The identification of floral visitation by pollinators provides an opportunity to improve our understanding of the fine-scale ecological interactions between plants and pollinators, contributing to biodiversity conservation and promoting ecosystem health. In this review we outline the various methods which can be used to identify floral visitation, providing a comparison between molecular and non-molecular methods. We review the literature covering the ways in which DNA metabarcoding has been used to answer ecological questions relating to plant use by pollinators and discuss the findings of this research. We present detailed methodological considerations for each step of the metabarcoding workflow, from sampling through to amplification and finally bioinformatic analysis. Detailed guidance is provided to researchers for utilization of these techniques, emphasizing the importance of standardization of methods and improving the reliability of results. Future opportunities and directions of using molecular methods to analyze plant-pollinator interactions are then discussed.

The modifications in planting structure and continuous land use adjustments have significant implications for the structure, diversity, and functioning of arthropod communities in farmland landscapes, particularly regarding the biological control of pest insects by natural enemy species. Therefore, there is an urgent need to comprehensively and accurately assess insect community diversity in croplands. In this study conducted between 2022-2023, we utilized Malaise traps and sweep netting to collect insects from three different double-cropping paddy fields. We extracted DNA from preservative ethanol of Malaise traps and tissue samples of selected parasitoid wasps, followed by amplification using DNA barcoding primers to prepare high-throughput sequencing libraries. As a result, we annotated a total of 4956 operational taxonomic units (OTUs) belonging to 174 genera and 32 families of parasitoid wasps. Our findings demonstrate that the ethanol filter method efficiently captures a wide range of information diversity. However, it demonstrates a lower resolution and may result in a reduction of species abundance. Furthermore, we have identified additional insect species in the parasitoid samples. This demonstrates that high throughput sequencing from adult parasitoid wasps can effectively detect host specie, enabling a comprehensive understanding of host species and providing novel insights into food web construction.

Paddy fields are classified as wetland environments, and they comprise a freshwater ecosystem. They are ecologically important habitats and breeding grounds for many aquatic insects, amphibians, and reptiles. However, the paddy field ecosystem is constantly threatened by climate change and indiscriminate use of pesticides. The meta-barcode analysis of eDNA (environmental DNA) method is highly effective in accumulating information on many organisms that inhabit the paddy field ecosystem because it can explore organisms that are difficult to identify with traditional sampling methods. The eDNA method is very effective for accumulating information on many organisms in the rice field ecosystem. It can indirectly identify the existence of taxa no longer found in the target ecosystem due to behavioral characteristics like amphibia. In the metabarcoding results of this study, genes of more than five species of frogs were found, but it was impossible to confirm all frog taxa, frog's morphological pictures, and croak sounds. On the other hand, some frog taxa had been found only in the metabarcoding analysis. The eDNA of frogs found only in metabarcoding is estimated to have been introduced from nearby areas inhabited by frogs rather than the target region. Due to the powerful analytical resolution of eDNA metabarcoding, this eDNA-based paddy field search is expected to help investigate the biodiversity in agricultural ecosystems.

In this study we sought to investigate the impact of urbanization, presence of concrete river bottom, and nutrient pollution on microbial communities along the L.A. River. Six molecular markers were evaluated for identification of bacteria, plants, fungi, fish, and invertebrates in 90 samples. PCA (principal components analysis) was used with PAM (partitioning around medoids) clustering to reveal community structure and an NB (Negative binomial) model in DESeq2 was used for differential abundance analysis. PCA and factor analysis exposed the main axes of variation but were sensitive to outliers. Differential abundance of Proteobacteria was associated with soft bottom sites, and there was an apparent balance in the abundance of organisms responsible for nitrogen cycling. Nitrogen cycling was explained by differential abundance of ammonia oxidizing archaea, the complete ammonia oxidizers Nitrospira sp., nitrate reducing bacteria Marmoricola sp., and nitrogen fixing bacteria Devosia sp. which were differentially abundant at soft-bottom sites (p adj < 0.002). In contrast, differential abundance of several Cyanobacteria and other anoxygenic phototrophs was associated with the concrete bottom sites, which suggested the accumulation of excess nitrogen. The soft bottom sites tended to be represented by differential abundance of aerobes, whereas the concrete-associated species tended to be alkaliphilic, saliniphilic, calciphilic, sulfate dependent, and anaerobic. In Glendale Narrows, downstream from multiple water reclamation plants, there were differential abundance of cyanobacteria and algae, however indicator species for low nutrient environments and ammonia-abundance were also present. There was differential abundance of ascomycetes associated with Arroyo Seco and a differential abundance of Scenedesmaceae green algae and cyanobacteria in Maywood, in the analysis which compared suburban with urban river communities. The proportion of Ascomycota to Basidiomycota within the LA River differed from the expected proportion based on published worldwide freshwater and river 18S data; the shift in community structure was most likely associated with the extremes of urbanization. This study indicates that extreme urbanization can result in overrepresentation of cyanobacterial species that could cause reductions in water quality and safety.

