Cynara cardunculus L. var. scolymus L. Landrace "Carciofo Ortano" as a Source of Bioactive Compounds.

The preservation of agricultural biodiversity and socioeconomic development are relevant both to enhance domestic production and to support innovation. In the search for new biomolecules, we have focused on the "Carciofo Ortano" landrace, growth in the northern part of the Lazio region. Artichoke cultivation generates substantial by-products, including leaves, stems, and roots, which could serve as valuable sources of biomolecules and prebiotic dietary fiber. To valorize the leaf waste of the "Carciofo Ortano" landrace, a multidisciplinary approach was applied. Chemical analysis using HPLC-DAD identified mono-O- and di-O-caffeoylquinic acids and the sesquiterpene cynaropicrin in all artichoke leaf extracts. SPME-GC/MS analyses detected aliphatic alcohols in the fresh leaf samples. Antiproliferative and cytotoxic studies on cancer (SH-SY5Y, MCF-7, MDA) and normal (MCF-10A) human cell lines revealed that leaf extracts induced a selective dose and time-dependent biological effect. While showing slight activity against environmental bacterial strains, artichoke leaf extracts exhibited significant antifungal activity against the phytopathogenic fungus Alternaria alternata. Overall, the results highlight the potential of "Carciofo Ortano" cultivation by-products as a rich source of biomolecules with versatile applications in humans, animals, and the environment.

Lignin Nanoparticles with Entrapped Thymus spp. Essential Oils for the Control of Wood-Rot Fungi.

After decades of utilization of fossil-based and environmentally hazardous compounds for wood preservation against fungal attack, there is a strong need to substitute those compounds with bio-based bioactive solutions, such as essential oils. In this work, lignin nanoparticles containing four essential oils from thyme species (Thymus capitatus, Coridothymus capitatus, T. vulgaris, and T. vulgaris Demeter) were applied as biocides in in vitro experiments to test their anti-fungal effect against two white-rot fungi (Trametes versicolor and Pleurotus ostreatus) and two brown-rot fungi (Poria monticola and Gloeophyllum trabeum). Entrapment of essential oils provided a delayed release over a time frame of 7 days from the lignin carrier matrix and resulted in lower minimum inhibitory concentrations of the essential oils against the brown-rot fungi (0.30-0.60 mg/mL), while for the white-rot fungi, identical concentrations were determined compared with free essential oils (0.05-0.30 mg/mL). Fourier Transform infrared (FTIR) spectroscopy was used to assess the fungal cell wall changes in the presence of essential oils in the growth medium. The results regarding brown-rot fungi present a promising approach for a more effective and sustainable utilization of essential oils against this class of wood-rot fungi. In the case of white-rot fungi, lignin nanoparticles, as essential oils delivery vehicles, still need optimization in their efficacy.

A Rapid Method for Extracting High-Quality DNA from Soils.

Metagenomics offers the possibility to study the microbial community of soils at large scale, allowing the characterization also of unculturable microorganisms. Availability of high-quality DNA is a prerequisite of this approach. However, since soils have highly heterogeneous physicochemical properties, several parameters need to be considered for the development of the best molecular practices. A method for isolation of high-molecular-weight and good-quality metagenomic DNA from different soil samples is described here. The protocol combines physical and chemical strategies to ensure efficient cell lysis and precipitation of humic impurities-free DNA suitable for downstream processing for metagenomics study.

Molecular markers dataset to assess the genetic diversity of oriental plane trees from historical sites in Lazio (central Italy).

