Genetic Structure and Variation in Aggressiveness in European and Australian Populations of the Grapevine Dieback Fungus, Eutypa lata

Eutypa lata is an ascomycete fungus causing a severe dieback in grapevine. The genetic structure of populations of E. lata from seven regions in Australia, France, Italy and Spain was examined using 20 random amplified polymorphic DNA (RAPD) markers. In some regions, populations were subdivided and a total of 14 samples were analysed. A total of 231 RAPD haplotypes were found among the 240 isolates. Vegetative compatibility testing further demonstrated that isolates of the same haplotype were genetically distinct. Gene diversity was the highest in the population from northern Italy and lowest in the Alsace region in France. Linkage disequilibrium between pairs of putative loci was very low and most of the multilocus analyses were consistent with the hypothesis of random association of the loci. This suggests that random mating occurred in every population and that the sexual stage shapes the genetic structure of E. lata populations in the regions sampled. Only 6% of the total variability was attributable to differences between populations. Nevertheless, significant differences in allele frequency appeared with respect to six RAPD markers indicating some genetic differentiation between populations. This differentiation appeared attributable to differences between the Italian and Spanish populations and the other populations. We thus hypothesize that a restriction of gene flow exists within Europe. The population from Australia was genetically closer to the French and Spanish populations than to that from Italy. Genetic diversity is associated with considerable variation in aggressiveness, which was assessed on cuttings in the greenhouse in six populations. All populations included a range of isolates differing in aggressiveness, but the Italian population seemed to have more isolates with low aggressiveness.     

Dieback symptoms on olive trees caused by the fungus Eutypa lata1

The fungus Eutypa lata was isolated from olive trees for the first time in 1988 from two young 3-year-old trees. It was evident that the infection was initiated from the graft union. The trees were also infected by Armillaria mellea. In a second case in 1991, extensive cankers were observed along the branches which often were girdled and died. The infected area extended from the bark into the sapwood and even into the heartwood. Cultures made on potatodextrose agar from the margin of necrotic and healthy tissues resulted in typical colonies of the fungus E. lata, as well as of Verticillium dahliae. The colonies of E. lata produced the characteristic pycnidia of the anamorph Libertella blepharis 3–4 weeks later. Pathogenicity tests made on olive, apricot, almond and walnut trees resulted in the death of the inoculated twigs or in canker formation and extensive brown discoloration of the wood.     

Detection of Eutypa lata and Eutypella vitis in Grapevine by Nested Multiplex Polymerase Chain Reaction

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.     

Pseudomonas syringae pv. coryli, the Causal Agent of Bacterial Twig Dieback of Corylus avellana

ABSTRACT Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.     

Morphological and molecular identification of Eutypa lata on grapevine in Serbia

Journal of Plant Diseases and Protection - During a survey from 2004 to 2016, symptoms of grapevine dieback were observed in six vineyards in Serbia. Symptoms initially appeared as small,...     

Genetic structure of the fungal grapevine pathogen Eutypa lata from four continents

The generalist ascomycete fungus Eutypa lata causes Eutypa dieback of grapevine (Vitis vinifera) worldwide. To decipher the cosmopolitan distribution of this fungus, the population genetic structure of 17 geographic samples was investigated from four continental regions (Australia, California, Europe and South Africa), based on analysis of 293 isolates genotyped with nine microsatellite markers. High levels of haplotypic richness (R = 0·91–1) and absence of multilocus linkage disequilibrium among loci supported the preponderance of sexual reproduction in all regions examined. Nonetheless, the identification of identical multilocus haplotypes with identical vegetative compatibility groups, in some vineyards in California and South Africa, suggests that asexual dispersal of the fungus among neighbouring plants could be a rare means of disease spread. The greatest levels of allelic richness (A = 4·89–4·97) and gene diversity (H = 0·66–0·69) were found in Europe among geographic samples from coastal areas surrounding the Mediterranean Sea, whereas the lowest genetic diversity was found in South Africa and Australia (A = 2·78–3·74; H = 0·49–0·57). Samples from California, Australia and South Africa, which had lower genetic diversity than those of Europe, were also characterized by demographic disequilibrium and, thus, may represent founding populations of the pathogen. Low but significant levels of genetic differentiation among all samples (D_EST = 0·12, P = 0·001; F_ST = 0·03, P = 0·001) are consistent with historical gene flow preventing differentiation at continental scales. These findings suggest that global, human‐mediated spread of the fungus may have resulted in its current global distribution.     

