carrying the AvrB gene (Pst AvrB), induced a strong increase in fluorescence 3–4 h post-infiltration (hpi) which peaked 7–8 hpi. The increase in HKGreen-2 fluorescence was inhibited by co-injecting the peroxynitrite-scavenger urate together with the pathogen, and was almost completely eliminated by co-infiltrating urate with HKGreen-2, confirming that HKGreen-2 fluorescence in planta is induced specifically by peroxynitrite. This establishes a link between peroxynitrite synthesis and tyrosine nitration, and we therefore propose that peroxynitrite transduces the NO signal by modifying protein functions.

Host range evaluation and morphological characterization of Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew in Malaysia

A total of 29 isolates of Pseudoperonospora cubensis were collected from various cucurbit farms in West Malaysia. Sporangia of 13 isolates had the ability to germinate at 14°C and were used for host range (pathotype) study using leaf disc assay on a set of twelve cucurbit cultivars. Twelve different pathotypes of P. cubensis were determined and this demonstrated that P. cubensis isolates from cucurbit farms in West Malaysia are highly variable. Based on the host range study, majority of P. cubensis isolates were categorized into medium and high pathogenicity groupings and this fact shows the potential of this pathogen in invading cucurbit fields in the tropical regions. However, the pathotypes could not be differentiated based on morphological characterization of the sporangia and sporangiophores. Therefore, molecular characterization of the 13 isolates of P. cubensis will be useful to study the relationships among them. Key word : Oomycete, differential hosts, compatibility rating, pathogenic variation, sporangial germination.

Identification of Dictyothrips betae as the vector of Polygonum ring spot virus

Dictyothrips betae (Thysanoptera: Thripidae) is the predominant thrips species on Polygonum convolvulus and Polygonum dumetorum plants infected with a recently described tospovirus species, Polygonum ring spot virus (PolRSV). Laboratory transmission experiments (leaf disk assays) with adults collected directly in the field demonstrated the competence of this thrips to transmit PolRSV, although only at a rate of 4%. However, this increased to 16% using newly emerged larvae fed on infected leaves. Frankliniella occidentalis and Thrips tabaci failed to transmit PolRSV in leaf disk assays. Reverse transcription‐polymerase chain reaction (RT‐PCR) with specific primers for the N protein and Western blot analysis of adult thrips to detect the N protein confirmed the presence of the virus in D. betae individuals after feeding for at least 5 days on healthy plants. For molecular identification purposes partial sequences of mitochondrial cytochrome c oxidase subunit I (COI), nuclear 28S ribosomal DNA and the elongation factor‐1α (EF‐1α) from D. betae were cloned. COI sequence was also used for deriving a phylogenetic tree, including D. betae. The results confirmed a relationship between this species and tospovirus‐transmitting insects of the genus Thrips.