First report of the parasitoid wasp Trissolcus basalis (Wollaston, 1858) in Austria (Hymenoptera: Scelionidae)

Im Sommer 2021 wurden parasitierte Eigelege der Grünen Reiswanze Nezara viridula (Linnaeus, 1758) in Wiener Privatgärten festgestellt. Die morphologischen und molekularbiologischen Untersuchungen ergaben, dass es sich um den Eiparasitoiden Trissolcus basalis (Wollaston, 1858) handelt. Dies ist der erste Nachweis von Trissolcus basalis aus Nezara viridula Eigelegen in Österreich.Trissolcus basalis (Wollaston, 1858) was recorded for the first time in Austria. The scelionid wasp parasitized egg masses of the southern green stink bug Nezara viridula (Linnaeus, 1758), which were collected in private gardens in Vienna in summer 2021

Molekularbiologische Diagnostik von Pflanzenkrankkeiten

In derAbteilung für Molekularbiologische Diagnose von Pflanzenkrankheiten der AGES werden molekularbiologische Diagnoseverfahren eingesetzt um gesetzliche Kontroll- und Vollzugsaufgaben zu erfüllen, aber auch um Untersuchungen auf pflanzliche Schaderreger für private Kunden durchzuführen. Verwendet werden unterschiedliche PCR Techniken um Phytoplasmen, Bakterien, Pilze, Viren, Viroide, Insekten und Nematoden zu diagnostizieren. Die Vorteile der PCR Techniken werden dargestellt. Stichwörter: PCR, RT-PCR, qPCR, RT-qPCR, DNA-BarcodingMolecular diagnosis of plant diseasesThe Department for Molecular Diagnostics of Plant Diseases of the Austrian Agency for Health and Food Safety (AGES) uses molecular diagnostic tools to support control and execution tasks regulated by law, and to test private plant samples for plant pathogens. Different PCR techniques are used to investigate for phytoplasma, bacteria, fungi, virus, viroids, insects and nematods. The advantages of molecular tools are summarized. Keywords: PCR, RT-PCR, qPCR, RT-qPCR, DNA-barcodin

Tracking the dissemination of Erwinia amylovora in the Eurasian continent using a PCR targeted on the duplication of a single CRISPR spacer

Fire blight is the most devastating disease affecting pome fruit production globally. The pathogen is native to North America and was imported to western Europe in the 1950s, progressively spreading over the continent in the ensuing decades. Previous phylogenetic studies have revealed the extreme genetic homogeneity of the pathogen outside its center of origin, which makes epidemiological studies difficult. These are generally only possible using hypervariable regions of the genome such as those represented by CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), which are, however, not practical to sequence due to their size and variability. Here, we present a simple PCR assay targeting the duplication of a single CRISPR spacer in Erwinia amylovora that was found to be an important marker to discriminate between two main European populations of the pathogen. We implemented the assay on a total of 582 isolates to follow the spread of fire blight across the continent over several decades and, wherever possible, within single countries. The results obtained point to the occurrence of two major separate introduction events for E. amylovora in Europe that occurred approximately 20 years apart, and confirmed the existence of two principal distribution areas located in Northeastern Europe and the Eastern Mediterranean Basin from which the pathogen moved on to colonize the Eurasian continent

Comparison of the performance of the main real-time and conventional PCR detection tests for ‘Candidatus Liberibacter’ spp., plant pathogenic bacteria causing the Huanglongbing disease in Citrus spp

