Leaf Disc Assay Research Articles

Transmission Efficiency of Papaya ringspot virus by Three Aphid Species

The transmission efficiency of Papaya ringspot virus (PRSV) by three aphid vectors (i.e., Aphis gossypii, A. craccivora, and Myzus persicae) was studied. Efficiency was measured by single-aphid inoculation, group inoculation (using five aphids), duration of virus retention, and the number of plants following a single acquisition access period (AAP) to which the aphids could successfully transmit the virus. Single-aphid inoculation studies indicated that M. persicae (56%) and A. gossypii (53%) were significantly more efficient in transmitting PRSV than A. craccivora (38%). Further, in the former two species, the time required for initiation of the first probe on the inoculation test plant was significantly shorter compared to A. craccivora. PRSV transmission efficiency was 100% in all three species when a group of five aphids were used per plant. There was a perceptible decline in transmission efficiency as the sequestration period increased, although M. persicae successfully transmitted PRSV after 30 min of sequestration. A simple leaf-disk assay technique was employed for evaluating the transmission efficiency of three species of aphids. The results of leaf-disk assays also indicated that A. gossypii (48%) and M. persicae (56%) were more efficient PRSV vectors than A. craccivora. Using leaf-disk assays, the ability of individual aphids to inoculate PRSV serially to a number of plants was studied. Following a single AAP on an infected leaf, M. persicae was more efficient than the other two species with 52.5% transmission after the first inoculation access period (IAP). However, its inoculation efficiency significantly decreased with the second and subsequent IAPs. A. gossypii was able to transmit PRSV sequentially up to four successive leaf disks, but with significantly declining efficiency. Since A. gossypii is reported to be the numerically dominant vector in south India in addition to being a more efficient vector capable of inoculating PRSV to multiple plants, it should be the target vector for control strategies.

Persistence of Phenylamide Insensitivity in Pseudoperonospora humuli.

Downy mildew, caused by Pseudoperonospora humuli, is an important disease of hop in most production regions in the northern hemisphere. Insensitivity to phenylamide fungicides was detected in isolates of P. humuli in production regions in Oregon and Idaho in 1992, and these fungicides since have been used on a limited basis. In this study, the prevalence of phenylamide insensitivity among isolates of P. humuli collected from 2005 to 2007 in the northwestern United States was quantified using a leaf disk assay with a discriminating dose (25 μg/ml) of either metalaxyl or mefenoxam with inoculum derived from 201 systemically infected diseased shoots (basal spikes) collected from 6, 10, and 11 hop yards in Idaho, Washington, and Oregon, respectively. A subset of 47 basal spike isolates and 42 monosporic isolates collected from two yards in Idaho and nine yards in Oregon during 2006 and 2007 were assayed using a dilution series of metalaxyl to determine the effective dose that inhibited 50% of the incidence of sporulation (ED50). Insensitivity to mefenoxam was detected in 31 of 74 (41.9%) basal spikes collected from 13 hop yards. Insensitivity to the related compound metalaxyl was detected in 52 of 80 (65%) spikes collected from nine hop yards, including four hop yards in Washington. Log ED50 values ranged from -2.25 to 2.67 for basal spike isolates and -2.27 to 2.98 for monosporic isolates and had a similar distribution. Log ED50 values for monosporic isolates and entire basal spike isolates were significantly associated. However, the slope of the regression line was less than 1, indicating that the log ED50 values obtained from entire basal spike isolates were greater than the corresponding log ED50 values obtained from a monosporic isolate obtained from that spike. This research suggests that insensitivity to phenylamide fungicides is a stable phenotype in P. humuli. Management of downy mildew should rely on measures other than these fungicides in yards or regions where phenylamide insensitivity is prevalent, which is now known to include certain hop yards in Idaho, Oregon, and Washington.