Aquaponics are efficient systems that associate aquatic organisms’ production and plants by recirculating water and nutrients between aquaculture and hydroponic tanks. In this study, we have characterised the bacterial communities in the fresh water aquaponics system that can mineralise polysaccharides and phytate by producing carbohydrate degrading enzymes and phytases, by 16S rRNA gene sequencing and in vitro culture techniques. Around 20% of the operational taxonomic units (OTUs) identified were previously reported to carry fibre-degrading enzymes putative genes, namely β-glucanase (1%), xylanase (5%) or cellulases (17%). Ten % of the OTUs were previously reported to carry putative genes of phytases with different catalytic mechanisms, namely β-propeller (6%), histidine acid phytases (3%) and protein tyrosine phytase (<1%). Thirty-eight morphologically different bacteria were isolated from biofilms accumulated in fish and plant compartments, and identified to belong to the Bacilli class. Among these, seven could produce xylanase, 8 produced β-glucanase, 14 produced cellulase, and 11 isolates could secrete amylases. In addition, Staphylococcus sp. and Rossellomorea sp. could produce consistent extracellular phytate-degrading activity. The PCR amplification of β-propeller genes both in environmental samples and in the isolates obtained showed that this is the most ecologically relevant phytase type in the aquaponics systems used. In summary, the aquaponics system is abundant with bacteria carrying enzymes responsible for plant-nutrient mineralisation.

East Lake in Wuhan, China, harbors a high number of freshwater fish species of great conservation value, concurrently serving as vital resources for local livelihoods. However, the ecosystem is threatened by an array of anthropogenic activities, thus requiring consistent monitoring of the local fish community to enable more efficacious conservation management. In place of conventional surveying methods, we undertook the first analysis of the fish distribution within East Lake via metabarcoding of environmental DNA (eDNA). The accuracy and efficacy of eDNA metabarcoding relies heavily upon selecting an appropriate primer set for PCR amplification. Given the varying environmental conditions and taxonomic diversity across distinct study systems, it remains a challenge to propose an optimal genetic marker for universal use. Thus, it becomes necessary to select PCR primers suitable for the composition of fish in the East Lake. Here, we evaluated the performance of two primer sets, Mifish-U and Metafish, designed to amplify 12S rRNA barcoding genes in fishes. Our results detected a total of 116 taxonomic units and 51 fish species, with beta diversity analysis indicating significant differences in community structure diversity between the 6 sampling locations encompassing East Lake. While it was difficult to accurately compare the species-level discriminatory power and amplification bias of the two primers, Mifish outperformed Metafish in terms of taxonomic specificity for fish taxa and reproducibility. These findings will assist with primer selection for eDNA-based fish monitoring and biodiversity conservation in the East Lake and other freshwater ecosystems.

Crop output is directly impacted by plant infections (with fungi as the major pathogen), making accurate diagnosis of these threats crucial. Developing technology and multidisciplinary approaches are turning to genomic analyses in addition to traditional culture methods in diagnosing fungal plant diseases. The metagenomic next-generation sequencing (mNGS) method is preferred for genotyping identification of organisms, identification at the species level, illumination of metabolic pathways, and determination of microbiota. Moreover, the data obtained so far show that this new approach shows promise as an emerging new trend in fungal disease detection. Another approach covered by mNGS technologies, known as metabarcoding, enables use of specific markers specific to a genetic region and allows for genotypic identification by facilitating the sequencing of certain regions. Although the core concept of mNGS remains constant across applications, the specific sequencing methods and bioinformatics tools used to analyze the data do differ. In this review, we focus on how mNGS technology, including metabarcoding, is applied in fungal pathogenesis and its promising developments for the future.