The dataset presented in this article is related to the research paper titled "Dimensional and genetic characterization of the last oriental plane trees (Platanus orientalis L.) of historical sites in Lazio (central Italy)" (Ciaffi et al., 2022). Indeed, the molecular analyses reported in that article consisted in a comparison of Italian veteran plane trees with 12 certified accessions of P. orientalis, P. occidentalis and their hybrids P. acerifolia (4 individuals per species). First, LEAFY gene analyses allowed identifying 32 P. orientalis and two P. acerifolia in four sites of the province of Rome, confirming also that the two representative trees from the two gardens of the province of Viterbo belong to P. orientalis. Second, the use of Simple Sequence Repeat (SSR) and Inter Simple Sequence Repeat (ISSR) molecular markers provided useful information regarding the genetic relationships within and among all the historical sites. Owing to the use of SSR and ISSR molecular markers, a dataset of parameters related to the genetic diversity of the same plant material was obtained and presented in this article. For SSR markers, seven loci previously developed for P. occidentalis (Lang, 2010) and two specifically developed for P. orientalis (Rinaldi et al., 2019) were employed. For ISSR markers, DNA samples were amplified with eight primers before used for the determination of genetic stability of micro-propagated plantlets of P. acerifolia (Huang et al., 2009) and for the genetic characterization of plane trees within the formal gardens of Villa Lante of Bagnaia and Palazzo Farnese (Viterbo, Italy) (Ciaffi et al., 2018). To the best of our knowledge, this is the first report on the genetic diversity data for veteran oriental plane trees within heritage sites, which will offer helpful information for their management and conservation.

Seed quantity affects the fungal community composition detected using metabarcoding.

Pest introductions via trade in tree seed may result from a lack of adequate survey and validation protocols. Developing better diagnostic protocols to identify potentially harmful pests and pathogens in forest tree seed is of critical importance. High-throughput sequencing-based barcoding and metabarcoding provide effective tools for screening potentially harmful organisms in various plant materials, including seeds. However, the sample size needed to detect the total microorganism diversity of a community is a major challenge in microbiome studies. In this work, we examined how increasing sample size (ranging between 100 and 1000 seeds) influences diversity of fungal communities detected by high throughput sequencing in Pinus sylvestris seeds. Our results showed that as sample size increased, fungal alpha diversity also increased. Beta-diversity estimators detected significant differences between the mycobiota from different samples. However, taxonomic and functional diversity were not correlated with sample size. In addition, we found that increasing the number of PCR replicates resulted in a higher abundance of plant pathogens. We concluded that for the purpose of screening for potentially harmful pathogens using HTS, greater efforts should be made to increase the sample size and replicates when testing tree seed.

The Never-Ending Presence of Phytophthora Species in Italian Nurseries.

Plant trade coupled with climate change has led to the increased spread of well-known and new Phytophthora species, a group of fungus-like organisms placed in the Kingdom Chromista. Their presence in plant nurseries is of particular concern because they are responsible for many plant diseases, with high environmental, economic and social impacts. This paper offers a brief overview of the current status of Phytophthora species in European plant nurseries. Focus was placed on Italian sites. Despite the increasing awareness of the risk of Phytophthora spread and the management strategies applied for controlling it, the complexity of the Phytophthora community in the horticulture industry is increasing over time. Since the survey carried out by Jung et al. (2016), new Phytophthora taxa and Phytophthora-host associations were identified. Phytophthorahydropathica, P. crassamura, P. pseudocryptogea and P. meadii were reported for the first time in European plant nurseries, while P. pistaciae, P. mediterranea and P. heterospora were isolated from Italian ornamental nurseries. Knowledge of Phytophthora diversity in plant nurseries and the potential damage caused by them will help to contribute to the development of early detection methods and sustainable management strategies to control Phytophthora spread in the future.

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods.

Nuts of the sweet chestnut (Castanea sativa) are a widely appreciated traditional food in Europe. In recent years producers and consumers reported a drop of nut quality due to the presence of rot diseases caused by Gnomoniopsis smithogilvyi. Early detection of this pathogen is fundamental to the economic viability of the chestnut industry. In the present study, we developed three molecular methods based on real-time portable LAMP, visual LAMP and qPCR assays for G. smithogilvyi. The molecular assays were specific for G. smithogilvyi and did not amplify the other 11 Gnomoniopsis species and 11 other fungal species commonly associated with chestnuts. The detection limit of both the qPCR and real-time portable LAMP (P-LAMP) assays was 0.128 pg/µL, while the visual LAMP (V-LAMP) assay enabled the detection up to 0.64 pg/µL. By using these newly developed molecular tools, the pathogen was detected in symptomatic and asymptomatic nuts, but not in leaves. The reliability of these molecular methods, including the P-LAMP assay, was particularly useful in detecting G. smithogilvyi of harvested nuts in field, even in the absence of rot symptoms.

Effect of gaseous ozone treatment on the aroma and clove rot by Fusarium proliferatum during garlic postharvest storage.