Variation in Pathogenicity and Genetic Structure in the Eutypa lata Population of a Single Vineyard

ABSTRACT Fifty-five isolates of Eutypa lata were collected in 1994 from a single vineyard, each from a different vine that showed either shoot and foliar symptoms of Eutypa dieback or only abnormalities during the period of 1990 to 1994. These isolates showed a large variation in pathogenicity after inoculation on cuttings in the greenhouse. Variability also was observed for cultural traits and radial growth rate on potato dextrose agar (PDA) medium, but no relation was found between these characteristics and pathogenicity. The isolates were paired on PDA medium, and a barrage reaction was observed for all pairings, indicating the isolates were vegetatively incompatible. Thirty-two random amplified polymorphic DNA (RAPD) markers were used to study the genetic relatedness of the isolates and the genetic structure of the population. Fifty-five different RAPD patterns were observed, confirming the genetic uniqueness of each isolate. Gametic disequilibrium values were calculated among all pairs of the 32 putative RAPD loci, and only one value was significant. Unweighted pair-group method with arithmetic averages analysis of distance data, as well as a heterogeneity statistic (Fst), did not indicate any population substructure. The results strongly suggest that isolates originated from a random-mating population and that the disease was propagated in this vineyard by ascospores produced from diverse outside sources. Southern hybridization performed for five markers indicated that the two-allele assumption made to interpret RAPD data may be violated for markers that are similar in size. However, the exclusion of such markers did not change the conclusions of the study.     

Identification of grapevine marker genes for early,non‐destructive Eutypa lata infection diagnosis

Eutypa lata is the causal agent of eutypa dieback, a highly damaging trunk disease affecting all grape‐growing areas, with currently neither an efficient curative treatment nor an early non‐destructive diagnostic method. The present work was carried out to discover grapevine genes expressed in response to the presence of E. lata that could be useful to develop an early (before visible foliar symptoms) and non‐destructive (using grapevine leaves) diagnostic tool. Microarray analyses were carried out from (i) infected plants showing characteristic E. lata foliar and vascular symptoms and positive pathogen recovery from vascular lesions (S⁺R⁺), (ii) infected plants showing no symptoms (S^?R⁺), and (iii) symptomless plants with negative pathogen recovery (S^?R^?). Vineyard and greenhouse‐grown plants, naturally or artificially infected respectively, and uninoculated controls were characterized and leaf RNA was hybridized with 15k operon grapevine oligonucleotide microarrays. Among the grapevine genes differentially expressed between S^?R⁺ and S^?R^? plants in greenhouse and vineyard conditions, 10 were highlighted as robust candidate genes for diagnosis: seven were specifically involved in response to infection and three were associated with symptom absence. Five were confirmed to be effective diagnostic marker genes usable in a qRT‐PCR‐based test performed on RNA extracted from grapevine leaves cultivated in either greenhouse or vineyard conditions. Furthermore, their expression profiles in response to infection with E. lata or other major grapevine fungi (Erysiphe necator, Plasmopara viticola, Botrytis cinerea) could be distinguished. The usefulness of these genes to develop an early and non‐destructive method for diagnosis of E. lata infection is discussed with regard to the advantages and drawbacks of previous E. lata diagnostic studies.     