Amphibian populations have been globally declining since at least 1990. In temperate forests of eastern North America, vernal pools offer important breeding habitats to many amphibians but are usually not considered for forest management because of their small size and temporary status. Effective monitoring and management of amphibians rely on accurate knowledge of their spatiotemporal distributions, which is expensive to collect due to the amount of fieldwork re-quired. In this study, we tested whether eDNA metabarcoding could identify the same amphibian communities as traditional inventory protocols. We collected eDNA samples in twelve vernal pools in the spring of 2019 and identified their communities by metabarcoding. At each pool, three traditional amphibian inventory methods were used in May and June 2019: call surveys using acoustic recorders, trapping, and active search surveys. In total, 13 amphibian species were detected, most of them being detected by both eDNA and traditional methods. We found that species ecology and behavior are key factors of its detectability by a specific method. As eDNA metabarcoding is comparatively inexpensive and presents a higher repeatability, we conclude that eDNA sampling should be considered for integration as a standard monitoring tool, after an initial assessment of amphibian diversity.

Soil-borne oomycetes include devastating plant pathogens that cause substantial losses in the agricultural sector. To better manage this important group of pathogens, it is critical to understand how they respond to common agricultural practices, such as tillage and crop rotation. Here, a long-term field experiment was established using a split-plot design with tillage as the main plot factor (conventional tillage [CT] vs. no till [NT], 2 levels) and rotation as the subplot factor (monocultures of soybean, corn, or wheat, and corn-soybean-wheat rotation, 4 levels). Post-harvest soil oomycete communities were characterized over three consecutive years (2016-2018) by metabarcoding the Internal Transcribed Spacer 1 (ITS1) region. The community contained 292 Amplicon Sequence Variants (ASVs) and was dominated by Globisporangium spp. (85.1% in abundance, 203 ASV) and Pythium spp. (10.4%, 51 ASV). NT decreased diversity and community compositional structure heterogeneity, while crop rotation only affected the community structure under CT. The interaction effects of tillage and rotation on most oomycetes species accentuated the complexity of managing these pathogens. Soil and crop health represented by soybean seedling vitality was lowest in soils under CT cultivating soybean or corn, while grain yield of the three crops responded differently to tillage and crop rotation regimes.

Systematics plays the most crucial role in biodiversity conservation which is at stake due to anthropogenic activities and environmental degradations. The ever-increasing decline of classical taxonomic expertise drives the need to develop molecular marker-based tools for quick, efficient, and reliable detection of organisms, to assess their ecological impacts for deepening our understanding of systematic and evolutionary relationships between organisms which is central to biology. The pace of alpha taxonomy has quickened by its integration with an increasingly fashionable and novel concept called DNA barcoding which utilizes a short genetic marker or barcode to categorize species for enhanced biodiversity assessment. As a supplementary but not complete alternative of systematics research, DNA barcoding, however, not error-free, brings precision in identification by solving existing problems of classical taxonomy and phylogenetics, irrespective of the growth stage of organisms, particularly for known taxa rather than unknown ones. Mitochondrial gene Cytochrome C Oxidase 1 (COI) serves as a universal animal barcode but there is no such universal barcode for plants and developing a suitable one is more challenging. With the recent advancement of Next Generation Sequencing (NGS), DNA metabarcoding technology is advancing rapidly. Still, ambiguity and error prevail with the correct identification of species due to some problems. After extensive analysis of the existing DNA barcoding papers, this review discusses commonly used DNA barcodes in plants and animals, their roles, advantages, limitations to solve existing problems of conservation biology and add the author’s views and recommendations.

The nature of the relationship between the communities of microorganisms making up the microbiota in and on a host body has been increasingly explored in recent years. Microorganisms, including bacteria, archaea, viruses, parasites, and fungi, have often long co-evolved with their hosts. In human, the structure and diversity of microbiota vary according to the host’s immunity, diet, environment, age, physiological and metabolic status, medical practices (e.g. antibiotic treatment), climate, season, and host genetics. The recent advent of next generation sequencing (NGS) technologies enhanced observational capacities and allowed for a better understanding of the relationship between distinct microorganisms within microbiota. The interaction between the host and their microbiota has become a field of research into microorganisms with therapeutic and preventive interest for public health applications. This review aims at assessing the current knowledge on interactions between prokaryotic and eukaryotic communities. After a brief description of the metagenomic methods used in the studies analysed, we summarise the findings of available publications describing the interaction between the bacterial communities and protozoa, helminths, and fungi, either in vitro, in experimental models, or in humans. Overall, we observed the existence of a beneficial effect in situations where some microorganisms can improve the health status of the host, while the presence of other microorganisms has been associated with pathologies, resulting in an adverse effect on human health.