It is known that garlic bulbs preserved with traditional methods undergo considerable losses, ranging from 25 to 40%. A frequent cause of these losses is associated with the development of pathogenic fungi, such as those of the genus Fusarium. The effect of ozone on post-harvest garlic bulbs was evaluated. Garlic cloves inoculated with Fusarium proliferatum F21 and F22 strains, were exposed to a continuous gaseous ozone flow (2.14 µg m-3), during 4 days, 20 h a day. After ozone-treatment, the garlic samples were moved at 22 °C to mimic retail conditions (shelf life). The changes in several quality parameters such as fungal decay and aroma were evaluated on garlic samples, as whole bulbs, cloves with and without tunic, through a sensorial descriptive test, SPME analysis in GC/MS and microbiological approaches. The data collected showed that ozone treatment did not affect the aromatic profile of garlic. A significant detrimental effect of ozone treatment on garlic decay was observed. Our results encourage the use of gaseous ozone treatment for containing garlic fungal decay during its storage.

Ozone gas as a storage treatment to control Gnomoniopsis castanea, preserving chestnut quality.

BACKGROUND: Chestnuts are gluten-free, low-fat, cholesterol-free products. Postharvest decay reduces chestnut shelf life and can cause severe economic losses. In this study we investigated the effect of ozone (O3 ) gaseous treatment on chestnut rot caused by Gnomoniopsis castanea and the quality parameters of chestnuts. RESULTS: The results showed that ozone treatment (150 ppb during the day, and 300 ppb during the night) reduced the decay of chestnuts and had a fungistatic effect on isolates of G. castanea. The exposure of chestnuts to ozone did not alter weight losses, sugar content and titratable acidity. The concentration of total phenolics decreased during the storage period, both for treated and untreated nuts. However, after 150 days of treatment the polyphenol content of the chestnuts exposed to ozone was significantly higher than in control nuts. CONCLUSIONS: Our results suggested that ozone is an appropriate and economical tool to maximize the quality of chestnut shelf life, enabling it to be stored for long periods. © 2019 Society of Chemical Industry.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

Commodity risk assessment of black pine (Pinus thunbergii Parl.) bonsai from Japan.

The EFSA Panel on Plant health was requested to deliver a scientific opinion on how far the existing requirements for the bonsai pine species subject to derogation in Commission Decision 2002/887/EC would cover all plant health risks from black pine (Pinus thunbergii Parl.) bonsai (the commodity defined in the EU legislation as naturally or artificially dwarfed plants) imported from Japan, taking into account the available scientific information, including the technical information provided by Japan. The relevance of an EU-regulated pest for this opinion was based on: (a) evidence of the presence of the pest in Japan; (b) evidence that P. thunbergii is a host of the pest and (c) evidence that the pest can be associated with the commodity. Sixteen pests that fulfilled all three criteria were selected for further evaluation. The relevance of other pests present in Japan (not regulated in the EU) for this opinion was based on (i) evidence of the absence of the pest in the EU; (ii) evidence that P. thunbergii is a host of the pest; (iii) evidence that the pest can be associated with the commodity and (iv) evidence that the pest may have an impact in the EU. Three pests fulfilled all four criteria and were selected for further evaluation (Crisicoccus pini, Sirex nitobei and Urocerus japonicus). For the selected 19 pests, the risk mitigation measures proposed in the technical dossier were evaluated. Limiting factors on the effectiveness of the measures were documented. For each of the 19 pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including any uncertainties. For all evaluated pests, the median likelihood of the pest freedom is 99.5% or higher and within the 90% uncertainty range it is 99% or higher.

Data set useful for the micropropagation and the assessment of post-vitro genetic fidelity of veteran trees of P. orientalis L.

The data presented in this article are related to the research article titled "Conservation of veteran trees within historical gardens (COVE): a case study applied to Platanus orientalis L. in central Italy" (Ciaffi et al., 2018) [1]. This article reports data on the composition of the substrates used in the different steps of Platanus orientalis micropropagation: establishment of in vitro culture, multiplication, elongation and rooting. Moreover, molecular data were used to assess the genetic fidelity of the micropropagated plants respect mother plants after three year of in vitro cultivation. Fifteen ISSR markers, used in "Determination of genetic stability of long-term micropropagated plantlets of Platanus acerifolia using ISSR markers" (Huang et al., 2009) [2] on P. acerifolia and in "Variant identification in Platanus occidentalis L. using SNP and ISSR markers" (Lee et al., 2012) [3] on P. occidentalis, were successfully employed in the present study on P. orientalis. The plant material was collected from the Renaissance garden of Villa Lante in Viterbo, Italy. It is envisioned that these data set will provide useful information for the conservation of veteran oriental plane trees of historical gardens.

