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Abstract Background The gut virome is an integral component of the gut microbiome, playing a crucial role in maintaining gut health. However, accurately depicting the entire gut virome is challenging due to the inherent diversity of genome types (dsDNA, ssDNA, dsRNA, and ssRNA) and topologies (linear, circular, or fragments), with subsequently biases associated with current sequencing library preparation methods. To overcome these problems and improve reproducibility and comparability across studies, universal or standardized virome sequencing library construction methods are highly needed in the gut virome study. Results We repurposed the ligation-based single-stranded library (SSLR) preparation method for virome studies. We demonstrate that the SSLR method exhibits exceptional efficiency in quantifying viral DNA genomes (both dsDNA and ssDNA) and outperforms existing double-stranded (Nextera) and single-stranded (xGen, MDA + Nextera) library preparation approaches in terms of minimal amplification bias, evenness of coverage, and integrity of assembling viral genomes. The SSLR method can be utilized for the simultaneous library preparation of both DNA and RNA viral genomes. Furthermore, the SSLR method showed its ability to capture highly modified phage genomes, which were often lost using other library preparation approaches. Conclusion We introduce and improve a fast, simple, and efficient ligation-based single-stranded DNA library preparation for gut virome study. This method is compatible with Illumina sequencing platforms and only requires ligation reagents within 3-h library preparation, which is similar or even better than the advanced library preparation method (xGen). We hope this method can be further optimized, validated, and widely used to make gut virome study more comparable and reproducible. Video Abstract

Soil microbes are increasingly recognised as key contributors to wine terroir, playing crucial roles in soil health and nutrient cycling. Fungi and prokaryotes interact with soils, influencing physical and chemical properties and mediating nutrient availability for roots. These microorganisms also impact vine performance and wine quality. Viticulture is expanding to higher elevations due to their cooler temperatures; Mendoza's mountainous regions are of particular interest being, characterised by vineyards with heterogenous soil stoniness. However, the effect of this variability on soil-associated microbial communities remains unclear. This study explores microbial populations (alpha and beta diversity, taxonomical composition, and their relationship with soil physicochemical properties) in soils of contrasting stoniness, at different depths, vintages and sample types. A Malbec vineyard with heterogenous soil stoniness was selected, with two experimental sites 30 m apart containing stony soil (SS; 77 % stoniness) and non-stony soil (NS; 0 % stoniness), respectively, and which were managed identically. Samples were collected from two depths (0.3 m and 0.6 m) during two vintages (2017 and 2018), from bulk and rhizosphere. Amplicon sequencing targeted the V3-V4 region of the 16S rRNA gene (prokaryotes) and the ITS1 region (fungi). Results showed that soil type significantly influences fungal populations, with less effect on prokaryotes. Vintage, reflecting annual changes in weather and viticultural practices, was the most significant factor affecting microbial communities. Depth was particularly important for fungi, while the sampling type (bulk or rhizosphere) had no significant impact on the microbiome within the same soil profile. Certain soil components were found to influence microbial communities: pH affected prokaryotes, while calcareous content specifically influenced the Proteobacteria phylum. Additionally, five fungal orders were more abundant in the stony soils, though some remained unidentified. These findings provide a baseline for understanding microorganisms in contrasting soils types of a high-elevation vineyard, and they highlight the role of microbial diversity in supporting unique soil-plant-environment interactions.

Abstract Background The interplay between gut microbiota (GM) and the metabolization of dietary components leading to the production of short-chain fatty acids (SCFAs) is affected by a range of factors including colonic pH and carbohydrate source. However, there is still only limited knowledge on how the GM activity and metabolite production in the gastrointestinal tract could be influenced by pH and the pH gradient increases along the colon. Results Here we investigate the effect of pH gradients corresponding to levels typically found in the colon on GM composition and metabolite production using substrates inulin, lactose, galactooligosaccharides (GOS), and fructooligosaccharide (FOS) in an in vitro colon setup. We investigated 3 different pH regimes (low, 5.2 increasing to 6.4; medium, 5.6 increasing to 6.8 and high, 6.0 increasing to 7.2) for each fecal inoculum and found that colonic pH gradients significantly influenced in vitro simulated GM structure, but the influence of fecal donor and substrate was more pronounced. Low pH regimes strongly influenced GM with the decreased relative abundance of Bacteroides spp. and increased Bifidobacterium spp. Higher in vitro simulated colonic pH promoted the production of SCFAs in a donor- and substrate-dependent manner. The butyrate producer Butyricimonas was enriched at higher pH conditions, where also butyrate production was increased for inulin. The relative abundance of Phascolarctobacterium, Bacteroides, and Rikenellaceae also increased at higher colonic pH, which was accompanied by increased production of propionate with GOS and FOS as substrates. Conclusions Together, our results show that colonic substrates such as dietary fibres influence GM composition and metabolite production, not only by being selectively utilized by specific microbes, but also because of their SCFA production, which in turn also influences colonic pH and overall GM composition and activity. Our work provides details about the effect of the gradients of rising pH from the proximal to distal colon on fermenting dietary substrates in vitro and highlights the importance of considering pH in GM research.

Abstract Following a request from the European Commission, the EFSA Panel on Plant Health performed a quantitative risk assessment for the EU of Phlyctinus callosus (Coleoptera: Curculionidae), a polyphagous pest occurring in Australia, New Zealand and South Africa. The current risk assessment focused on potential pathways for entry, the climatic conditions allowing establishment, the expected spread capacity and the impact considering a time horizon of 10 years (2023–2032). The Panel identified the import of apples, cut flowers and table grapes as the most relevant entry pathways. Over the next 10 years, an annual median estimate of approximately 49.5 (90% certainty range, CR, ranging from 4.0 to 881.2) potential P. callosus founder populations are expected. When the probability of establishment is considered and climatic indicators are used to define the areas in the EU where establishment is possible, the model estimated a median of 1 founder population every 1.3 years (90% CR: 1 every 30.8 years to 23.3 per year) in the scenario where the areas are defined by the union of all the climatic indicators and 1 founder population every 11.9 years (90% CR: 1 every 256.6 years to 2.5 per year) in the scenario where establishment is possible only in the areas defined by the climatic indicator of minimum soil temperature. The estimated number of founder populations per year is mostly driven by the probability of establishment in the rural areas, infestation rate in table grapes and the probability of transfer to a suitable host in the rural area. The risk of entry for cut flowers and apples is substantially lower than the risk from the table grapes. If such founder populations were to establish, P. callosus is estimated to spread by natural dispersal and common agricultural practices at a rate of 15.5 m/year (90% CR 5.1–46.8 m/year) after a lag phase of 4.0 years (90% CR 1.3–8.7 years). The impact, expressed as percentage loss of the production directly attributable to P. callosus in the areas where establishment is possible and assuming farmers do not apply specific control measures was estimated at 0.5% (90% CR 0.01%–2.8%) for cut flowers/foliage, 5.2% (90% CR 2.2%–11.7%) for apples and 2% (90% CR 1.3%–5.2%) for table grapes. Options for risk reduction are discussed, but their effectiveness is not quantified.