Plants especially in their natural habitat are considered part of a rich ecosystem that includes many various microorganisms in the soil. The current study aimed to identify the bacterial profile of agriculture-related soil samples using the metabarcoding technique to compare and explore relevant rhizosphere bacteria associated with plant cultivations in newly reclaimed land and habitual cultivated ones. Total environmental DNA was extracted from rhizosphere and non-cultivated samples derived from three land types in Egypt. The bacterial 16S rDNA was amplified and sequenced by NGS technology to profile each sample. The microbial profile was characterized by statistical and literature-based methods. Among all samples, the most identified phyla were Actinobacteriota (28%), followed by Proteobacteriota (26%), Firmicutes (14%), Ac-idobacteriota and Chloroflexi (7%), Gemmatimonadota (5%), Bacteriodota and Crenarchaeota (3%), and Myxococcota (2%), in addition to 37 other phyla with <1% counts. A total of 74 OTU was unique to the plant rhizosphere area and classified as Bacteriodota (5.1%:0.3%), Firmicutes (2.4%:0.1%), and Proteobacteria (3.5%:2%) phyla in agriculture and reclaimed lands, respectively. Moreover, the rhizosphere profile included a large portion of uncultured and unidentified bac-terial species, which opened a window to further analysis. Our analysis provides a key Knowledge about the rhizosphere microbiome and highlights its possible use to create microbial-based bi-ofertilizers targeting plant performance in contrast to traditional fertilizers and their side effect on the agriculture sector.

A decade after environmental scientists integrated high-throughput sequencing technologies in their toolbox, the genomics-based monitoring of anthropogenic impacts on biodiversity and ecosystems is yet to be implemented by regulatory frameworks. Despite the broadly acknowledged potential of environmental DNA and RNA to cost-efficiently and accurately monitor biodiversity, technical limitations and conceptual issues still stand in the way of its routine application by end-users. In addition, the multiplicity of potential implementation strategies may contribute to a perception of the methodology as being premature or “in development”, hence restraining regulators from binding these tools into legal frameworks. This review focuses on the strengths and limitations of genomics-based strategies that have emerged over the past ten years and have been classified for this purpose into three broad strategies: (A) Taxonomy-based approaches that focus on known bio-indicators or the diversity of taxonomically described taxa, (B) De novo approaches that do not require well-established taxonomy, and (C) Function-based approaches that rely on community-wide metrics, where taxa are interchangeable, or on functional profiles instead of compositional turnovers. We finally propose a roadmap for the implementation of environmental genomics into routine monitoring programs that leverage recent analytical advancements, upon which some critical limitations are alleviated.

In the cold deserts of the McMurdo Dry Valleys (MDV) the suitability of soil for microbial life is determined by both contemporary processes and legacy effects. Climatic changes and accompanying glacial activity have caused local extinctions and geochemical changes to soil ecosystems over several million years, while high elevation refugia may have escaped these disturbances and existed under relatively stable conditions. This study describes the impact of historical glacial and lacustrine disturbance events on microbial communities across the MDV. Soil bacterial communities from 17 sites representing either putative refugia or sites disturbed during the Last Glacial Maximum (LGM) (22-17kya) were characterized using 16S metabarcoding. Regardless of geographic distance, several putative refugia sites at elevations above 600 meter displayed highly similar microbial communities. At a regional scale, community composition was found to be influenced by elevation and geographic proximity more so than soil geochemical properties. These results suggest that despite the extreme conditions, diverse microbial communities exist in these putative refugia that have presumably remained undisturbed at least through the last glacial maximum. We suggest that similarities in microbial communities can be interpreted as evidence for historical climate legacies on an ecosystem-wide scale.