Variation in susceptibility of grapevine pruning wound to infection by Eutypa lata in south-western France

Grapevine cultivars Cabernet Sauvignon (susceptible to Eutypa dieback), and Merlot (tolerant), were pruned three times during the dormant season (mid-December, mid-January and mid-February) and wounds on the 1-year-old canes were inoculated weekly with ascospores of Eutypa lata after pruning. No differences in susceptibility to infection were observed between cultivars, although in the vineyard they differed in symptom expression. Infection by E. lata varied with pruning date and the age of the pruning wound, and was higher and of longer duration with early pruning (December). At low temperature, infection of the pruning wounds by E. lata was increased, whereas the growth of other microorganisms was reduced. Moderate temperatures encouraged the growth of other microorganisms, notably Rhodotorula sp. This natural colonizer of grapevine pruning wounds was able to reduce the infection capacity of E. lata . It was more effective when inoculation with E. lata was carried out with low numbers of ascospores and when it was delayed until 14 days after application of the wound colonizer, infection being reduced by 95–100%.     

Pathogenesis of Eutypa lata in grapevine: identification of virulence factors and biochemical characterization of cordon dieback

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.     

Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

ABSTRACT Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.     

The Biological Control of Fusarium oxysporum,the Causal Agent of Potato Rot

This study was carried out in vivo and in vitro to search the biological control possibilities of soil borne dry rot causal agent Fusarium oxysporum causing yield losses in potato. In this study, 2 Pantoea agglomerans (BRTB and RK-92), 2 Bacillus pumilus (RK-103 and TV-67C), 7 Bacillus subtilis (BAB-140, TV-12H, TV-6F, EK?7, TV-17C, CP?1 and TV-125A), 3 Bacillus megaterium (TV-103B, TV-87A and TV-91C), 1 Ochrobactrum anthropi (A-16B), 1 Agrobacterium radiobacter (A-16) and 1 Bacillus thuringiensis subsp. kurstaki (BAB-410) bacterial and 2 Trichoderma harzianum (ET 4 and ET 14) fungal isolates tested efficacy in previous studies were used. In pot trial, the experiment was established with the most effective five bacterial strains (BRTB 66.22%; RK-103, 50.90%; BAB-140 50.00%; TV-103B 49.10%; TV-12H 48.65% and TV-6F 48.20%) and two fungal isolates (ET 4; 69.44%, ET 14; 66.66%). BRTB, the most effective bacterial strain, prevented completely the development of the pathogen. Based on the application time of BRTB, infection was not observed on seedlings on growing from tubers inoculated with pathogen 4?h after dipping into the bacterial solution. In storage treatments, BRTB was the most efficacy isolate when compared with others. As a result, BRTB strain of Pantoea agglomerans can be candidate in the biological control of F. oxysporum.

     

Vegetative Compatibility Groups in Colletotrichum coccodes, the Causal Agent of Black Dot on Potato

ABSTRACT Black dot of potato, caused by Colletotrichum coccodes, is a disease of growing economic importance, but the degree of genetic diversity and pathogenic differentiation among isolates is unknown. Using nitrate auxotrophic (Nit) mutants, we characterized vegetative compatibility groups (VCG) diversity for C. coccodes for 110 isolates originating from Israel, The Netherlands, and France. We recovered frequencies of nit1 and NitM mutant classes at 38.5 and 7.2%, respectively, and selected 12 isolates as tester isolates. Using these testers, we defined four multimember VCGs at 7.3, 35.5, 20.0, and 10.0% frequency in this sample. Thirty isolates (27.3% of all tested isolates) could not be assigned to any of the major groups, and showed only self-compatibility. The frequency of recovery of Nit mutant sectors was highest in isolates from VCG4, with 50.9 and 13.6% recovery for nit1 and NitM, respectively. However, we did not detect differences in the frequency of mutant classes among the three countries of origin. In pathogenicity tests, isolates from VCG3 were the most aggressive to potato, as expressed by high stem colonization levels and sclerotia density on root and crown. These results suggest that there is significant VCG diversity in this species and that this VCG diversity may be correlated with pathogenic characteristics or specialization.     

