Comparison of rapid detection assays for grapevine leafroll disease associated closteroviruses

Three rapid detection assays (ELISA, dsRNA analysis and ISEM) were compared for their sensitivity, specificity, and simplicity in the detection of grapevine leafroll associated closteroviruses (GLRaV). Each was found to have advantages and disadvantages for routine testing. ELISA is sensitive and easy to use, but different antisera are needed to detect different GLRaV types. Because mixing or blending of antisera can produce good results in a single ELISA test, each antiserum does not need to be used separately unless it is important to determine the type of GLRaV present. DsRNA analysis can detect all the types of GLRaV tested but has a relatively low sensitivity and is labor intensive, which makes it unsuitable for testing large numbers of samples. Furthermore, dsRNA does not give unequivocal diagnosis of GLRaV infections. ISEM is sensitive and rapid. However, like ELISA, this technique requires an antiserum to each GLRaV type tested and an electron microscopy. Our recommendation is that ELISA should be used with multiple antisera for large scale testing programs. Samples for which ELISA results are inconclusive should be retested with ISEM and/or dsRNA. When the disease status of an individual sample must be determined conclusively, a few grams of tissue should be processed to concentrate the virus and then subjected to ELISA and examination by electron microscopy with negative staining. A dsRNA analysis should be carried out as well

Xylella fastidiosa in Olive in Apulia: Where We Stand

A dramatic outbreak of Xylella fastidiosa decimating olive was discovered in 2013 in Apulia, Southern Italy. This pathogen is a quarantine bacterium in the European Union (EU) and created unprecedented turmoil for the local economy and posed critical challenges for its management. With the new emerging threat to susceptible crops in the EU, efforts were devoted to gain basic knowledge on the pathogen biology, host, and environmental interactions (e.g., bacterial strain(s) and pathogenicity, hosts, vector(s), and fundamental drivers of its epidemics) in order to find means to control or mitigate the impacts of the infections. Field surveys, greenhouse tests, and laboratory analyses proved that a single bacterial introduction occurred in the area, with a single genotype, belonging to the subspecies pauca, associated with the epidemic. Infections caused by isolates of this genotype turned to be extremely aggressive on the local olive cultivars, causing a new disease termed olive quick decline syndrome. Due to the initial extension of the foci and the rapid spread of the infections, eradication measures (i.e., pathogen elimination from the area) were soon replaced by containment measures including intense border surveys of the contaminated area, removal of infected trees, and mandatory vector control. However, implementation of containment measures encountered serious difficulties, including public reluctance to accept control measures, poor stakeholder cooperation, misinformation from some media outlets, and lack of robust responses by some governmental authorities. This scenario delayed and limited containment efforts and allowed the bacterium to continue its rapid dissemination over more areas in the region, as shown by the continuous expansion of the official borders of the infected area. At the research level, the European Commission and regional authorities are now supporting several programs aimed to find effective methods to mitigate and contain the impact of X. fastidiosa on olives, the predominant host affected in this epidemic. Preliminary evidence of the presence of resistance in some olive cultivars represents a promising approach currently under investigation for long-term management strategies. The present review describes the current status of the epidemic and major research achievements since 2013

Identification of the agent of grapevine fleck disease

An antiserum against an Italian isolate of grapevine phloem-limited isometric virus (GPLIV) was used in an ELISA survey carried out for assessing the natural distribution of the virus and its association with fleck disease. A total of 591 vines of Vitis rupestris were checked for the presence of GPLIV. Of 150 plants with fleck symptoms, 138 (92 %) were ELISA-positive and 12 (8 %) negative. Of 441 symptomless V. rupestris, 435 (98,6 %) were ELISA-negative and 6 (1,4 %) positive for GPLIV. The virus was detected in about 30 % of 694 vines of different origin grown in Apulia (Southern Italy). The highest infection (53 %) was in a commercial vineyard of cv. Italia and the lowest (8 %) in a plot of certified and visually selected rootstocks. Fleck-infected, but not fleck-free V. rupestris contained virus particles and vesiculated inclusion bodies in phloem tissues. LN 33 plantlets derived from in vitro culture of meristem tips from ELISA-positive fleck-infected mother plants were found to be free from GPLIV, as ascertained by ELISA and thin-sectioning. These vines failed to induce fleck symptoms when grafted on V. rupestris. It is concluded that GPLIV is the agent of fleck and, therefore, it should be renamed grapevine fleck virus (GFKV)

Cost-effectiveness of intravitreal therapy with both anti-VEGF and Dexamethasone implant in patients with Diabetic Macular Edema