Effect of water on germination of Plasmopara viticola oospores

The effect of moisture in grape leaf litter holding overwintering Plasmopara viticola oospores was investigated. Oospores were incubated under different regimes of water activity (aW 0·991 to 0·123) for 2 to 15 days and their ability to germinate and cause infection was determined using a sensitive leaf disk assay. Reduction of aW caused a significant shift in the infection dynamics, with maximum effect when aW ≤ 0·56. Dynamics of aW in the leaf litter under natural conditions were estimated from moisture data using a Chen‐Clayton equation. Daily patterns of leaf litter moisture (M in % weight) were determined in non rainy periods between mid February and mid June, while the Chen‐Clayton equation was calculated using data of aW and M measured in both sorption and desorption conditions, at different temperatures. Water activity was highest at 08·00 hours, decreased progressively until 14·00 hours, and then increased. Water activity was favourable for oospores to develop in about 25% of the measurements, all made between 18·00 hours and 08·00 hours. A close relationship was found between vapour pressure deficit (VPD in hPa) and aW of the leaf litter, so that when VPD is lower than 2·13 hPa there is sufficient water for oospores to develop. Results showed that leaf litter moisture due to water from the atmosphere makes oospore development possible during non rainy periods.

Multiple ALS Mutations Confer Herbicide Resistance in Waterhemp (Amaranthus tuberculatus)

In a survey of herbicide responses among Illinois waterhemp half-sib populations, several were observed with differential responses to imazethapyr and thifensulfuron, two acetolactate synthase (ALS)–inhibiting herbicides. Plants from two waterhemp populations were verified resistant to imazethapyr, but susceptible to chlorimuron, using a nondestructive leaf-disc assay. Sequencing of the ALS gene revealed that imazethapyr-resistant waterhemp plants from both populations had inferred amino acid substitutions at position 653 of ALS. Depending on the population, the serine at position 653 of ALS was substituted with either asparagine (S653N) or threonine (S653T). Waterhemp lines were derived from each population to create uniformly imidazolinone-resistant (IR) waterhemp biotypes, designated IR-62 and IR-101. ALS-inhibitor responses of each IR biotype were compared with a previously identified ALS inhibitor–resistant biotype with a tryptophan to leucine substitution at position 574 (W574L) and an herbicide-susceptible control. Whole-plant dose–response experiments with waterhemp biotypes containing W574L, S653N, or S653T mutations indicated that each biotype was resistant to imazethapyr, but only the biotype with a W574L mutation was resistant to thifensulfuron. In vitro ALS-activity assays revealed unique patterns of cross-resistance among protein extracts derived from each biotype in response to imazethapyr, thifensulfuron, cloransulam, and pyrithiobac. In conclusion, three different forms of target-site–based resistance to ALS inhibitors have been identified in waterhemp.

Evaluation for Resistance to Powdery Mildew in Cornus Species and Hybrids Using a Leaf Disk Assay

Abstract Using a leaf disk assay, eight cultivars or/and breeding lines in Cornus florida L., C. kousa (Buerger ex Miq.) Hance, five cultivars in C. kousa × C. florida, one cultivar in C. kousa × C. nuttallii Aud. and one cultivar in (C. kousa × C. nuttallii) × C. kousa were evaluated for resistance to powdery mildew [Erysiphe pulchra (Cooke and Peck) U. Braun & S. Takamatsu (syn. Microsphaera pulchra Cooke and Peck)]. Flowering dogwoods (C. florida) cultivars were more susceptible than the other species and hybrids with the exception of the C. florida × C. kousa hybrid ‘Ruth Ellen’. Resistance in kousa dogwoods and hybrids was manifested as restriction of hyphal growth and inhibition of sporulation. Although mildew colonies and sporulation were detected on leaves of all flowering dogwood cultivars, ‘Karen's Appalachian Blush’ and ‘Worlds Fair’ were more resistant than other cultivars. These dogwoods had significantly lower values of percent geminated conidia with branched hyphae, infection efficiency, sporulation and delayed latent period. The leaf disk assay provides a laboratory procedure to screen new cultivars and lines of dogwoods for resistance to powdery mildew.

Integration of Copper-Based and Reduced-Risk Fungicides for Control of Blumeriella jaapii on Sour Cherry.