Sensitivity of Mycosphaerella fijiensis, Causal Agent of Black Sigatoka of Banana, to Propiconazole

ABSTRACT One hundred monoascosporic isolates of Mycosphaerella fijiensis were collected in February and November 1994 from each of two banana (Musa spp.) plantations in Costa Rica. Locations at San Pablo and Coopecariari had been sprayed with propiconazole for the past 7 years to control black Sigatoka. One hundred monoascosporic isolates from a third location, San Carlos, with no history of fungicide use, also were tested for sensitivity to propiconazole. Fifty percent effective concentration (EC(50)) values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. In the February sample, the mean EC(50) values for San Pablo and Coopecariari populations were 0.06 and 0.05 mug a.i. ml(-1), respectively, which were not statistically different (P = 0.05). The mean EC(50) value of the population at San Carlos was 0.008 mug a.i. ml(-1), which was significantly lower (P = 0.001) than the mean EC(50) values obtained at San Pablo and Coopecariari. Frequency distributions of EC(50) values of isolates from the three populations collected in February showed that 80% of isolates from San Pablo and Coopecariari had EC(50) values greater than the highest EC(50) value from San Carlos, indicating a significant shift in reduced sensitivity to propiconazole. Isolates collected in November 1994, after eight treatments of propiconazole at San Pablo and Coopecariari, showed a significant increase in mean EC(50) values compared with the means observed in February. The high proportion of isolates with reduced sensitivity to propiconazole may account for the unsatisfactory control of black Sigatoka between 1992 and 1993 in the two banana plantations at San Pablo and Coopecariari.     

Characterization, In Vitro Culture, and Molecular Analysis of Thecaphora solani, the Causal Agent of Potato Smut

ABSTRACT The fungus Thecaphora solani (syn.: Angiosorus solani), the causal agent of potato smut, was cultivated in vitro for the first time. Teliospores obtained from galls of infected potato plants were used to inoculate commonly used solid and liquid media. The teliospores produced two kinds of vegetative tissue depending on the nutrient status of the media. A very slow radial-growing, hyaline, and septate mycelium, as usually seen in most of the in vitro-cultivated filamentous fungi, was obtained in wateragar medium after 30 to 40 days. On the other hand, a white, sponge-like mycelial mass was obtained in HCM + 1% activated charcoal, and on common potato dextrose agar or malt-yeast-peptone solid or liquid media, after 40 to 50 days under lab conditions. The identity among teliospores and the sponge-like mycelial mass was corroborated by DNA fingerprinting and partial sequencing of the large subunit (LSU) rDNA region. The sexual cycle of the pathogen was completed under lab conditions based on the development of teliospores on the sponge-like mycelial mass. The first attempt to reproduce the disease under controlled conditions was successful, inducing a gall in a cv. Desirée potato explant cultivated in vitro inoculated with radial-growing mycelia. Phylogenetic analysis of LSU rDNA data of the genus Thecaphora and other smut fungi confirmed the initial classification of the pathogen as T. solani.     

Genetic Diversity of Xanthomonas campestris pv. vitians, the Causal Agent of Bacterial Leafspot of Lettuce