The sentinel tree nursery as an early warning system for pathway risk assessment: Fungal pathogens associated with Chinese woody plants commonly shipped to Europe.

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

Pest risk assessment of Atropellis spp. for the EU territory.

Following a request from the European Commission, the EFSA Plant Health (PLH) Panel performed a risk assessment for Atropellis spp. in the EU focusing on the risk of entry, the host range and the potential impacts. Atropellis is a fungal pathogen of several Pinus spp. in North America. The pathogen has not been reported from Europe and is a quarantine pest regulated in Annex IIAI of Council Directive 2000/29/EC on plants (other than fruit and seeds), isolated bark and wood of Pinus. The main pathways of entry considered were Pinus plants, wood and isolated bark. Given the ban of importing Pinus plants from outside Europe into the EU and the lack of information on EU imports of isolated Pinus bark, only the wood pathway was assessed quantitatively. The conclusion of the assessment of entry for scenario A0 (current regulatory situation) is that the risk of entry of Atropellis spp. is close to zero. This conclusion is expected to apply also in the case of removing the specific Atropellis regulations, because of the remaining generic Pinus requirements, as well as in a scenario with additional risk reduction options. The uncertainty associated with this assessment is relatively limited, given that all the quartiles of the estimated distribution of the number of potential founder populations are close to zero. For the North American Pinus spp. known to be susceptible and widely planted in the EU (mainly P. contorta and P. strobus), the damage observed in North America (loss of wood quality, stem deformations, mortality in young stands, environmental consequences) is expected in the EU to a similar (or higher) degree, should the pathogen be introduced. Similar impacts are expected on the European Pinus spp. known to be host of Atropellis spp. These include widespread and locally abundant species such as P. nigra, P. sylvestris and P. pinaster. There are, however, large uncertainties associated with this impact assessment due to the unknown susceptibility of several other Pinus spp. present in Europe. There is a need for research on the susceptibility to Atropellis spp. of those European Pinus spp.

Morphological and molecular characterisation of Geosmithia species on European elms.

Species of the genus Geosmithia are associated with insect species, mainly bark beetles. On Ulmus spp., the same beetles are also vectors of Ophiostoma ulmi s.l., the agent of Dutch elm disease (DED), a worldwide elm disease. Aim of this paper is to characterise Geosmithia species associated with elms and/or elm beetles in Europe. Seventy-two strains representative of all morphological taxonomic units were used to build a phylogenetic tree based on ITS, ß-tubulin and elongation factor 1-α gene regions. On the basis of molecular and morpho-physiological traits, seven taxonomic entities were identified. In addition to the species previously known our results assigned strains previously identified as Geosmithia pallida to two separate taxa: Geosmithia sp. 2 and Geosmithia sp. 5. Two new species, Geosmithia omnicola and Geosmithia ulmacea, are described. Two strains were assigned to the partially described species Geosmithia sp. 20. Geosmithia species living on Ulmus do not discriminate between elm species, but between different environments. The association between Ulmus and Geosmithia is common, stable, and seems to be related to specific vectors. The relationship between Geosmithia and Ophiostoma would deserve further investigation, as these fungi share the same vectors and habitat for a significant part of their life cycles.

Ozone fumigation for safety and quality of wine grapes in postharvest dehydration.

This paper proposes postharvest ozone fumigation (as a method) to control microorganisms and evaluate the effect on polyphenols, anthocyanins, carotenoids and cell wall enzymes during the grape dehydration for wine production. Pignola grapes were ozone-treated (1.5 g/h) for 18 h (A=shock treatment), then dehydrated or ozone-treated (1.5 g/h) for 18 h and at 0.5 g/h for 4 h each day (B=long-term treatment) during dehydration. Treatment and dehydration were performed at 10 °C. No significant difference was found for total carotenoid, total phenolic and total anthocyanin contents after 18 h of O3 treatment. A significant decrease in phenolic and anthocyanin contents occurred during treatment B. Also carotenoids were affected by B ozone treatment. Pectin methylesterase (PME) and polygalacturonase (PG) activities were higher in A-treated grapes during dehydration. Finally, ozone reduced fungi and yeasts by 50%. Shock ozone fumigation (A treatment) before dehydration can be used to reduce the microbial count during dehydration without affecting polyphenol and carotenoid contents.