Environmental DNA (eDNA) sampling has emerged as a powerful approach for monitoring biodiversity in the context of diverse terrestrial and aquatic ecosystems, and has broad potential for both environmental surveys and species management. eDNA studies focusing on plant and animal species have capitalized on analytical methodologies developed originally for metagenomic studies of microorganisms, and on technological improvements in sample collection. However, while eDNA studies have proliferated rapidly, efforts to standardize sampling and analytical methods are still nascent. Critically, fundamental data concerning the distribution and persistence of eDNA in different ecological contexts are lacking, complicating both confidence in species detection and comparability between studies. We propose that key deficits could be addressed by using well-designed synthetic DNA standards or tracers, which are widely used in fields ranging from medical diagnostics to hydrology. In-field application of a standardized mixture of barcoded synthetic DNA fragments of varying lengths and concentrations that are recovered concommitantly with eDNA sampling should provide critical yet missing data on environmental DNA distribution and turnover, as well as an internal molecular standard to enhance both species detection and comparability between eDNA studies.

A comparative study of organic and conventional farming systems was conducted in almond orchards to determine the effect of management practices on their fungal and bacterial communities. Soils from two orchards under organic (OM) and conventional (CM), and nearby nonmanaged soil (NM) were analyzed and compared. Several biochemical and biological parameters were measured (soil pH, electrical conductivity, total nitrogen, organic material, total phosphorous, total DNA, and fungal and bacterial DNA copies). Massive parallel sequencing of regions from fungal ITS rRNA and bacterial 16S genes was done to characterize their diversity in the soil. We report a larger abundance of bacteria and fungi in soils under OM, with a more balanced fungi:bacteria ratio, compared to bacteria-skewed proportions under CM and NM. The fungal phylum Ascomycota corresponded to around the 75% relative abundance in the soil, whereas for bacteria, the phyla Proteobacteria, Acidobacteriota and Bacteroidota integrated around 50% of their diversity. Alpha diversity was similar across practices, but beta diversity was highly clustered by soil management. Linear discriminant analysis effect size (LEfSE) identified bacterial and fungal taxa associated to each type of soil management. Analyses of fungal functional guilds revealed 3-4 times larger abundance of pathogenic fungi under CM compared to OM and NM treatments. Among them, the genus Cylindrocarpon was more abundant under CM and Fusarium under OM.

The purpose of this paper is to elucidate the roles that microbes may be playing in the rootzone of the medicinal plant Datura inoxia. We hypothesized that rhizospheric and endophytic microbes would be found that were capable of performing the same secondary metabolic functions of the plant rootzone they inhabited. We also hypothesized that the microbial functions would be co-operative with and supportive to plant secondary metabolite production, for example, by providing precursors to important plant bioactive molecules. The methods employed were mi-crobial barcoding, tests of essential oils against antibiotic resistant bacteria and other soil bacterial isolates, 16S Next Generation Sequencing (NGS) metabarcoding, and Whole Genome Shotgun (WGS) taxonomic and functional. A few of the main bacterial genera of interest that were dis-covered in the Datura root microbiome were Flavobacterium, Chitinophaga, Pseudomonas, Strepto-myces, Rhizobium, and Bacillus. In the context of known interactions, and current results, plants and microbes influence the flavonoid biosynthetic pathways of one other, in terms of the regulation of the phenylpropanoid pathway. This is important because these compounds are phyto-protective antioxidants and are precursors to many aromatic bioactive compounds that are relevant to human health. There was strong evidence to support the notion that synergistic production of plant de-rived secondary metabolites by microbes occurred, as well as the ability for the compounds to enter plant cells. There are possible biopharmaceutical and agricultural applications of the natural interplay that was discovered during this study of the Datura inoxia rhizosphere.

(1) Introduction: Karstic springs are used worldwide by rural communities as sources of fresh water for human use and livestock survival. In Romania, 1/3 of the population has no direct access to the public water supply. The present study is part of a country-wide project on developing simple, quick and cheap methods for seasonal environmental and microbiological monitoring of karstic springs used as drinking water supply by rural populations. Critical steps for the monitoring workflow consist in the evaluation of water quality and choosing of suitable membrane filters to efficiently capture environmental DNA for further microbial diversity estimation by 16S rRNA gene-based metabarcoding; (2) Methods: Several commercial membrane filters of different composition and pore sizes were tested on the water sampled from three karstic springs in Romania, followed by water chemistry and whole community 16S rRNA gene-based metabarcoding analysis; (3) Results: We found that the different types of applied membrane filters provide a varying recovery of diversities and abundances of both overall and pathogenic bacteria; and (4) Conclusions: The result of the experiment with different filters shows which are the best for amplicon-based metabarcoding monitoring of karst springs.