Abstract The EFSA Panel on Plant Health performed a pest categorisation of Monema flavescens (Lepidoptera, Limacodidae), following the commodity risk assessment of Acer palmatum plants grafted on A. davidii from China, in which M. flavescens was identified as a pest of possible concern to the European Union. This species can be identified by morphological taxonomic keys and by barcoding. The adults of the overwintering generation emerge from late June to late August. The eggs are laid in groups on the underside of the host‐plant leaves, on which the larvae feed throughout their six to eight larval instars. Pupation occurs in ovoid cocoons at the junction between twigs and branches, or on the trunk. Overwintering occurs as fully grown larvae or prepupae in their cocoon. There are one or two generations per year. M. flavescens is polyphagous and feeds on broadleaves; it has been reported on 51 plant species belonging to 24 families. It mainly occurs in Asia (Bhutan, China, the Democratic People's Republic of Korea, Japan, Nepal, the Republic of Korea), Russia (Eastern Siberia) and Taiwan. It is also present in the USA (Massachusetts). The pest's flight capacities are unknown. The main pathway for entry and spread is plants for planting with cocoons attached. This is partially closed by prohibition of some hosts. In several EU member states climatic conditions are conducive for establishment and many host plants are widespread. Introduction of M. flavescens may result in defoliations influencing tree health and forest diversity. The caterpillars also have urticating spines affecting human health. Phytosanitary measures are available to reduce the likelihood of entry, establishment and spread, and there is a definite potential for classical biological control. Recognising that natural enemies prevent M. flavescens being regarded as a pest in Asia, there is uncertainty regarding the magnitude of potential impact in EU depending on the influence of natural enemies. All criteria assessed by EFSA for consideration as a potential quarantine pest are met.

Abstract The EFSA Panel on Plant Health performed a pest categorisation of Matsucoccus matsumurae (Hemiptera: Matsucoccidae), the Massonian pine bast scale, for the EU territory. This pest categorisation was initiated following the commodity risk assessment of artificially dwarfed plants from China consisting of Pinus parviflora (Japanese white pine) grafted on P. thunbergii (Japanese black pine) performed by EFSA, in which M. matsumurae was identified as a pest of possible concern. However, its identity is not firmly established due to uncertainty regarding its taxonomic relationship with Matsucoccus pini (Green), a species widespread in Europe. M. matsumurae occurs in western China and has been reported as a pest of P. massoniana (Chinese red pine) and P. thunbergii. These hosts occur in the EU as ornamental/amenity trees. Other scales in the Matsucoccus genus feed on a variety of Pinus species and the host range of M. matsumurae could be wider than is currently recorded. The scale has one or two generations per year. All stages occur on the branches and stems of hosts with developing nymphs and adult females feeding through the bark on host phloem vessels. Symptoms include the yellowing/browning of host needles, early needle drop, desiccation of shoots and bark necrosis. The most serious infestations occur in hosts that are 8–25 years old and there can be some host mortality. In principle, host plants for planting and plant products such as cut branches and wood with bark could provide entry pathways into the EU. However, prohibitions on the import of Pinus from non‐European third countries regulate these pathways. In China, M. matsumurae occurs in regions with temperate humid conditions and hot summers. These conditions are also found in parts of southern EU. Were M. matsumurae to establish in the EU, it is conceivable that it could expand its host range; however, this remains uncertain. Some uncertainty exists over the magnitude of potential impacts. M. matsumurae satisfies the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest, assuming M. pini is not a synonym, which is a key uncertainty.

Abstract The European Commission requested the EFSA Panel on Plant Health to conduct a pest categorisation of Coniella castaneicola (Ellis & Everh) Sutton, following commodity risk assessments of Acer campestre, A. palmatum, A. platanoides, A. pseudoplatanus, Quercus petraea and Q. robur plants from the UK, in which C. castaneicola was identified as a pest of possible concern to the EU. When first described, Coniella castaneicola was a clearly defined fungus of the family Schizoparmaceae, but due to lack of a curated type‐derived DNA sequence, current identification based only on DNA sequence is uncertain and taxa previously reported to be this fungus based on molecular identification must be confirmed. The uncertainty on the reported identification of this species translates into uncertainty on all the sections of this categorisation. The fungus has been reported on several plant species associated with leaf spots, leaf blights and fruit rots, and as an endophyte in asymptomatic plants. The species is reported from North and South America, Africa, Asia, non‐EU Europe and Oceania. Coniella castaneicola is not known to occur in the EU. However, there is a key uncertainty on its presence and geographical distribution worldwide and in the EU due to its endophytic nature, the lack of systematic surveys and possible misidentifications. Coniella castaneicola is not included in Commission Implementing Regulation (EU) 2019/2072 and there are no interceptions in the EU. Plants for planting, fresh fruits and soil and other growing media associated with infected plant debris are the main pathways for its entry into the EU. Host availability and climate suitability in parts of the EU are favourable for the establishment and spread of the fungus. Based on the scarce information available, the introduction and spread of C. castaneicola in the EU is not expected to cause substantial impacts, with a key uncertainty. Phytosanitary measures are available to prevent its introduction and spread in the EU. Because of lack of documented impacts, Coniella castaneicola does not satisfy all the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest.