Practical resistance to sterol demethylation inhibitor (DMI) fungicides among populations of Blumeriella jaapii, the cherry leaf spot (CLS) pathogen, was documented in 2005. In the present study, strategies to reduce selection for DMI-resistant strains of B. jaapii and adapt to possible restrictions on the use of chlorothalonil are described. Ten field trials were conducted on the sour cherry cultivars Balaton and Montmorency to test the efficacy of integrating respiration-inhibitor and copper-based fungicides into spray programs. Programs that included up to three sprays of copper-based fungicides were among the most effective for controlling CLS, although leaf phy-totoxicity was sometimes observed. Under high disease pressure, eliminating chlorothalonil compromised CLS control. 'Balaton' and 'Montmorency' did not differ in the percentage of leaves with CLS or defoliation resulting from CLS. The physical modes of action of representative DMI, QoI, and copper-based fungicides were evaluated in a leaf disk assay. Trifloxystrobin, a QoI fungicide, provided the best protection against infection by B. jaapii. All fungicides were more effective than water when applied 46 h postinfection, although differences were not statistically significant in one of two trials. Tebuconazole, a DMI, was the only fungicide that was more effective than water in preventing resporulation from existing lesions in both trials. Isolates of B. jaapii, which varied in DMI-sensitivity, all were sensitive to copper in vitro.

Comparative Efficacy of Systemic Acquired Resistance-Inducing Compounds Against Rust Infection in Sunflower Plants

ABSTRACT Four inducers of systemic acquired resistance (SAR) were examined for their efficacy in controlling rust infection caused by Puccinia helianthi in sunflower plants. Of the four compounds, DL-3-amino-n-butanoic acid (DL-beta-aminobutyric acid [BABA]) was the most effective and sodium salicylate (NaSA) was the least effective in protecting against rust. In leaf disk assays, full protection was obtained with BABA at 25 mug/ml, benzodiathiazol-S-methyl ester (BTH) at 100 mug/ml, 2,6-di-chloroisonicotinic acid (INA) at 100 mug/ml, and NaSA at >200 mug/ml. L-2-amino-n-butanoic acid (AABA) was partially effective, whereas N-methyl-BABA and 4-aminobutnoic acid (GABA) were ineffective. The R-enantiomer of BABA, but not the S-enantiomer, was more effective than the racemic mixture. In intact plants, BABA applied as a foliar spray or a root dip, before or after (up to 48 h) inoculation, provided significant protection for 8 days. BTH, INA, and NaSA were less protective and more phytotoxic compared with BABA. BABA did not affect urediospore germination, germ tube growth, appressorial formation, or initial ingress of P. helianthi, but strongly suppressed mycelial colonization in the mesophyll and, consequently, pustule and urediospore formation. No accumulation of defense compounds (phenolics, lignin, or callose) was detected in BABA-treated inoculated or noninoculated plants. This is the first report on the activity of BABA against an obligate Basidomycete pathogen in planta.

Shikimate Accumulation in Sunflower, Wheat, and Proso Millet after Glyphosate Application

Experiments were conducted to examine the utility of a spectrophometric leaf disc assay for detecting shikimate accumulation after glyphosate application in sunflower, proso millet, and wheat. The assay was conducted on both greenhouse- and field-grown plants. Glyphosate was applied at five rates ranging from 840 to 53 g ae ha−1. Shikimate accumulation data were generated at 1, 4, 7, and 14 d after application (DAA). Sunflower accumulated shikimate more rapidly and at lower glyphosate rates than the other two species. At 14 DAA, glyphosate at the two highest rates remained detectable in all three species. Plants receiving lower glyphosate doses (210, 105, and 53 g ae ha−1) had begun to grow out of the injury, or at least the shikimate levels in the plants were no longer significantly different than that present in the untreated controls. This spectrophotometric assay is both rapid and simple, with respect to other means of detecting shikimate, and it can be used to detect glyphosate drift. For it to be used by crop managers, samples from potentially drift-affected crops should be taken as soon as possible after the suspected drift event or immediately after the appearance of glyphosate injury.