Purpose: The aim of this study was to evaluate the cost-effectiveness of intravitreal therapy (IVT) with both anti-VEGF and Dexamethasone implant in patients with Diabetic Macular Edema (DME) during two years’ follow-up. Methods: A retrospective review of 191 patients (382 eyes) with type I and II diabetes and DME was performed. Pre-IVT and final best correct visual acuity (BCVA), central macular thickness (CMT), intraocular pressure (IOP), number and type of IVT,number of examinations, and fluorescein angiography were assessed. Based on surgery procedure other than IVT, patients were divided into 5 groups. To avoid bias, we analysed only patients who had undergone cataract surgery before (group 1) or during enrolment (group 2). Results: 41 eyes from Group 1 and 48 eyes from group 2 were evaluated. Median BCVA ranged between 20/80 and 20/63 Snellen (P = 0.008) in Group 1 and from 20/63 to 20/40 Snellen (P = 0.0035) in Group 2, while improvement up to 1 Snellen line was observed in 58.5 and 68.75% of eyes in Group 1 and 2, respectively.In terms of median CMT, a statistically reduction (P = 0.0007) was found in Group 2 (-85 μm), whereas no statistical differences were found in Group 1. The two groups showed no statistically significant difference in median IOP. The estimated cost per eye was €7803 in Group 1 and €8988 Group 2, whereas the mean cost per patient was €15190 and €16580 in Group 1 and 2, respectively. Analysis between groups did not show any statistical difference in the considered parameters. Conclusions: In this study, despite the high treatment cost, vision improvement in DME patients undergoing IVT was disappointing. Our results emphasise the need for a better understanding of the cost-effectiveness of DME treatmen

Nomenclature of grapevine leafroll-associated putative closteroviruses

Comparative immunoenzymatic (ELISA), immunoelectron microscopic (IEM) and immunoblotting tests were carried out with antisera produced in different laboratories and commercial diagnostic kits on closterolike viruses reported in the literature under the name of grapevine corky bark-associated virus (GCBaV) and grapevine leafroll-associated viruses IIa and IIb (GLRaV IIa and GLRaV IIb). The results of these studies have established that GCBaV is the same as GLRaV IIb and that both viruses are apparently identical to an isolate of GLRaV-2 identified in France, whose designation as the authentic GLRaV 2 is proposed. GLRaV IIa is serologically distinct from all known clostero-like viruses of the grapevine and, therefore, the provisional name of grapevine leafroll-associated Virus 6 (GLRaV-6) is suggested for it

Pseudococcus affinis MASK., new vector of grapevine trichoviruses A and B

Research Note Grapevine trichovirus A (GVA) and grapevine trichovirus B (GVB) were successfully transferred with bulk transmission trials under controlled conditions, from infected grapevines to herbaceous hosts by Pseudococcus affinis MASK., a pseudococcid mealybug that may attack grapevines. P. affinis is the fourth mealybug species capable of vectoring GVA and GVB, confirming that transmission by mealybugs of grapevine trichoviruses may not be species-specific

Some properties of a hitherto undescribed filamentous virus of the grapevine

An apparently new, non mechanically-transmissible clostero-like virus, for which the name ''grapevine leafroll-associated virus 7('') (GLRaV-7) is proposed, was found in Albanian grapevine accessions. Virus particles were filamentous, had conspicuous cross banding and a length of 1500-1700 nm. Virions had coat protein subunits with an estimated M(r) of ca. 37 kDa and a ssRNA genome with size of ca. 19.5 kb as deduced from the estimate of dsRNA (ca. 19.5 kbp) extracted from grapevine tissues. A virus-specific antiserum was raised, which decorated virions at a dilution of 1:1000. This antiserum did not recognize particles of any of the six grapevine leafroll-associated clostero-like viruses (GLRaV-1 to -6) known to date, nor of grapevine trichovirus A (GVA) and B (GVB). Grapevine indicators graft-inoculated with material from accessions containing GLRaV-7 reacted with mild leafroll-like symptoms. In a survey in which 2226 vines from 30 different countries were examined by ELISA, GLRaV-7 was found in 141 plants from Albania, Greece, Hungary, Egypt, and Italy

On the possible relationship between Kober stem grooving and grapevine virus A

Investigations were carried out to establish possible correlations of two diseases of the rugose wood complex, i.e. Rupestris stem pitting (RSP) and Kober stem grooving (KSG) with grapevine virus A (GVA) and grapevine leafroll associated viruses I (GLRaV I) and III (GLRaV III). To this purpose 84 clonal accessions of different wine grape cultivars were analyzed by ELISA and by indexing onto the indicators Vitis rupestris, Kober 5BB and LN 33. The results obtained clearly indicated that none of the viruses taken into consideration is apparently involved in the etiology of RSP. Conversely, a remarkably close association of GVA with KSG was discovered

Properties of a new isolate of grapevine leafroll-associated virus 2

A new isolate of grapevine leafroll-associated virus 2 (GLRaV-2-H4) was recovered by mechanically inoculating herbaceous hosts with concentrated tissue extracts from a North American accession of Vitis rupestris. Contrary to the Semillon isolate of GLRaV-2, isolate H4 elicited necrotic local lesions in Nicotiana clevelandii and infected systemically N. occidentalis inducing very severe symptoms. The migration rate of dissociated capsid protein of GLRaV-2-H4 in SDS-PAGE differed slightly from that of GLRaV-2-Sem. The coat protein sequence of GLRaV-2-H4 differed by about 12 % at the nucleotide level from capsid proteins of the other two GLRaV-2 isolates that have been sequenced to date. No serological differences could be detected. Isolate H4 is a biological variant of GLRaV-2, which can be distinguished from other mechanically transmitted isolates of the same virus because of differences in type and reactions of the herbaceous host range and in molecular traits of the coat protein cistron

A non-mechanically transmissible isometric virus associated with asteroid mosaic of the grapevine

Research Not