Abstract The EFSA Panel on Plant Health performed a pest categorisation of Popillia quadriguttata (Coleoptera: Scarabaeidae), following a commodity risk assessment of bonsai Pinus parviflora grafted onto P. thunbergii from China, in which P. quadriguttata was identified as a pest of possible concern for the territory of the European Union. This is a univoltine polyphagous pest that occurs in eastern Asia from Vietnam northwards through eastern China and Taiwan, South Korea and into Far East Russia. Hosts include species of fruit trees within the genera Malus and Prunus, trees of forestry and environmental importance such as Quercus and Ulmus, shrubs such as Wisteria, soft fruit such as Rubus, grasses, including amenity turf and field crops such as potatoes, maize and soybean. Adults feed on host leaves, tender stems, flower buds, flowers and fruits; larvae feed on host roots. In northern China P. quadriguttata is a major pest of soybean; in South Korea, P. quadriguttata is one of the most serious insect pests of golf course turf. P. quadriguttata could enter the EU on various pathways including infested soil and growing media accompanying host plants for planning. Biotic factors (host availability) and abiotic factors (climate suitability) suggest that large parts of the EU would be suitable for establishment. Local spread would be mainly via natural dispersal of adults. Long distance spread would be facilitated by the movement of eggs, larvae and pupae infesting soil especially with plants for planting; adults could spread on plants for planting without soil. Economic and or environmental impacts would be expected on a range of plants if P. quadriguttata were to establish in the EU. Phytosanitary measures are available to reduce the likelihood of its introduction. P. quadriguttata satisfies all of the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

Abstract The EFSA Panel on Plant Health performed a pest categorisation of Bailey's rust mite, Calepitrimerus baileyi Keifer (Acariformes: Eriophyidae), following the commodity risk assessment of Malus domestica plants from Türkiye performed by EFSA, in which C. baileyi was identified as a pest of possible concern to the European Union. This mite is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072. The eriophyid is known to occur in Africa, America, Asia, Europe (Greece and Serbia) and Oceania on Malus spp., which is the only confirmed host genus for C. baileyi. Plants for planting of Malus spp. are the main potential pathway for entry into the EU. However, plants for planting of the genus Malus Mill. are considered as high‐risk plants (EU 2018/2019) and therefore prohibited from entering the EU unless granted a country‐specific derogation. This is the case for the import of Malus spp. plants for planting from Serbia ((EU) 2020/1361 corrected by 2022/1309). Therefore, this derogation could provide a plausible entry pathway for C. baileyi into the EU. Climatic conditions and the ample availability of the host, Malus spp., in the EU are conducive for establishment, as proven by the occurrence of C. baileyi in Greece. However, the species is not reported as having an impact in Greece, despite reports of damage outside the EU. Measures to prevent further entry and spread of C. baileyi in the EU are available. C. baileyi satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest. However, uncertainties about the distribution of C. baileyi within the EU and its impact on apples in the EU are considered key and affect the confidence of conclusions for this categorisation.

Abstract The EFSA Panel on Plant Health performed a pest categorisation of Shirahoshizo patruelis (Voss, 1937) (Coleoptera: Curculionidae), following the commodity risk assessment of bonsai plants from China consisting of Pinus parviflora grafted on P. thunbergii, in which S. patruelis was identified as a pest of possible concern to the European Union (EU). This categorisation refers to S. flavonotatus, which is the pest's current valid scientific name. It is native to China and has never been recorded in the EU. It completes from 2 to 3 generations per year. Eggs are laid in cracks and crevices of trunks and branches with bark thickness of approximately 0.6–1.2 cm. The pest overwinters as an adult or as a mature larva under the bark. Plants for planting, wood with bark and wood products provide pathways for entry. Although the weevil has been reported to carry the nematode Bursaphelenchus xylophilus, it is not considered a vector. Climatic conditions and availability of host plants in some EU countries would allow S. flavonotatus to establish and spread. Impact on Pinus spp. is anticipated. Recognising that the weevil is reported to attack both weakened and healthy trees, there is uncertainty on the magnitude of impact. Its recorded capacity to attack non‐Asian Pinus species also indicates its ability to adapt and expand the range of trees it can utilise as hosts, which could include European Pinus species. Phytosanitary measures are available to reduce the likelihood of entry and spread. S. flavonotatus meets the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

Abstract Following a request from the European Commission, the EFSA Panel on Plant Health performed a quantitative risk assessment for the EU of African Leucinodes species (Lepidoptera: Crambidae), which are fruit and shoot borers, especially of eggplant type fruit. The assessment focused on (i) potential pathways for entry, (ii) distribution of infested imports within EU, (iii) climatic conditions favouring establishment, (iv) spread and (v) impact. Options for risk reduction are discussed, but their effectiveness was not quantified. Leucinodes spp. are widely distributed across sub‐Saharan Africa but are little studied and they could be much more widespread in Africa than reported. Much African literature erroneously reports them as Leucinodes orbonalis which is restricted to Asia. The import of eggplant type fruit from sub‐Saharan Africa consists of special fruit types and caters mostly to niche markets in the EU. The main pathway for entry is fruit of Solanum aethiopicum and exotic varieties of eggplant (S. melongena). CLIMEX modelling was used with two possible thresholds of ecoclimatic index (EI) to assess establishment potential. Climates favouring establishment occur mostly in southern Europe, where, based on human population, 14% of the imported produce is distributed across NUTS2 regions where EI ≥ 30; or where 23% of the produce is distributed where EI ≥ 15. Over the next 5 years, an annual median estimate of ~ 8600 fruits, originating from Africa, and infested with African Leucinodes spp. are expected to enter EU NUTS2 regions where EI ≥ 15 (90% CR ~ 570–52,700); this drops to ~ 5200 (90% CR ~ 350–32,100) in NUTS2 regions where EI ≥ 30. Escape of adult moths occurs mostly from consumer waste; considering uncertainties in pathway transfer, such as adult emergence, mate finding and survival of progeny, the annual median probability of a mated female establishing a founder population in NUTS regions where EI ≥ 15 was estimated to be 0.0078 (90% CR 0.00023–0.12125). This equates to a median estimate of one founder population ~ every 128 years (90% CR approximately one every 8–4280 years). Using an EI ≥ 30, the median number of founder populations establishing in the EU annually is 0.0048 (90% CR 0.0001–0.0739), equating to a median estimate of one founder population approximately every 210 years (90% CR approximately one every 14–7020 years). Under climate change for the period 2040–2059, the percent of infested produce going to suitable areas would be increased to 33% for EI ≥ 15 and to 21% for EI ≥ 30. Accordingly, the waiting time until the next founder population would be reduced to median estimates of 89 years for EI ≥ 15 (90% CR ~ 6–2980 years) and 139 years for EI ≥ 30 (90% CR 9–4655 years). If a founder population were to establish, it is estimated to spread at a rate of 0.65–7.0 km per year after a lag phase of 5–92 years. Leucinodes spp. are estimated to reduce eggplant yield by a median value of 4.5% (90% CR 0.67%–13%) if growers take no specific action, or 0.54% (90% CR between 0.13% and 1.9%) if they do take targeted action, matching previous estimates made during a risk assessment of L. orbonalis from Asia.