Agrobacterium-mediated Genetic Transformation in Cucumber (Cucumis sativus L.)

The present study was undertaken to develop efficient transformation protocol for cucumber cv. Poinsett 76 using Agrobacterium strain EHA 105. Five-day-old mature cotyledon explants was used for transformation study. The infected explants were co-cultivated for 2 days in MS medium containing BA (1.0 mg Lˉ1). The selection of transformed shoots was carried out in MS medium fortified with BA (1.0 mg Lˉ1), Cefotaxime (300 mg Lˉ1) and PPT (2.0 mg Lˉ1). The transformed shoots were elongated in MS medium containing BA (1.0 mg Lˉ1), Cefotaxime (300 mg Lˉ1), PPT (2.0 mg Lˉ1) along with GA3 (0.5 mg Lˉ1). The rooting of elongated shoots was achieved in MS medium with BA (1.0 mg Lˉ1), Cefotaxime (300 mg Lˉ1), PPT (2.0 mg Lˉ1) and IBA (0.6mg Lˉ1). The transient GUS expression assay and leaf disc assay were carried out in order to find transformed shoots. The molecular confirmation of transformed shoots revealed the foreign gene integration into cucumber genome.

Identification and Characterization of Plant Genes Involved inAgrobacterium-Mediated Plant Transformation by Virus-Induced Gene Silencing

Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.

Insecticidal and antifeedant activities of proteins secreted by entomopathogenic fungi against Spodoptera littoralis (Lep., Noctuidae)

Abstract: Entomopathogenic fungi are a poorly exploited source of insecticidal proteins, which may be used for the development of new natural insecticides and as alternative molecules for transgenic deployment. The crude soluble protein extracts in Adamek's liquid medium of 25 fungal isolates belonging to the fungal species Metarhizum anisopliae, Beauveria bassiana, Beauveria brongniartii and Scopulariopsis brevicaulis were screened by per os toxicity on Spodoptera littoralis larvae. Whilst extracts from two M. anisopliae and two B. bassiana isolates gave significant mortalities when applied either on alfalfa leaf discs or incorporated into artificial diet, the one from M. anisopliae 01/58‐Su isolate was the only most toxic that showed promise for S. littoralis control. In leaf disc assays, this extract exhibited strong dose‐related toxic and antifeedant activity against the larvae. At 10, 20 and 40 μg protein/insect, the extract gave 61.3%, 96.6% and 100.0% mortality, respectively, and average survival time of 5.7, 4.3 and 3.1 days respectively. Not only the antifeeding index was dose‐related, but it significantly increased over time in a dose‐related manner. Longer exposure times led to a dose‐related significant increase in larval mortality. The exposure times for 50% mortality were 91.3 h and 62.1 h for 20 and 40 μg protein/insect respectively. The crude extract when exposed to higher temperature or protease treatment lost toxicity, indicating that toxicity was protein mediated. In addition, the liquid medium composition did not influence its insecticidal activity. The effects of the protein extract on midgut cells of second instar larvae of S. littoralis were investigated by using both light and electron microscopy. A progressive bleeding of the midgut epithelium into the gut lumen was observed along with lysis of the epithelium and deterioration of the microvilli.

