High-throughput identification technologies provide efficient tools for understanding the ecology and functioning of microorganisms. Yet, these methods have been only rarely used for monitoring and testing ecological hypotheses in plant pathogens and pests in spite of their immense importance in agriculture, forestry and plant community dynamics. The main objectives of this manuscript are the following: (a) to provide a comprehensive overview about the state-of-the-art high-throughput quantification and molecular identification methods used to address population dynamics, community ecology and host associations of microorganisms, with a specific focus on antagonists such as pathogens, viruses and pests; (b) to compile available information and provide recommendations about specific protocols and workable primers for bacteria, fungi, oomycetes and insect pests; and (c) to provide examples of novel methods used in other microbiological disciplines that are of great potential use for testing specific biological hypotheses related to pathology. Finally, we evaluate the overall perspectives of the state-of-the-art and still evolving methods for diagnostics and population-and community-level ecological research of pathogens and pests. K E Y W O R D S community ecology, crop disease, forest pathology, high-throughput sequencing, metabarcoding, metagenomics, molecular diagnostics, plant pathogens, sentinel plantations play a key role in maintaining diversity and primary productivity in ---

Standardized tools are needed to identify and prioritize the most harmful non-native species (NNS). A plethora of assessment protocols have been developed to evaluate the current and potential impacts of non-native species, but consistency among them has received limited attention. To estimate the consistency across impact assessment protocols, 89 specialists in biological invasions used 11 protocols to screen 57 NNS (2614 assessments). We tested if the consistency in the impact scoring across assessors, quantified as the coefficient of variation (CV), was dependent on the characteristics of the protocol, the taxonomic group and the expertise of the assessor. Mean CV across assessors was 40%, with a maximum of 223%. CV was lower for protocols with a low number of score levels, which demanded high levels of expertise, and when the assessors had greater expertise on the assessed species. The similarity among protocols with respect to the final scores was higher when the protocols considered the same impact types. We conclude that all protocols led to considerable inconsistency among assessors. In order to improve consistency, we highlight the importance of selecting assessors with high expertise, providing clear guidelines and adequate training but also deriving final decisions collaboratively by consensus.

Forest decline is nowadays a major challenge for ecosystem sustainability. Dehesas, which consists of savannah-like mediterranean ecosystems, are threatened by the holm oak decline in the south-west of Iberian peninsula. Phytophthora cinnamomi is considered the main agent of holm oak root rot, but little is known about the relationship between diversity of soilborne microbial community and the decline syndrome of holm oak. It would be hypothesized that the changes in the structure and functionality of the soil microbiome might influence tree health status through changes in richness and diversity of beneficial organisms such as mycorrhizal species, or fungal plant pathogens such as Fusarium spp. or Alternaria spp. total DNA of soil samples from declined oak dehesas was extracted and analyzed through metabarcoding techniques, to evaluate the specific composition and diversity of the fungal and oomycete communities and their relationship with the disease symptoms. the fungal community included a wide range of pathogens and abundance of ectomycorrhizal key taxa related with low defoliation degree. Phytophthora cinnamomi and Pythium spiculum did not appear among the most abundant oomycetes, nor were they related directly to defoliation levels. Moreover, a particular taxon belonging to the genus Trichoderma was strongly correlated with the scarcity of pathogenic Phytophthora spp. the diversity and composition of fungal and oomycete communities were related to the severity of the decline symptoms. the metabarcoding study of microbiome represents a powerful tool to develop biocontrol strategies for the management of the holm oak root rot.

Introduction of and invasion by alien plant pathogens represents the main cause of emerging infectious diseases affecting domesticated and wild plant species worldwide. The trade in living plants is the most common pathway of introduction. Many of the alien tree pathogens recently introduced into Europe were not previously included on any quarantine lists. To help determine the potential risk of pest introduction through trading of ornamental plants, a sentinel nursery was established in Beijing, China in 2008. The sentinel nursery planting included four of the most common ornamental woody species shipped to Europe including Ilex cornuta var. fortunae, Zelkova schneideriana, Fraxinus chinensis and Buxus microphylla. Symptoms developing on these species within the sentinel nursery were detected in 2013 and consisted of necrotic spots on leaves, canker and stem necrosis, shoot blight and shoot necrosis. Fungi associated with the trees and their symptoms included Alternaria alternata detected from all hosts; Diaporthe liquidambaris and Diaporthe capsici from bark and leaf necrosis of Zelkova schneideriana; Botryosphaeria dothidea and Nothophoma quercina from stem cankers on Fraxinus chinensis and leaf necrosis on Ilex cornuta; and Pseudonectria foliicola from leaf necrosis on Buxus microphylla. Next generation sequencing analysis from asymptomatic tissues detected eighteen OTU’s at species level among which some taxa had not been previously recorded in Europe. These results clearly demonstrate that looking at trees of internationally traded species in the region of origin can reveal the presence of potentially harmful organisms of major forestry, landscape or crop trees. Results of this study also provide an indication as to how some disease agents can be introduced using pathways other than the co-generic hosts. Hence, sentinel nurseries represent one potential mechanism to address the current lack of knowledge about pests in the countries from where live plants are shipped and the threats they represent to native flora and crops in importing countries.