Abstract Following a request from the European Commission, the EFSA Panel on Plant Health performed a quantitative risk assessment for the EU of Retithrips syriacus (Mayet) (Thysanoptera: Thripidae), a polyphagous thrips, regarded as a tropical/subtropical pest occurring in several countries of Africa, South America, Asia and in the EU in Cyprus. The current risk assessment focused on potential pathways for entry, the climatic conditions allowing establishment, the expected spread capacity and the impact considering a time horizon of 10 years (2023–2032). The Panel identified the import of cut roses, persimmons, table grapes, as well as plants for planting of the genera Acalypha and Terminalia from third countries and those of Persea americana (avocado) from Israel as the most relevant entry pathways to consider. Over the next 10 years, an annual median estimate of 95 (90% Certainty Range, CR, ranging from 13 to 1832) potential R. syriacus founder populations per year are expected to successfully transfer to a suitable host in the EU NUTS2 regions where the climatic conditions are predicted as suitable for establishment; this value drops to a median of 4.6 founder populations per year (90% CR: 1 every 1.9 years – 85.6 per year) after considering the actual probability of establishment of a potential founder population. The estimated number of founder population per year is mostly driven by the import of cut roses and plants for planting. If such founder populations were to establish, R. syriacus is estimated to spread at a median rate of 0.05 km/year (90% CR 0.02–2.30 km/year) after a median lag phase of 1.1 years (90% CR 0.3–3.3 years). The overall impact on yield (expressed as % of the total agricultural production) directly attributable to R. syriacus when considering: (i) the main R. syriacus hosts in the EU, (ii) the areas of the EU where establishment is possible, (iii) the current agricultural practices and (iv) the evidence of impact from the countries where the pest is established for a long time, was estimated at 0.065% as the median value of the uncertainty distribution (90% CR 0.001%–0.571%). Options for risk reduction are discussed, but the effectiveness was not quantified.

Abstract Following a request from the European Commission, the EFSA Panel on Plant Health performed a quantitative risk assessment of Leucinodes orbonalis (Lepidoptera: Crambidae), the eggplant fruit and shoot borer, for the EU. The assessment focused on potential pathways for entry, climatic conditions favouring establishment, spread and impact. Options for risk reduction are discussed but effectiveness was not quantified. L. orbonalis is a key pest of eggplant (aubergine/brinjal) in the Indian subcontinent and occurs throughout most of southern Asia with records mostly from India and Bangladesh. The main pathway of entry is fruit of solanaceous plants, primarily exotic varieties of eggplant, Solanum melongena and turkey berry, S. torvum. The trade in both commodities from Asia is small but nevertheless dwarfs the trade in other Solanum fruits from Asia (S. aethiopicum, S. anguivi, S. virginianum, S. aculeatissimum, S. undatum). Other Solanum fruits were therefore not further assessed as potential pathways. The trade in eggplant from Asia consists of special fruit types and caters mostly to niche markets in the EU, while most eggplant consumed in Europe is produced in southern European and northern African countries, where L. orbonalis does not occur. Using expert knowledge elicitation (EKE) and pathway modelling, the Panel estimated that approximately 3–670 infested fruit (90% certainty range, CR) of S. melongena or fruit bunches of S. torvum enter into regions of the EU that are suitable for L. orbonalis establishment each year. Based on CLIMEX modelling, and using two possible thresholds of ecoclimatic index (EI) to indicate uncertainty in establishment potential, climates favouring establishment occur mostly in southern Europe, where, based on human population, approximately 14% of the imported produce is distributed across NUTS2 regions where EI ≥ 30; or 23% of the produce is distributed where EI ≥ 15. Escape of adult moths occurs mostly from consumer waste. By analysing results of different scenarios for the proportion of S. melongena and S. torvum in the trade, and considering uncertainties in the climatic suitability of southern Europe, adult moth emergence in areas suitable for establishment is expected to vary between 84 individuals per year and one individual per 40 years (based on 90% CR in different scenarios). In the baseline scenario, 25% of the solanaceous fruit from Asia is S. torvum, 75% is S. melongena and EI ≥ 30 is required for establishment. After accounting for the chances of mating, host finding and establishment, the probability of a mated female establishing a founder population in the EU is less than 1 in 100,000 to about 1 event per 622 years (90% CR in baseline scenario). The waiting time until the first establishment is then 622 to more than 100,000 years (CR). If such a founder population were established, the moth is estimated to spread at a rate of 0.65–7.0 km per year after a lag phase of 5–92 years. The impact of the insect on the production of eggplant is estimated to be 0.67%–13% (CR) if growers take no specific action against the insect and 0.13%–1.9% if they do take targeted actions. Tomato (S. lycopersicum) and potato (S. tuberosum) are hosts of L. orbonalis, but the insect does not develop to maturity in tomato fruit, and it does not feed on potato tubers under field conditions; hence, damage to potato can only occur due to feeding on shoots. Tomato and potato are not preferred hosts; nevertheless, impact can occur if populations of L. orbonalis are high and preferred hosts are not available. The Panel did not assess this damage due to insufficient information.

Abstract The European Commission requested the EFSA Panel on Plant Health to conduct a pest categorisation of Garella musculana (Erschov) (Lepidoptera: Nolidae), following a commodity risk assessment of Juglans regia plants for planting from Türkiye, in which G. musculana was identified as a pest of possible concern to the European Union (EU). Commonly known as the Asian walnut moth, this pest is native to Central Asia and develops on shoots, buds and fruits of Juglans species such as the English walnut, J. regia and the black walnut, J. nigra. Other reported host plants, such as Prunus dulcis and Populus spp., still require confirmation. The pest was first recorded in the EU (Bulgaria) in 2016 and was then reported in Romania in 2018 and Italy in 2021. This moth completes from one to four generations per year depending on environmental conditions (from valley to mountain forests and orchards up to an altitude of 2100 m). Eggs are laid in groups of 2–3 on young nuts or on buds of 1‐year‐old shoots. Neonate larvae usually enter the young nut through the peduncle. After fully exploiting one nut, the larva continues feeding in another one. Development takes 25–40 days. Larvae of the autumn generation do not enter the nuts, and so feed only in the pericarp. Larvae also often feed inside 1‐year‐old shoots or leaf axils. Larvae develop within the host but exit to pupate under loose bark or in deep cracks of bark. The pest overwinters at the larval or pupal stages. Plants for planting, cut branches and infested nuts provide pathways for entry. Climatic conditions and availability of host plants in southern and central EU MSs have allowed this species to establish and spread in Bulgaria, Romania and Italy. Adults can fly and the pest could spread naturally within the EU. Impact on Juglans spp. cultivated for fruit, timber and ornamental purposes is anticipated. Phytosanitary measures are available to reduce the likelihood of entry and further spread of G. musculana. This species meets the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