Eukaryotic enteric pathogens (EEP) are a public health issue in tropical areas. Yet, their interactions with the gut mycobiota remain poorly understood. We conducted a cross-sectional study in Malian children to analyze the impact of EEP on the gut fungal community. EEP were assessed by qPCR and the gut mycobiota was characterized by ITS1-2 metabarcoding in stool samples collected from 296 children. The 100 controls, in whom no EEP was detected, were compared to: a) 196 children with ≥1 EEP; b) 91 with only Blastocystis; c) 35 with only Giardia intestinalis; and d) 12 with another (&lt;1% each) EPP. The gut fungal community structure was homogenous in each children’s group. Linear size-effect discriminant analysis highlighted five relatively more abundant species, including Fusarium longipes and Penicillium caseifulvum, in children with ≥1 EEP, whereas 28, including Aspergillus sydowii and Microdochium colombiense were more abundant in controls. Fusarium, Pyxidiophora, and Stereum abundance was higher in Blastocystis-infected children, whereas Ogataea and Allocryptovalsa were more abundant in controls. Sordariales and Mortierellales abundance was higher in Giardia intestinalis-infected children, whereas Agaricales and Capnodiales abundance was higher in controls. In conclusion, EEP do not significantly alter the gut fungal community structure, and further studies are warranted to confirm our findings that particular taxa are associated with susceptibility or resistance to specific EEP.

Because data on the fungal gut community structure of African children are scarce, we aimed to describe by reanalysing rRNA ITS1 and ITS2metabarcoding data from a study designed to assess the influence of microbiota in malaria susceptibility in Malian children from the Dogon country. More specifically, we aimed to establish the core gut mycobiome and compare the gut fungal community structure of breastfed children, aged 0-2 years, with other age groups. Briefly, DNA was extracted from 296 children’s stool samples. Both rRNA ITS1 and ITS2 genomic barcodes were amplified and subjected to Illumina MiSeq sequencing. The ITS2 barcode generated 1,975,320 reads and 532 operational taxonomic units (OTUs), while the ITS1 barcode generated 647,816 reads and 532 OTUs. The alpha diversity was significantly higher by using the ITS1 compared to the ITS2 barcode (p

Zooplankton and ichthyoplankton community assessments depend on species diagnostics, yet morphological identifications are time consuming, require taxonomic expertise, and are hampered by lack of diagnostic characters –particularly for larval stages. Metabarcoding can identify multiple species in communities from short DNA sequences in comparison to reference databases. To evaluate species resolution across phylogenetic groups and food webs of zooplankton and ichthyoplankton, we compare five metabarcode mitochondrial (mt)DNA markers from gene regions of (a)cytochrome c oxidase subunit I, (b) cytochrome b, (c) 16S ribosomal RNA, and (d) 12S ribosomal RNA for DNA extracted from net tows in the Northeastern Pacific Ocean’s Salish Sea across seven sites and two seasons. Species resolved by metabarcoding are compared to invertebrate morphological identifications and biomass estimates. Results indicate that species resolution for different zooplankton and ichthyoplankton taxa can markedly vary among gene regions and markers, and in comparison to morphological identifications. Thus, researchers seeking “universal” metabarcoding should take caution that several markers and gene regions likely will be needed; all will miss some taxa and yield incomplete overlap. Species resolution requires careful attention to taxon marker selection and coverage in reference sequence repositories. In summary, combined multi-marker metabarcode and morphological approaches improve broadscale zooplankton diagnostics.

eDNA extracted from the gut contents of filter feeders with unique feeding habits can be used to identify biodiversity in aquatic ecosystems. In this study, we used eDNA from the gut contents of the clam Corbicula fluminea to examine estuarine ecosystem biodiversity. The field survey was conducted at three study sites in the Nakdong River Estuary, which is characterised by closed estuarine features resulting from the presence of an estuarine barrage. The collected C. fluminea samples were dissected to separate the gut contents, and the extracted eDNA was amplified using the 18S V9 primer targeting all eukaryotes. The amplified DNA was sequenced using next generation sequencing (NGS) techniques, and BLASTn was performed based on the NCBI database. We obtained 21 unique operational taxonomic units (OTUs), including fish (approximately 9.52%), copepods (approximately 14.29%), and green algae (approximately 23.81%), which represented a wide range of habitats such as marine, brackish, freshwater, and terrestrial environments. These results suggest that various organisms living in aquatic ecosystems can be identified through eDNA from the gut contents of C. fluminea and support the potential application of bivalves as eDNA samplers in diverse aquatic ecosystems.