(126) Morphology and Physiology of Sugar Transport in Apple Leaves

Sorbitol is the predominant phloem-translocated carbohydrate in apple. The pathway—either apoplastic or symplastic—of sugar transport from photosynthetic cells to the phloem is not established. Furthermore, the presence of absence of phloem loading has not been tested. This study characterized the morphology and physiology of sugar movement to the phloem in apple leaves. An electron micrographic survey of apple leaf minor vein morphology was performed. Plasmodesmata were abundant and found at the interfaces of each cell type from mesophyll to sieve elements, indicating a symplastic sugar pathway. We also tested for a phloem loading mechanism. First, 14C-labeled sorbitol and sucrose were introduced exogenously to leaf discs to determine if they are loaded into veins from the apoplast. Although leaf discs floated on a solution containing either sugar actively accumulated label, the labeling pattern was diffuse, with no accumulation in minor veins. The addition of the sulfhydryl reagent PCMBS to the leaf disc assay inhibited sugar uptake. We also attempted plasmolysis of apple leaf sections to measure the solute concentration difference between photosynthetic mesophyll cells and cells of the minor vein phloem. Apple leaf pieces fixed in a solution containing 1.5 mol/kg osmoticum did not plasmolyze. We conclude that although active uptake of both sorbitol and sucrose takes place in apple leaves, apoplastic phloem-loading is absent. Considering the high sugar concentration and the symplastic connectivity among leaf cell types, we propose that sugars are instead enter the phloem after moving down—rather than against—a concentration gradient.

Frankliniella cephalica, a New Vector for Tomato spotted wilt virus.

Frankliniella cephalica (Crawford) is an invasive species of thrips found in the islands of Yaeyama in the Okinawa Prefecture, Japan. During the late 1990s to early 2000s, a species of thrips was isolated from wild flowers of Bidens pilosa L. and Ipomoea batatas L. growing close to cultivated fields. They were subsequently identified as F. cephalica using fine morphological characteristics with the help of Steve Nakahara (U.S. Department of Agriculture, Beltsville, MD) and Laurence Mound (CSIRO, Australia). Voucher specimens were deposited in the Laboratory of Insect Resources, Faculty of Agriculture, Tokyo University of Agriculture by Shuji Okajima (2). We investigated the ability of F. cephalica to vector Tomato spotted wilt virus (TSWV) by experimentally determining virus transmission efficiency. Newly hatched larvae as much as 12 h old underwent a viral acquisition-access period (AAP) of 24 h, during which they fed on the leaves of Datura stramonium infected with TSWV-O, a Japanese type isolate. Transmission efficiency of adults 4 days after emergence from molt (14 days after the AAP) was determined by a petunia leaf disk assay (3) in which the adults were individually allowed to feed for successive 24-h inoculation access periods (IAP) on two different leaf disks of Petunia × hybrida cv. Polo Blue. Transmission of the virus by the adults was considered positive if at least one of the leaf disks showed viral necrotic spot. We tested 20 randomly selected leaf disks with clear necrotic spots using a simplified rapid immunofilter paper assay. All selected disks were positive for TWSV. The transmission efficiencies were 24.6% for female (n = 57) and 54.4% for male (n = 125) adults. The efficiency was significantly different between sexes (Fisher's exact probability test, P < 0.001). We also examined changes in the virus infection site at different developmental stages in thrips using immunofluorescence microscopy with a polyclonal antibody to N protein of the virus (4). After a 6-h AAP feeding by first instar larvae, the virus was found initially to infect the epithelial cells and then spread throughout the midgut tissue in the second instar larvae 5 days after acquisition of the virus. In viruliferous adults, the virus was present in the salivary glands and on the basement membrane of the midgut tissue. These data indicate that F. cephalica is a new insect vector for TSWV. F. cephalica is a major insect pest of tropical crops in tropical and subtropical coastal belts (1). The presence of a thrips vector in weed hosts surrounding cultivated fields might increase the chance of crops in this habitat becoming infected with viruses.

Leaf Disk Infection by Colletotrichum acutatum and Its Relation to Fruit Rot in Diverse Blueberry Germplasm

Response to foliar infection by Colletotrichum acutatum Simmonds ex Simmonds was assayed in a diverse group of 149 blueberry cultivars and selections using a detached leaf-disk assay. Disks were inoculated and incubated for one week, then were digitally imaged, and images analyzed for percent leaf decay. Infection percentages across cultivars averaged 32%, and ranged from 8% to 79%. The lowest levels of foliar infection were seen in the cultivars, Burlington, Sharpblue, and Berkeley. Foliar responses were compared to anthracnose fruit rot susceptibility data from a previous study. Several clones were observed to have low levels of both foliar and fruit infection. Cultivars with particularly good resistance to both phases included `Sharpblue', `Sunshine Blue', `Legacy', `Little Giant', `Flordablue', `Elliott', `Blue Ridge', `Blue Rose', and `November Glow'. Little correlation was observed between foliar response and fruit response to anthracnose infection (r = 0.15). Since C. acutatum overwinters primarily in vegetative tissue, breeding new cultivars with foliar resistance may assist in reducing inoculum levels of this disease under field conditions.