Abstract The EFSA Panel on Plant Health performed a pest categorisation of Dendrolimus punctatus (Lepidoptera: Lasiocampidae), following a commodity risk assessment of bonsai Pinus parviflora grafted onto P. thunbergii from China, in which D. punctatus was identified as a pest of possible concern to the European Union (EU). D. punctatus, also known as the Masson pine caterpillar, is present in China, Taiwan, Vietnam, India and has recently spread to Japanese islands close to Taiwan. Larval feeding on the needles of Pinus elliottii, P. luchuensis, P. massoniana, P. merkusii and P. tabulaeformis causes important damage. D. punctatus larvae can also feed on P. armandii, P. echinata, P. latteri, P. parviflora, P. sylvestris var. mongolica, P. taeda, P. taiwanensis and P. thunbergii, but full development on these hosts is uncertain. The pest has three to five generations per year; winter is spent as larvae on branch tips, on tree trunks and in the soil. The females lay egg clusters on pine needles. Pupation occurs in cocoons attached to branches or needles. D. punctatus could enter the EU either as eggs, larvae or pupae in the foliage of plants for planting or cut branches, as larvae on wood with bark or as overwintering larvae in branches, crevices in the bark or in the litter of potted plants. However, Annex VI of 2019/2072 prohibits the introduction of D. punctatus hosts (Pinus spp.) from countries and areas where the pest occurs. There are climate zones where the pest occurs in Asia that also occur in the EU, though they are limited, which constitutes an uncertainty regarding establishment. The pest's main hosts are not grown in the EU. However, the fact that it attacks the North American Pinus echinata, P. elliottii and P. taeda in its Asian native area suggests a potential capacity to shift to pine species occurring in the EU territory. D. punctatus satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest. Whether the Pinus commonly found in Europe could act as hosts is unknown but is fundamental, affecting the criteria of establishment and magnitude of impact.

Abstract Following the commodity risk assessment of bonsai plants (Pinus parviflora grafted on Pinus thunbergii) from China performed by EFSA, the EFSA Plant Health Panel performed a pest categorisation of Pyrrhoderma noxium, a clearly defined plant pathogenic basidiomycete fungus of the order Hymenochaetales and the family Hymenochaetaceae. The pathogen is considered as opportunistic and has been reported on a wide range of hosts, mainly broad‐leaved and coniferous woody plants, causing root rots. In addition, the fungus was reported to live saprophytically on woody substrates and was isolated as an endophyte from a few plant species. This pest categorisation focuses on the hosts that are relevant for the EU (e.g. Citrus, Ficus, Pinus, Prunus, Pyrus, Quercus and Vitis vinifera). Pyrrhoderma noxium is present in Africa, Central and South America, Asia and Oceania. It has not been reported in the EU. Pyrrhoderma noxium is not included in Commission Implementing Regulation (EU) 2019/2072. Plants for planting (excluding seeds), bark and wood of host plants as well as soil and other growing media associated with plant debris are the main pathways for the entry of the pathogen into the EU. Host availability and climate suitability factors occurring in parts of the EU are favourable for the establishment and spread of the pathogen. The introduction and spread of the pathogen into the EU are expected to have an economic and environmental impact in parts of the territory where hosts are present. Phytosanitary measures are available to prevent the introduction and spread of the pathogen into the EU. Pyrrhoderma noxium satisfies all the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest.

Abstract Background There is an increasing interest in investigating the human gut virome for its influence on the gut bacterial community and its putative influence on the trajectory towards health or disease. Most gut virome studies are based on sequencing of stored fecal samples. However, relatively little is known about how conventional storage buffers and storage conditions affect the infectivity of bacteriophages and influence the downstream metavirome sequencing. Results We demonstrate that the infectivity and genome recovery rate of different spiked bacteriophages (T4, c2 and Phi X174) are variable and highly dependent on storage buffers. Regardless of the storage temperature and timespan, all tested phages immediately lost 100% (DNA/RNA Shield) or more than 90% (StayRNA and RNAlater) of their infectivity. Generally, in SM buffer at 4 °C phage infectivity was preserved for up to 30 days and phage DNA integrity was maintained for up to 100 days. While in CANVAX, the most effective buffer, all spiked phage genomes were preserved for at least 100 days. Prolonged storage time (500 days) at – 80 °C impacted viral diversity differently in the different buffers. Samples stored in CANVAX or DNA/RNA Shield buffer had the least shifts in metavirome composition, after prolonged storage, but they yielded more contigs classified as “uncharacterised”. Moreover, in contrast to the SM buffer, these storage buffers yielded a higher fraction of bacterial DNA in metavirome-sequencing libraries. We demonstrated that the latter was due to inactivation of the DNases employed to remove extra-cellular DNA during virome extraction. The latter could be partly avoided by employing additional washing steps prior to virome extraction. Conclusion Fecal sample storage buffers and storage conditions (time and temperature) strongly influence bacteriophage infectivity and viral composition as determined by plaque assay and metavirome sequencing. The choice of buffer had a larger effect than storage temperature and storage time on the quality of the viral sequences and analyses. Based on these results, we recommend storage of fecal virome samples at in SM buffer at 4 °C for the isolation of viruses and at – 80 °C for metagenomic applications if practically feasible (i.e., access to cold storage). For fecal samples stored in other buffers, samples should be cleared of these buffers before viral extraction and sequencing. Video Abstract

Abstract Background The influence of diet on immune function and resistance to enteric infection and disease is becoming ever more established. Highly processed, refined diets can lead to inflammation and gut microbiome dysbiosis, whilst health-promoting dietary components such as phytonutrients and fermentable fibres are thought to promote a healthy microbiome and balanced mucosal immunity. Chicory (Cichorium intybus) is a leafy green vegetable rich in fibres and bioactive compounds that may promote gut health. Results Unexpectedly, we here show that incorporation of chicory into semisynthetic AIN93G diets renders mice susceptible to infection with enteric helminths. Mice fed a high level of chicory leaves (10% dry matter) had a more diverse gut microbiota, but a diminished type-2 immune response to infection with the intestinal roundworm Heligmosomoides polygyrus. Furthermore, the chicory-supplemented diet significantly increased burdens of the caecum-dwelling whipworm Trichuris muris, concomitant with a highly skewed type-1 immune environment in caecal tissue. The chicory-supplemented diet was rich in non-starch polysaccharides, particularly uronic acids (the monomeric constituents of pectin). In accordance, mice fed pectin-supplemented AIN93G diets had higher T. muris burdens and reduced IgE production and expression of genes involved in type-2 immunity. Importantly, treatment of pectin-fed mice with exogenous IL-25 restored type-2 responses and was sufficient to allow T. muris expulsion. Conclusions Collectively, our data suggest that increasing levels of fermentable, non-starch polysaccharides in refined diets compromises immunity to helminth infection in mice. This diet-infection interaction may inform new strategies for manipulating the gut environment to promote resistance to enteric parasites.