Pyrosequencing of environmental soil samples reveals biodiversity of the Phytophthora resident community in chestnut forests.

Pyrosequencing analysis was performed on soils from Italian chestnut groves to evaluate the diversity of the resident Phytophthora community. Sequences analysed with a custom database discriminated 15 pathogenic Phytophthoras including species common to chestnut soils, while a total of nine species were detected with baiting. The two sites studied differed in Phytophthora diversity and the presence of specific taxa responded to specific ecological traits of the sites. Furthermore, some species not previously recorded were represented by a discrete number of reads; among these species, Phytophthora ramorum was detected at both sites. Pyrosequencing was demonstrated to be a very sensitive technique to describe the Phytophthora community in soil and was able to detect species not easy to be isolated from soil with standard baiting techniques. In particular, pyrosequencing is an highly efficient tool for investigating the colonization of new environments by alien species, and for ecological and adaptive studies coupled with biological detection methods. This study represents the first application of pyrosequencing for describing Phytophthoras in environmental soil samples.

Four phenotypically and phylogenetically distinct lineages in Phytophthora lateralis.

Until recently Phytophthora lateralis was known only as the cause of dieback and mortality of Chamaecyparis lawsoniana in its native range in the Pacific Northwest (PNW). Since the 1990s however disease outbreaks have occurred increasingly on ornamental C. lawsoniana in Europe; and in 2007 the pathogen was discovered in soil around old growth Chamaecyparis obtusa in Taiwan, where it may be endemic. When the phenotypes of over 150 isolates of P. lateralis from Taiwan, across the PNW (British Columbia to California) and from France, the Netherlands and the UK were compared three growth rate groups were resolved: one slow growing from Taiwan, one fast growing from the PNW and Europe, and one of intermediate growth from a small area of the UK. Within these growth groups distinct subtypes were identified based on colony patterns and spore metrics and further discriminated in a multivariate analysis. The assumption that the three main growth groups represented phylogenetic units was tested by comparative sequencing of two mitochondrial and three nuclear genes. This assumption was confirmed. In addition two phenotype clusters within the Taiwan growth group were also shown to be phylogenetically distinct. These four phenotypically and genotypically unique populations are informally designated as the PNW lineage, the UK lineage, the Taiwan J lineage, and the Taiwan K lineage. Their characteristics and distribution are described and their evolution, taxonomic, and plant health significance is discussed.

Phytophthora himalsilva sp. nov. an unusually phenotypically variable species from a remote forest in Nepal.

The Himalaya have received little investigation for Phytophthora species. In a remote forest in Western Nepal ten isolates of an unknown Phytophthora were recovered from the rhizosphere of Quercus, Castanopsis, Carpinus and Cupressus spp. The Phytophthora, formally named here as a P. himalsilva sp. nov., is homothallic with either amphigynous or paragynous antheridia and papillate, highly variable sporangia which may also be facultatively caducous. Based on ITS, ß-tubulin, and cox I sequences Phytophthora himalsilva falls within Phytophthora Clade 2c together with Phytophthora citrophthora, Phytophthora meadii, Phytophthora colocasiae, and Phytophthora botryosa. It is suggested that Clade 2c has radiated within Asia. Molecular and sporangial characters indicate that P. himalsilva and P. citrophthora may share a recent common ancestor although they have diverged in their breeding systems. Although highly local the P. himalsilva isolates exhibited significant variation in growth rates and optimum temperatures for growth. This may reflect adaptation to different niches within a heterogeneous sub-tropical to temperate forest environment. Their cox I polymorphisms were also rather variable, including possible clustering for subsite. The occurrence of a previously unknown Phytophthora in a remote forest in Nepal highlights once again the plant health risk associated with moving rooted plants and soil between different bio-geographical regions of the world and the need for rapid pathological screening of potential risk organisms.

Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum.

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.