Local Variation in Susceptibility of Colorado Potato Beetle (Coleoptera: Chrysomelidae) to Insecticide

The susceptibility status of Colorado potato beetle, Leptinotarsa decemlineata (Say), adults to phosalone was determined by dip and glass jar assay techniques. Bioassay results indicated a narrow variation in Colorado potato beetle insecticide susceptibility among sample sites. LC50 values were generally highest from specimens collected in field that received frequent phosalone applications for seven consecutive growing seasons. In five populations tested, LC50 values ranged from 503.72 to 827.95 ppm in dip test method. In glass jar technique, resistance ratio value of 1.72 for LC50 was obtained. A significant linear relationship between LC50 values of individual populations across test methods was detected. Both bioassay techniques were suitable for monitoring resistance to insecticide 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 dip method. Filter paper and leaf disk techniques for larvae were two bioassay methods used to determine phosalone susceptibility in L. decemlineata populations. Both bioassay techniques exhibited a similar level of susceptibility of the larvae to phosalone; however, the fiducial limit 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. Differences in LC50 ranking among fields between adults and larvae indicated a differential susceptibility to insecticide between life stages. Low LC50 values obtained from Colorado potato beetle in sample sites indicated that phosalone resistance was not severe in these fields. 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 phosalone, respectively.

Local Variation in Susceptibility of Colorado Potato Beetle (Coleoptera: Chrysomelidae) to Insecticide

The susceptibility status of Colorado potato beetle, Leptinotarsa decemlineata (Say), adults to phosalone was determined by dip and glass jar assay techniques. Bioassay results indicated a narrow variation in Colorado potato beetle insecticide susceptibility among sample sites. LC50 values were generally highest from specimens collected in field that received frequent phosalone applications for seven consecutive growing seasons. In five populations tested, LC50 values ranged from 503.72 to 827.95 ppm in dip test method. In glass jar technique, resistance ratio value of 1.72 for LC50 was obtained. A significant linear relationship between LC50 values of individual populations across test methods was detected. Both bioassay techniques were suitable for monitoring resistance to insecticide 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 dip method. Filter paper and leaf disk techniques for larvae were two bioassay methods used to determine phosalone susceptibility in L. decemlineata populations. Both bioassay techniques exhibited a similar level of susceptibility of the larvae to phosalone; however, the fiducial limit 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. Differences in LC50 ranking among fields between adults and larvae indicated a differential susceptibility to insecticide between life stages. Low LC50 values obtained from Colorado potato beetle in sample sites indicated that phosalone resistance was not severe in these fields. 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 phosalone, respectively.

Variation in Septoria musiva and Implications for Disease Resistance Screening of Poplars.

A set of isolates of Septoria musiva differed in aggressiveness in hybrid poplar leaf disk and stem assays and culture growth in vitro. Clone × isolate interactions were observed in one of the stem assay experiments, but not in the leaf disk assay experiments. Random amplified polymorphic DNA (RAPD) analyses were performed using 52 isolates of S. musiva collected from hybrid poplars and a native poplar species in Minnesota, Wisconsin, Iowa, South Dakota, and North Dakota. There was a large degree of genetic similarity, although each isolate had a unique RAPD pattern. No relationships among isolates were found for molecular genetic distance and host clone, parentage, or taxonomic classification section; location or date of collection; or the previously determined level of field resistance of the host clones to Septoria canker. Results of the stem and leaf disk assays indicate that it may not be necessary to choose the most aggressive isolate for disease resistance screening. It may be more useful to select isolates that will discriminate the greatest variation in levels of disease resistance among the clones that are being screened.