Abstract Following the EFSA commodity risk assessment of Malus domestica plants imported from Türkiye into the EU, the EFSA Panel on Plant Health performed a pest categorisation of Pratylenchus loosi (Nematoda: Pratylenchidae) for the EU. Pratylenchus loosi belongs to the order Rhabditida, subfamily Pratylenchidae. This nematode is not known to be present in the EU. The species is not included in the EU Commission Implementing Regulation 2019/2072. The pest occurs primarily in tropical, subtropical and warm temperate areas. It is widely distributed in Asian countries, with tea plants (Camellia sinensis) as the main host. The pest was reported from more than 60 plant species, but reports from hosts other than C. sinensis, e.g. citrus (Citrus spp.) and banana (Musa spp.), are associated with high uncertainty due to doubtful pest identification. Morphological and molecular methods are available for the identification of the pest. Pathways of entry are host plants for planting except seeds, as well as soil attached to plants for planting, machinery or footwear. Soil import to the EU is prohibited from third countries. The climatic preferences of P. loosi are compatible with the microclimatic conditions occurring in the areas of the EU where tea is grown outside. The impact of the nematode is primarily known for Asian countries, where it is a devastating pathogen on tea plants, but there is a key uncertainty on impacts on hosts other than tea. Considering the strong pathogenicity of the pest, its establishment in tea producing areas would have negative consequences for tea producers. Therefore, the Panel concludes that P. loosi satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

Abstract The EFSA Panel on Plant Health performed a pest categorisation of Malacosoma parallela (Staudinger) (Lepidoptera: Lasiocampidae) for the territory of the European Union, following commodity risk assessments of Berberis thunbergii, Malus domestica, Prunus persica and P. dulcis plants for planting from Türkiye, in which M. parallela came to attention as of possible concern. M. parallela is commonly known as the mountain ring silk moth and is a polyphagous leaf‐eating pest in west‐central Asia, primarily feeding on deciduous trees and shrubs, and known to cause serious damage to Malus, Prunus, and Quercus species. It is found at a range of altitudes from 130 m to 3000 m although most common above 1000 m. It is a univoltine species. Eggs are laid in masses on twigs and branches in the summer and larvae hatch the following spring to feed on buds and fresh leaves. Host plants can be completely defoliated. Plants for planting and cut branches provide pathways for entry, especially if infested with egg masses. Host availability and climate suitability suggest that parts of the EU would be suitable for establishment. Adults can fly and the pest could spread naturally within the EU although adults only live for a few days. Faster and more extensive spread is therefore more likely via egg masses moved on plants for planting. The introduction of M. parallela into the EU could lead to outbreaks causing damage to deciduous trees and shrubs in forests and orchards. Phytosanitary measures are available to inhibit the entry and spread of this species. M. parallela satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

Abstract Following the commodity risk assessments of bonsai plants from China consisting of Pinus parviflora grafted on P. thunbergii performed by EFSA, the EFSA Plant Health Panel performed a pest categorisation of Pestalotiopsis disseminata, a clearly defined plant pathogenic fungus of the family Pestalotiopsidaceae. The pathogen has been reported on herbaceous, woody and ornamental plants causing symptoms such as leaf blight, shoot blight, seedling blight, pod canker, pre‐ and post‐harvest fruit rot, and gummosis. Moreover, the fungus was reported as an endophyte on a wide range of asymptomatic hosts. The pathogen is present in Africa, North and South America, Asia, Europe and Oceania. It has been reported from the EU, with a restricted distribution (Portugal). There is a key uncertainty on the geographical distribution of P. disseminata in the EU and worldwide, because of the endophytic nature of the fungus, the lack of surveys and since the pathogen might have been misidentified based only on morphology and pathogenicity tests. The pathogen is not included in Commission Implementing Regulation (EU) 2019/2072. This pest categorisation focuses on those hosts that are relevant for the EU and for which there is robust evidence that the pathogen was formally identified by a combination of morphology, pathogenicity and multilocus sequence analysis. Plants for planting, fresh fruits, bark and wood of host plants as well as soil and other plant growing media are the main pathways for the entry of the pathogen into the EU. Host availability and climate suitability factors occurring in parts of the EU are favourable for the establishment of the pathogen. Despite the low aggressiveness observed in most reported hosts, and the fact that P. disseminata may colonise plants as an endophyte, its introduction and spread in the EU may have an economic and environmental impact (with a key uncertainty) where susceptible hosts are grown. Phytosanitary measures are available to prevent the introduction and spread of the pathogen. The Panel cannot conclude on whether P. disseminata satisfies all the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest, because of the key uncertainties on the restricted distribution in the EU and the magnitude of the impact.

Abstract Following an EFSA commodity risk assessment of bonsai plants (Pinus parviflora grafted on Pinus thunbergii) imported from China, the EFSA Plant Health Panel performed a pest categorisation of Pestalotiopsis microspora, a clearly defined plant pathogenic fungus of the family Pestalotiopsidaceae. The pathogen was reported on a wide range of monocotyledonous, dicotyledonous and gymnosperms, either cultivated or wild plant species, causing various symptoms such as leaf spot, leaf blight, scabby canker, fruit spot, pre‐ and post‐harvest fruit rot and root rot. In addition, the fungus was reported as an endophyte on a wide range of asymptomatic plant species. This pest categorisation focuses on the hosts that are relevant for the EU and for which there is robust evidence that the pathogen was formally identified by a combination of morphology, pathogenicity and multilocus sequencing analyses. Pestalotiopsis microspora was reported in Africa, North, Central and South America, Asia and Oceania. In the EU, it was reported in the Netherlands. There is a key uncertainty on the geographical distribution of P. microspora worldwide and in the EU, because of the endophytic nature of the fungus, the lack of surveys, and because in the past, when molecular tools were not fully developed, the pathogen might have been misidentified as other Pestalotiopsis species or other members of the Pestalodiopsidaceae family based on morphology and pathogenicity tests. Pestalotiopsis microspora is not included in Commission Implementing Regulation (EU) 2019/2072. Plants for planting, fresh fruits, bark and wood of host plants as well as soil and other growing media associated with plant debris are the main pathways for the entry of the pathogen into the EU. Host availability and climate suitability in parts of the EU are favourable for the establishment and spread of the pathogen. The introduction and spread of the pathogen into the EU are expected to have an economic and environmental impact where susceptible hosts are grown. Phytosanitary measures are available to prevent the introduction and spread of the pathogen into the EU. Unless the restricted distribution in the EU is disproven, Pestalotiopsis microspora satisfies all the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest.