Studies of Phytophthora impacts in forests generally focus on individual species without recogni-tion that Phytophthora occur in multispecies communities. We investigated community structure of Phytophthora species in the rhizosphere of Agathis australis (kauri) in Te Wao Nui o Tiriwa / Waitākere Ranges, New Zealand, in the context of kauri dieback disease expression. Soil sampling and tree health monitoring was conducted on 767 randomly selected mature kauri trees. Phy-tophthora species were detected using both soil baiting and DNA metabarcoding of eDNA. Four species were detected with soil baiting (P. agathidicida, P. cinnamomi, P. multivora, and P. pseudo-cryptogea) and an additional three species with metabarcoding (P. kernoviae, P. cactorum/P. aleatoria and an unknown clade 7 species). Phytophthora cinnamomi was the most abundant species and was distributed throughout the forest. Both P. multivora and P. agathidicida were limited to forest edges, suggesting more recent arrivals. P. agathidicida presence was strongly correlated with declining canopy health, confirming its role as the main river of kauri dieback. The limited distribution of P. agathidicida and infrequent detections (11.0% samples) suggests that that this species is spreading as an introduced invasive pathogen and provide hope that with strategic management uninfected areas of the forest can be protected. The frequent detections of P.cinnamomi and P. multivora, from symptomatic trees in the absence of P. agathidicida suggest more research is needed to understand their roles in kauri forest health.

Phytoplasma diseases pose a substantial threat to diverse crops of agricultural importance. Management measures are usually implemented only after the disease has already occurred. Early detection of such phytopathogens, prior to disease outbreak, has rarely been attempted but would be highly beneficial for phytosanitary risk assessment, disease prevention and mitigation. In this study we present the implementation of a recently proposed proactive disease management protocol (DAMA: Document, Assess, Monitor, Act) for a group of vector-borne phytopathogens. We used insect samples collected during a recent biomonitoring program in southern Germany to screen for the presence of phytoplasmas. Insects were collected with Malaise traps in different agricultural settings. DNA was extracted from these mass trap samples and subjected to PCR-based phytoplasma detection and COI sequence metabarcoding. In about 1% of the analyzed samples, phytoplasma DNA was detected. Phytoplasma group identification was performed by 16S rRNA gene sequence analysis and the detected phytoplasmas could be assigned to the 16SrI subgroups B and L. Insect species in the sample were identified by DNA metabarcoding. By using established databases, checklists, and archives, we documented historical associations and records of phytoplasmas and its hosts in the study region. For the assessment in the DAMA protocol, phylogenetic triage was performed in order to determine the risk for tri-trophic interactions (plant-insect-phytoplasma) and associated disease outbreaks in the study region. A phylogenetic heat map constitutes the basis for risk assessment and was used here to identify a minimum number of seven leafhopper species suggested to be monitored by stakeholders in this region. A proactive stance in monitoring changing patterns of association between hosts and pathogens can be a cornerstone in capabilities to prevent future phytoplasma disease outbreaks. To our knowledge, this is the first time that the DAMA protocol has been applied in the field of phytopathology and vector-borne plant diseases.

Apple replant disease is a severe problem in orchards and tree nurseries. Evidence for the involvement of a nematode-microbe disease complex was reported. To search for this complex, plots with a history of apple replanting, and control plots cultivated for the first time with apple were sampled in two fields in two years. Shoot weight drastically decreased with each replanting. Nematodes were extracted from soil samples by floatation-centrifugation, washed on a 20 µm-sieve, and used for DNA extraction. Nematode communities and co-extracted fungi and bacteria were analyzed by high-throughput sequencing of amplified ribosomal fragments. The nematode community and co-extracted fungal and bacterial communities significantly differed between replanted and control plots. Free-living nematodes of the genera Aphelenchus, Cephalenchus, and an unidentified Dorylaimida were associated with replanted plots, as indicated by linear discriminant analysis effect size. Among the co-extracted fungi and bacteria, Mortierella was most indicative of replanting. Some genera, mostly Rhabditis, indicated healthy control plots. Isolating and investigating the putative disease complexes will help to understand and alleviate stress-induced root damage of apple in replanted soil.