Differences in virulence of Diplocarpon earlianum isolates on selected strawberry cultivars

Eight isolates of Diplocarpon earlianum were evaluated for virulence, measured as the ability to produce percent leaf area with symptoms (LAS), on 18 cultivars of strawberry (Fragaria x ananassa). Significant differences were observed from isolate and cultivar effects, and isolate x cultivar interaction. Four of the eight isolates, on the average of 18 strawberry cultivars, had LAS ranging from 6.7-9.6%, and were classified as a group of low virulence. The remaining four isolates had LAS ranging from 25.9-45.8% and formed a high virulence group. Of the cultivars tested, 'Vibrant' was resistant and 'Micmac' was susceptible to all isolates in both virulence groups; 'Honeoye', 'Redcoat', 'Scotland', 'St. Clair' and 'Vantage' were resistant to the low virulence group, but had differential interactions to isolates in the high virulence group; the remaining 11 cultivars were susceptible to the high virulence group, but had differential interactions to isolates in the low virulence group. It is suggested that a number of different pathogen isolates are required to test strawberry genotypes for leaf scorch resistance. A leaf disk assay was developed in this study and can be used for laboratory evaluations of strawberry genotypes for susceptibility to D. earlianum.

Interaction between a Nanovirus-like Component and the &amp;lt;italic&amp;gt;Tobacco Curly Shoot Virus&amp;lt;/italic&amp;gt;/Satellite Complex

The biological role of DNA1, a nanovirus-like component shown to be associated with the begomovirus/satellite complex, has not yet been identified. Here, we demonstrated that DNA1 of Tobacco curly shoot virus isolate Y35 (TbCSV-Y35) attenuated leaf-curling symptoms induced by TbCSV-Y35 or TbCSV-Y35 plus Y35 DNAbeta in the early stage of symptom development and induced leaf cluster at a later stage of symptom development in Nicotiana benthamiana plants. The leaf disc assay demonstrated that TbCSV-Y35 DNA1 replicated autonomously. Southern blot analysis revealed that TbCSV-Y35 DNA1 reduced viral DNA accumulation. Viral DNA accumulation was not reduced when plants were co-inoculated with TbCSV-Y35 DNAbeta, but the TbCSV-Y35 DNAbeta level was dramatically reduced in the presence of TbCSV-Y35 DNA1. To determine whether the interaction between TbCSV/satellite complex and DNA1 had isolate specificity, DNA1 of TbCSV isolate Y132 was cloned and sequenced. It was found to have 75% nucleotide sequence identity with TbCSV-Y35 DNA1. Infectivity tests showed that TbCSV-Y132 DNA1 had no effect on the symptoms induced by TbCSV-Y35 or TbCSV-Y35 and Y35 DNAbeta in N. benthamiana plants, although Y132 DNA1 could replicate in these plants.

Specificity of accumulation and transmission of tomato spotted wilt virus (TSWV) in two genera, Frankliniella and Thrips (Thysanoptera: Thripidae)

The accumulation and transmission of tomato spotted wilt virus (TSWV) was examined in second instar larvae and adults of two thrips genera, Frankliniella and Thrips. The species tested were F. occidentalis (Pergande), F. intonsa (Trybom), T. tabaciLindeman, T. setosus Moulton, T. palmi Karny and T. hawaiiensis (Morgan). In a standard petunia leaf disc assay, the efficiencies of TSWV transmission by two species of Frankliniella were higher than those of any Thrips species in the adult stage. A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) showed that large amounts of the TSWV-nucleocapsid (N) protein were present in the ELISA-positive larvae of each species, with the exception of T. palmi. The ELISA titre of and the proportion of virus-infected individuals of the two Frankliniella species increased or did not significantly change from the larval to the adult stages, whereas those of the four Thrips species decreased significantly. These results show that the specificity of virus transmission by adult thrips is probably affected by the amount of viral N protein accumulation in the adults and that the accumulation pattern from the larval to the adult stages is in between the two genera tested in the present study.