A global analysis of vineyard soil microbial diversity provides insights into the biogeographical patterns that characterize wine regions worldwide, capturing the microbial dimension of viticultural terroirs.

Fungal pathogens involved in grapevine trunk diseases (GTDs) may infect grapevines throughout their lifetime, from nursery to vineyard, via open wounds in stems, canes or roots. In vineyards, pruning wound protection products (PWPPs) offer the best means to reduce the chance of infection by GTD fungi. However, PWPPs may affect non-target microorganisms that comprise the natural endophytic mycobiome residing in treated canes, disrupting microbial homeostasis and indirectly influencing grapevine health. Using DNA metabarcoding, we characterized the endophytic mycobiome of one-year-old canes of cultivars Cabernet Sauvignon and Syrah in two vineyards in Portugal and Italy and assessed the impact of established and novel PWPPs on the fungal communities of treated canes. Our results reveal a large fungal diversity (176 taxa), and we report multiple genera never detected before in grapevine wood (e.g., Symmetrospora and Akenomyces). We found differences in mycobiome beta diversity when comparing vineyards (p = 0.01) but not cultivars (p > 0.05). When examining PWPP-treated canes, we detected cultivar- and vineyard-dependent alterations in both alpha and beta diversity. In addition, numerous fungal taxa were over- or under-represented when compared to control canes. Among them, Epicoccum sp., a beneficial genus with biological control potential, was negatively affected by selected PWPPs. This study demonstrates that PWPPs induce alterations in the fungal communities of grapevines, requiring an urgent evaluation of their direct and indirect effects on plants health with consideration of factors such as climatic conditions and yearly variations, in order to better advise viticulturists and policy makers.

Drinking water resources, such as groundwater, are threatened by pollution. The pesticide metabolite 2,6-dichlorobenzamide (BAM) is one of the compounds frequently found in groundwater. Studies have attempted to add specific BAM-degrading bacteria to sand filters at drinking water treatment facilities. This biotechnology has shown great potential in removing BAM from contaminated water. However, the degradation potential was formerly lost after ~2–3 weeks due to a decrease of the degrader population over time. The aim of the present study was to overcome the constraints leading to loss of degraders from inoculated filters. Our approach was threefold: (1) Development of a novel inoculation strategy, (2) lowering the flowrate to reduce washout of cells, and (3) increasing the concentration of nutrients hereunder the pollutant in a smaller inlet water stream. The two latter were achieved via modifications of the inlet water by applying membrane treatment which, besides producing an ultra-pure water fraction, produced a residual water stream with nutrients including BAM concentrated in ~ten-fold reduced volume. This was done to alleviate starvation of degrader bacteria in the otherwise oligotrophic sand filters and to enable a decreased flowrate. By this approach, we achieved 100% BAM removal over a period of 40 days in sand filter columns inoculated with the BAM-degrader Aminobacter sp. MSH1. Molecular targeting of the degrader strain showed that the population of degrader bacteria persisted at high numbers throughout the sand filter columns and over the entire timespan of the experiment. 16S rRNA gene amplicon sequencing confirmed that MSH1 dominated the bacterial communities of the inoculated sand filter columns at experimental termination. The community composition of the indigenous prokaryotes, based on beta diversity, in the sand filter columns was governed by the feed water type i.e., membrane retentate or untreated water.

Abies nordmanniana is an economically important tree crop widely used for Christmas tree production. After initial growth in nurseries, seedlings are transplanted to the field. Rhizosphere bacterial communities generally impact the growth and health of the host plant. However, the dynamics of these communities during A. nordmanniana growth in nurseries, and during transplanting, has not previously been addressed. By a 16S rRNA gene amplicon sequencing approach, we characterized the composition and dynamics of bacterial communities in the rhizosphere during early plant growth in field and greenhouse nurseries and for plants transplanted from the greenhouse to the field. Moreover, the N-cycling potential of rhizosphere bacteria across plant age was addressed in both nurseries. Overall, a rhizosphere core microbiome of A. nordmanniana, comprising 19.9% of the taxa at genus level, was maintained across plant age, nursery production systems, and even during the transplantation of plants from the greenhouse to the field. The core microbiome included the bacterial genera Bradyrhizobium, Burkholderia, Flavobacterium, Pseudomonas, Rhizobium, Rhodanobacter, and Sphingomonas, which harbor several N-fixing and plant growth–promoting taxa. Nevertheless, both plant age and production system caused significant changes in the rhizosphere bacterial communities. Concerning community composition, the relative abundance of Rhizobiales (genera Rhizobium, Bradyrhizobium, and Devosia) was higher in the rhizosphere of field-grown A. nordmanniana, whereas the relative abundance of Enterobacteriales and Pseudomonadales (genus Pseudomonas) was higher in the greenhouse. Analysis of community dynamics across plant age showed that in the field nursery, the most abundant bacterial orders showed more dynamic changes in their relative abundance in the rhizosphere than in the bulk soil. In the greenhouse, age-dependent dynamics even occurred but affected different taxa than for the field-grown plants. The N-cycling potential of rhizosphere bacterial communities showed an increase of the relative abundance of genes involved in nitrogen fixation and denitrification by plant age. Similarly, the relative abundance of reported nitrogen-fixing or denitrifying bacteria increased by plant age. However, different community structures seemed to lead to an increased potential for nitrogen fixation and denitrification in the field versus greenhouse nurseries.

Soil microbiomes, as a primary reservoir for plant colonizing fungi and bacteria, play a major role in determining plant productivity and preventing invasion by pathogenic microorganisms. The use of 16S rRNA and ITS high-throughput amplicon sequencing for analysis of complex microbial communities have increased dramatically in recent years, establishing links between wine specificity and, environmental and viticultural factors, which are framed into the elusive terroir concept. Given the diverse and complex role these factors play on microbial soil structuring of agricultural crops, the main aim of this study is to evaluate how external factors, such as vintage, vineyard location, cultivar and soil characteristics, may affect the diversity of the microbial communities present. Additionally, we aim to compare the influence these factors have on the structuring of bacterial and fungal populations associated with Malbec grapevine rhizosphere with that of the more widespread Cabernet Sauvignon grapevine cultivar. Samples were taken from Malbec and Cabernet Sauvignon cultivars from two different vineyards in the San Juan Province of Argentina. Total DNA extracts from the rhizosphere soil samples were sequenced using Illumina's Miseq technology, targeting the V3-V4 hypervariable 16S rRNA region in prokaryotes and the ITS1 region in yeasts. The major bacterial taxa identified were Proteobacteria, Bacteroidetes and Firmicutes, while the major fungal taxa were Ascomycetes, Basidiomycetes, Mortierellomycetes and a low percentage of Glomeromycetes. Significant differences in microbial community composition were found between vintages and vineyard locations, whose soils showed variances in pH, organic matter, and content of carbon, nitrogen, and absorbable phosphorus.

