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High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health.

Simple Summary: The major insect pest of European plantation forests is the large pine weevil. Traditionally, this pest has been controlled by applications of synthetic pesticides. However, because of human health and environmental concerns, there is a need to develop alternative sustainable management strategies. One such strategy is the use of biological control agents. We trialled the efficacy of wild-collected insect-killing fungi and commercial round worms (nematodes) alone and in combination against immature stages of the large pine weevil, which live under the bark of freshly cut stumps. All treatments, as assessed by monitoring emergence with specialised traps, significantly reduced the weevil populations compared to the untreated controls, and the destructive sampling of stumps revealed strong associations between treatment type and infection outcomes. We also monitored non-target insects emerging from treated and untreated stumps. There was no effect on abundance, taxon richness or community composition of the control agents, indicating that they encountered negligible negative environmental consequences. The large pine weevil (Hylobius abietis L.) is a major pest in European and Asian coniferous forests, particularly in managed plantations where clear-felling practices create ideal conditions for its population growth. Traditional management practices involving synthetic insecticides have limited efficacy in terms of reducing pest populations and pose environmental risks. This study evaluated the effectiveness of a wild entomopathogenic fungus (EPF) and the commercial entomopathogenic nematode Steinernema carpocapsae (EPN) as biological control agents (BCAs) against H. abietis in clear-felled spruce plantations in Wales and Scotland. Field trials used a randomised block design with three treatments (EPN full dose, EPF full dose and a combination of EPF+EPN at half doses each) compared to a control. Emergence trapping and destructive sampling were employed to assess treatment efficacy. All treatments significantly reduced weevil emergence, with the mixed treatment showing the greatest impact. Destructive sampling revealed strong associations between treatment type and infection outcomes in H. abietis, with a small but significant relationship between weevil developmental stages and infection types. Importantly, the treatments had no significant impact on the total abundance or taxon richness of non-target invertebrates. These findings suggest that wild EPFs alone and combined with EPNs are effective and environmentally safe alternatives to synthetic insecticides for managing H. abietis populations in managed forests. [ABSTRACT FROM AUTHOR]

Phasmarhabditis hermaphrodita is a parasitic nematode of terrestrial gastropods that has been formulated into a successful biological control agent used on farms and gardens across Europe. There are reports of members of the Phasmarhabditis genus affecting the survival of non-target organisms under lab conditions e.g. earthworms. Therefore, we exposed two species of earthworm (Lumbricus terrestris and Eisenia hortensis) and insect (Galleria mellonella and Tenebrio molitor) to a range of doses of P. californica (0, 30 and 150 nematodes per cm2). P. californica did not affect earthworm or insect survival emphasising these nematodes are gastropod parasites that pose no risk to earthworms or insects (based on laboratory trials). [ABSTRACT FROM AUTHOR]

Many vector mosquito species have evolved resistance to chemical insecticides and the search for novel biological control strategies warrants further attention. Entomopathogenic nematodes (EPNs) (Heterorhabditis and Steinernema spp.) have been developed as biological control agents for use against agricultural pests but whether they could be used to control aquatic mosquito larvae warrants further research. We exposed Aedes aegypti and Ochlerotatus detritus larvae to commercially available (Steinernema feltiae, S. carpocapsae, S. kraussei and Heterorhabditis bacteriophora) and wild isolated (S. affine and S. glaseri) EPNs and monitored survival over 7 days. We also exposed EPNs to water with a range of salinities and pHs found in the marshland habitats of British mosquitoes. Ae. aegypti and O. detritus were killed by commercial EPNs, but wild strains were unable to kill Ae. aegypti yet did kill O. detritus. All EPNs were capable of tolerating a wide range of pHs but showed variable tolerance to different salinities. EPNs could be used as an alternative to chemical insecticides but target species and habitat may influence choice of EPN strain in control operations. [ABSTRACT FROM AUTHOR]

Cockroaches are major pests, vectors of pathogenic bacteria and induce allergies. Current control methods use chemical pesticides, but they can be ineffective and costly and there are reports of resistance in the field; hence new control methods are needed. There are conflicting reports about the susceptibility of cockroaches to entomopathogenic nematodes (EPNs); so we investigated if EPNs could kill several diverse cockroach species, including the Madagascan hissing roach (Gromphadorhina portentosa), the Lobster roach (Nauphoeta cinerea) andBlaptica dubia. Female adult cockroaches were exposed to either commercial products containingSteinernema krausseior a combination ofHeterorhabditisspp. andSteinernemaspp. at 50 and 150 nematodes per cm2for 21 days. We also monitored feeding and the numbers of infective juveniles that were produced from each cockroach corpse. We found thatS. krausseiwere harmless to all cockroach species (at both doses) but when exposed to a mixture ofHeterorhabditisspp. andSteinernemaspp.B. dubiadied after 6 days and its feeding was strongly inhibited. We also found that the mixture ofHeterorhabditisspp. andSteinernemaspp. could proliferate in the cadavers ofB. dubiawhilstS. krausseicould only reproduce inG. portentosaandB. dubiabut notN. cinerea. In conclusion,S. krausseiwas harmless to all three cockroach species butB. dubiawas killed when exposed toHeterorhabditisspp. andSteinernemaspp., highlighting the differences in the host range of EPNs. [ABSTRACT FROM PUBLISHER]