23 Pág.Huanglongbing disease affects the Rutaceae family and is associated with three phloem-limited bacterium species: Candidatus Liberibacter asiaticus, africanus and americanus. These species are considered quarantine pathogens in the world, and pose major risks for citrus production and industry. Due to the low titer and the uneven distribution of the bacteria within its host plant, conventional PCR detection protocols can lead to false negative results, especially for early detection. Herein, three real-time PCR diagnostic methods recommended by the EPPO and FAO for asiaticus and africanus species detection were evaluated for their performance and compared with a conventional duplex PCR. Assessments were done as part of an international cooperative project under the EUPHRESCO guidance. Intra-laboratory assessment of the analytical specificity and analytical sensitivity was performed on 33 target or non-target DNA samples and seven target DNA samples were used to determine the sensitivity. Thereafter, repeatability, reproducibility, and concordance odds ratio were assessed on 20 target or non-target DNA samples through a collaborative test performance study organized among eight international laboratories. Results showed that the Li protocol proved to be the best method for asiaticus and africanus species detection, along with the conventional duplex PCR; whereas the Morgan protocol showed high performance only for asiaticus species. Interlaboratory reproducibility was high, suggesting that these real-time PCR methods can be readily transferred to diagnostic laboratories.This research was funded by Anses - Plant Health Laboratory (LSV).Peer reviewe

Morphological and Molecular Identification Protocols for Bactrocera dorsalis: A Joint Validation Study

Bactrocera dorsalis is considered among the most destructive and economically important invasive fruit flies worldwide. Native to Southeast Asia, in just a few years, it has spread almost throughout all of sub-Saharan Africa, causing fruit loss in several commercially grown crops. In Europe, it is frequently intercepted as larva on imported fruits during phytosanitary inspections. In recent years, adult specimens have been caught in traps in Austria, Italy, and France, increasing the level of attention to this species. Rapid and unambiguous identification by European plant health laboratories is important to effectively prevent its introduction and establishment in Europe. The harmonized use of validated diagnostic protocols is essential. However, in entomology, unlike other disciplines, diagnostic protocols often lack validation data to support their suitability as adequate identification tools. In this study, tests from two international diagnostic protocols for the identification of B. dorsalis underwent a thorough validation process to assess their performance characteristics (analytical and diagnostic sensitivity, specificity, accuracy, repeatability, and reproducibility). A novel process of a joint validation of morphological and molecular identification protocols is reported. The novelty of this approach arises from the fact that morphological and molecular tests are validated in the same study, based on the same panel of samples, also allowing a two-way control on the assigned values of samples. Potential critical issues that could represent weaknesses of the protocols are also discussed in detail. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Performance of diagnostic tests for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) from woody samples

The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an interlaboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and realtime PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave false-positive results. The use of the most reliable detection test is suggested for routine analyses, but when Psa-free status needs to be accurately assessed, it is recommended that at least two detection tests are used. This work provides a wide comparison of the available diagnostic methods, giving new information for a possible revision of the official diagnostic protocols (e.g. European and Mediterranean Plant Protection Organization (EPPO) protocol PM7/120 for the detection of Psa)

Diet of Amazon river turtles (Podocnemididae): a review of the effects of body size, phylogeny, season and habitat

Amazon rivers can be divided into three groups (black, white and clear waters) according to the origin of their sediment, dissolved nutrient content, and vegetation. White water rivers have high sediment loads and primary productivity, with abundant aquatic and terrestrial plant life. In contrast, black water rivers are acid and nutrient-poor, with infertile floodplains that support plant species exceptionally rich in secondary chemical defences against herbivory. In this study, we reviewed available information on the diet of Amazon sideneck river turtles (Family Podocnemididae). Our aim was to test the relationship between water type and diet of podocnemidids. We also took into account the effects of season, size, age, sex and phylogeny. Based on our review, turtles of this family are primarily herbivorous but opportunistic, consuming from 46 to 99% (percent volume) of vegetable matter depending on species, sex, season and location. There was no significant correlation between the maximum carapace size of a species and vegetable matter consumed. When the available information on diet, size and habitat was arranged on the podocnemidid phylogeny, no obvious evolutionary trend was evident. The physicochemical properties of the inhabited water type indirectly influence the average volume of total vegetable matter consumed. Species with no specialised stomach adaptations for herbivory consumed smaller amounts of hard to digest vegetable matter (i.e. leaves, shoots and stems). We propose that turtles with specialized digestive tracts may have an advantage in black water rivers where plant chemical defences are more common. Despite limitations of the published data our review highlights the overall pattern of diet in the Podocnemididae and flags areas where more studies are needed. © 2016 Elsevier Gmb