The phyllosphere microbiome plays an important role in plant fitness. Recently, bacteriophages have been shown to play a role in shaping the bacterial community composition of the phyllosphere. However, no studies on the diversity and abundance of phyllosphere bacteriophage communities have been carried out until now. In this study, we extracted, sequenced, and characterized the dsDNA and ssDNA viral community from a phyllosphere for the first time. We sampled leaves from winter wheat (Triticum aestivum), where we identified a total of 876 virus operational taxonomic units (vOTUs), mostly predicted to be bacteriophages with a lytic lifestyle. Remarkably, 848 of these vOTUs corresponded to new viral species, and we estimated a minimum of 2.0 × 106 viral particles per leaf. These results suggest that the wheat phyllosphere harbors a large and active community of novel bacterial viruses. Phylloviruses have potential applications as biocontrol agents against phytopathogenic bacteria or as microbiome modulators to increase plant growth-promoting bacteria.

Phaeomoniella chlamydospora is a tracheomycotic fungus that colonizes the xylem of grapevines (Vitis vinifera L.), causing wood discoloration, brown wood streaking, gummosis, and wood necrosis, which negatively affect the overall health, productivity, and life span of vines. Current control strategies to prevent or cope with P. chlamydospora infections are frequently ineffective. Moreover, it is unclear how fungicides commonly applied in vineyards against downy and powdery mildew agents affect the wood mycobiome, including wood pathogens such as P. chlamydospora. In this study, we used next-generation sequencing to assess the effects of foliar spray of grapevines with inorganic (copper oxychloride and sulfur), synthetic (penconazole and fosetyl-aluminum), and natural (Blad) fungicides currently used against the downy and powdery mildews. The subjects of our investigation were (i) the resident wood mycobiome, (ii) the early colonization by a consortium of fungal wood endophytes (ACEA1), (iii) the wood colonization success of P. chlamydospora, and (iv) the in planta interaction between P. chlamydospora and ACEA1, under greenhouse conditions, in rooted grapevine cuttings of cv. Cabernet Sauvignon. The data obtained suggest that the resident mycobiome is affected by different fungicide treatments. In addition, the early colonization success of the endophytes composing ACEA1 varied in response to fungicides, with relative abundances of some taxa being overrepresented or underrepresented when compared with the control. The wood colonization by P. chlamydospora comported significant changes in the mycobiome composition, and in addition, it was greatly affected by the foliar spray with Blad, which decreased the relative abundance of this pathogen 12-fold (4.9%) when compared with the control (60.7%) and other treatments. The presence of the pathogen also decreased considerably when co-inoculated into the plant with ACEA1, reaching relative abundances between 13.9% and 2.0%, depending on the fungicide treatment applied. This study shows that fungicides sprayed to prevent infections of powdery and downy mildews have an effect on non-target fungi that colonize the endosphere of grapevines. We suggest two potential control strategies to fight P. chlamydospora, namely, the foliar spray with Blad and the use of ACEA1. Further studies to confirm these results are required.

Esca is a disease complex belonging to the grapevine trunk diseases cluster. It comprises five syndromes, three main fungal pathogenic agents and several symptoms, both internal (i.e., affecting woody tissue) and external (e.g., affecting leaves and bunches). The etiology and epidemiology of this disease complex remain, in part, unclear. Some of the points that are still under discussion concern the sudden rise in disease incidence, the simultaneous presence of multiple wood pathogens in affected grapevines, the causal agents and the discontinuity in time of leaf symptoms manifestation. The standard approach to the study of esca has been mostly through culture-dependent studies, yet, leaving many questions unanswered. In this study, we used Illumina® next-generation amplicon sequencing to investigate the mycobiome of grapevines wood in a vineyard with history of esca. We characterized the wood mycobiome composition, investigated the spatial dynamics of the fungal communities in different areas of the stem and in canes, and assessed the putative link between mycobiome and leaf symptoms. An unprecedented diversity of fungi is presented (289 taxa), including five genera reported for the first time in association with grapevines wood (Debaryomyces, Trematosphaeria, Biatriospora, Lopadostoma, and Malassezia) and numerous hitherto unreported species. Esca-associated fungi Phaeomoniella chlamydospora and Fomitiporia sp. dominate the fungal community, and numerous other fungi associated with wood syndromes are also encountered (e.g., Eutypa spp., Inonotus hispidus). The spatial analysis revealed differences in diversity, evenness and taxa abundances, the unique presence of certain fungi in specific areas of the plants, and tissue specificity. Lastly, the mycobiome composition of the woody tissue in proximity to leaves manifesting ‘tiger stripes’ symptoms of esca, as well as in leaf-symptomatic canes, was highly similar to that of plants not exhibiting any leaf symptomatology. This observation supports the current understanding that leaf symptoms are not directly linked with the fungal communities in the wood. This work builds to the understanding of the microbial ecology of the grapevines wood, offering insights and a critical view on the current knowledge of the etiology of esca.

Soil DNA extraction encounters numerous challenges that can affect both yield and purity of the recovered DNA. Clay particles lead to reduced DNA extraction efficiency, and PCR inhibitors from the soil matrix can negatively affect downstream analyses when applying DNA sequencing. Further, these effects impede molecular analysis of bacterial community compositions in lower biomass samples, as often observed in deeper soil layers. Many studies avoid these complications by using indirect DNA extraction with prior separation of the cells from the matrix, but such methods introduce other biases that influence the resulting microbial community composition. To address these issues, a direct DNA extraction method was applied in combination with the use of a commercial product, the G2 DNA/RNA Enhancer, marketed as being capable of improving the amount of DNA recovered after the lysis step. The results showed that application of G2 increased DNA yields from the studied clayey soils from layers from 1.00 to 2.20 m. Importantly, the use of G2 did not introduce bias, as it did not result in any significant differences in the biodiversity of the bacterial community measured in terms of alpha and beta diversity and taxonomical composition. Finally, this study considered a set of customised lysing tubes for evaluating possible influences on the DNA yield. Tubes customization included different bead sizes and amounts, along with lysing tubes coming from two suppliers. Results showed that the lysing tubes with mixed beads allowed greater DNA recovery compared to the use of either 0.1 or 1.4 mm beads, irrespective of the tube supplier. These outcomes may help to improve commercial products in DNA/RNA extraction kits, besides raising awareness about the optimal choice of additives, offering opportunities for acquiring a better understanding of topics such as vertical microbial characterisation and environmental DNA recovery in low biomass samples.