ABSTRACT Bacterial leafspot of lettuce (BLS), caused by Xanthomonas campes-tris pv. vitians, has become more prevalent in many lettuce-growing areas of the world over the past decade. To gain insight into the nature of these outbreaks, the genetic variation in X. campestris pv. vitians strains from different geographical locations was examined. All strains were first tested for pathogenicity on lettuce plants, and then genetic diversity was assessed using (i) gas-chromatographic analysis of bacterial fatty acids, (ii) polymerase chain reaction analysis of repetitive DNA sequences (rep-PCR), (iii) DNA sequence analysis of the internal transcribed spacer region 1 (ITS1) of the ribosomal RNA, (iv) restriction fragment length polymorphism (RFLP) analysis of total genomic DNA with a repetitive DNA probe, and (v) detection and partial characterization of plasmid DNA. Fatty acid analysis identified all pathogenic strains as X. campestris, but did not consistently identify all the strains as X. campestris pv. vitians. The rep-PCR fingerprints and ITS1 sequences of all pathogenic X. campestris pv. vitians strains examined were identical, and distinct from those of the other X. campestris pathovars. Thus, these characteristics did not reveal genetic diversity among X. campestris pv. vitians strains, but did allow for differentiation of X. campestris pathovars. Genetic diversity among X. campestris pv. vitians strains was revealed by RFLP analysis with a repetitive DNA probe and by characterization of plasmid DNA. This diversity was greatest among strains from different geographical regions, although diversity among strains from the same location also was detected. The results of this study suggest that these X. campestris pv. vitians strains are not clonal, but comprise a relatively homogeneous group.     

Vegetative Compatibility Groups in Cercospora kikuchii, the Causal Agent of Cercospora Leaf Blight and Purple Seed Stain in Soybean

ABSTRACT Nitrogen nonutilizing (Nit) mutants were used to assess vegetative compatibility of 58 isolates of Cercospora kikuchii, 55 of which were isolated from soybean plants in Louisiana. Two isolates were vegetatively self-incompatible. Of 56 self-compatible isolates, 16 were assigned to six multimember vegetative compatibility groups (VCGs), 01 to 06, with 2 or 3 isolates in each VCG. The other 40 isolates each belonged to a distinct VCG. All six multimember VCGs contained isolates from different soy bean cultivars, and three included isolates from different locations. Only one of six multimember VCGs included isolates both from soybean leaves and seed, while the other five included isolates from only leaves or seed. The likelihood of tissue specificity or preference was discussed. All isolates and tested Nit mutants produced cercosporin on potato dextrose agar under light. Significantly different amounts of cercosporin were produced among wild-type isolates, and two Nit mutants produced significantly more cercosporin than their wild-type counterparts. All isolates produced typical Cercospora leaf blight symptoms on soybean plants in greenhouse pathogenicity tests.     

Influence of Environmental Conditions on Spore Production and Budding in Taphrina deformans, the Causal Agent of Peach Leaf Curl

ABSTRACT Environment-controlled studies were carried out to determine the growth of Taphrina deformans under different conditions of temperature, humidity, and nutrient availability similar to those found on plant surfaces during the peach-growing season. Both ascospores and blastospores were able to bud at all temperatures tested (5 to 37 degrees C), with the optimum at 14 and 21 degrees C, respectively. Temperature <20 degrees C favored ascospore production and release, with the optimum at 10 degrees C. Budding was approximately two-and-a-half times higher in a film of water than on a dry substrate, with 100% relative humidity and blastospores also maintained a certain budding ability at lower humidity levels (minimum tested = 47%). Both spore types did not bud after approximately 50 to 55 h in the absence of external nutrients. In the presence of a periodically renewed carbon source, such as simple sugars, at concentrations that typically are present on peach plant surfaces, the fungus maintained its budding capability over time. Results showed that T. deformans is able to bud profusely under a wide range of environmental conditions that occur on peach tree surfaces. This work supports the hypothesis that T. deformans is a part of the normal epiphytic mycoflora of peach trees throughout the entire growing season.     

Detection of Grapevine A vitivirus in Tunisian grapevines

Grapevine A vitivirus (GVA), Grapevine B vitivirus (GVB), Grapevine leaf-roll associated closterovirus 3 (GLRaV3) and Grapevine fanleaf nepovirus (GFLV) were detected by DAS-ELISA in samples of grapevine showing symptoms of rugose wood from different viticultural areas of Tunisia. After optimization of experimental conditions, PCR and IC-RT–PCR were found to be more efficient and sensitive than DAS-ELISA for the detection of GVA.