Milestones in the development and applications of plant virus vector as gene silencing platforms.

One of the main post-genomics challenges facing scientists remains the identification of gene function in a large number of plant species. Plant viruses offer great potential in linking genes to phenotypes through epigenetic expression or knockdown of selected genes. The past decade has seen the development and ever increasing applications of a gene knockdown technique termed virus-induced gene silencing (VIGS). VIGS recapitulates an RNA-mediated antiviral defense mechanism, mediating a homology-based post-transcriptional degradation of selected plant RNAs, leading to a loss-of-function phenotype. Due to its rapidity and increasing number of virus vectors developed as gene silencing platforms, VIGS has become a powerful technology to determine the function of genes in an increasing number of crop species, where the routinely available transgenesis or mutagenesis approaches are often not amenable to large genomes and complex genetic backgrounds.

Commodity risk assessment of Petunia spp. and Calibrachoa spp. unrooted cuttings from Guatemala.

The European Commission requested the EFSA Panel on Plant Health to evaluate the probability of entry of pests (likelihood of pest freedom at entry), including both, regulated and non-regulated pests, associated with unrooted cuttings of the genera Petunia and Calibrachoa produced under physical isolation in Guatemala. The relevance of any pest for this opinion was based on evidence following defined criteria, based on the methodology used for high-risk plants adapted for the specificity of this assessment. Nineteen EU regulated pests (Bemisia tabaci, pepper golden mosaic virus, pepper huasteco yellow vein virus, tomato severe leaf curl virus, tomato yellow leaf curl virus, tomato spotted wilt virus, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, Bactericera cockerelli, Eotetranichus lewisi, Epitrix subcrinita, Epitrix cucumeris, Helicoverpa zea, Chloridea virescens, Spodoptera ornithogalli, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Xanthomonas vesicatoria) and one EU non-regulated (Phenacoccus solenopsis) pest fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Guatemala were evaluated taking into account the possible limiting factors, and an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The limited and partially conflicting information provided in the dossier contributes to the wide estimates of pest freedom. The estimated degree of pest freedom varies among the pests evaluated, with Ralstonia spp. (R. solanacearum and R. pseudosolanacearum) being the pest most frequently expected on the imported cuttings. The expert knowledge elicitation indicated, with 95% certainty, that between 9916 and 10,000 bags containing unrooted cuttings per 10,000 would be free of Ralstonia spp.

Commodity risk assessment of Petunia spp. and Calibrachoa spp. unrooted cuttings from Kenya.

The European Commission requested the EFSA Panel on Plant Health to evaluate the probability of entry of pests (likelihood of pest freedom at entry), including both regulated and non-regulated pests, associated with unrooted cuttings of the genera Petunia and Calibrachoa produced under physical isolation in Kenya. The relevance of any pest for this opinion was based on evidence following defined criteria, based on the methodology used for High-Risk Plants adapted for the specificity of this assessment. Fourteen EU-regulated pests (Bemisia tabaci, cowpea mild mottle virus, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, potato leafroll virus, potato spindle tuber viroid, Ralstonia pseudosolanacearum, R. solanacearum, Scirtothrips dorsalis, tomato mild mottle virus, tomato spotted wilt virus, tomato yellow leaf curl virus and Xanthomonas vesicatoria) and six EU non-regulated pests (Aleurodicus dispersus, pepper veinal mottle virus, Nipaecoccus viridis, Phenacoccus solenopsis, Tetranychus neocaledonicus and tomato yellow ring virus) fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Kenya were evaluated, taking into account the possible limiting factors. Additionally, an expert judgement is given on the likelihood of pest freedom, taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The estimated degree of pest freedom varies among the pests evaluated, with T. neocaledonicus being the pest most frequently expected on the imported cuttings. The Expert Knowledge Elicitation indicated, with 95% certainty, that between 9942 and 10,000 bags containing unrooted cuttings of Petunia spp. and Calibrachoa spp. per 10,000 would be free of T. neocaledonicus.

The TGB1 movement protein of Potato virus X reorganizes actin and endomembranes into the X-body, a viral replication factory.

Potato virus X (PVX) requires three virally encoded proteins, the triple gene block (TGB), for movement between cells. TGB1 is a multifunctional protein that suppresses host gene silencing and moves from cell to cell through plasmodesmata, while TGB2 and TGB3 are membrane-spanning proteins associated with endoplasmic reticulum-derived granular vesicles. Here, we show that TGB1 organizes the PVX "X-body," a virally induced inclusion structure, by remodeling host actin and endomembranes (endoplasmic reticulum and Golgi). Within the X-body, TGB1 forms helically arranged aggregates surrounded by a reservoir of the recruited host endomembranes. The TGB2/3 proteins reside in granular vesicles within this reservoir, in the same region as nonencapsidated viral RNA, while encapsidated virions accumulate at the outer (cytoplasmic) face of the X-body, which comprises a highly organized virus "factory." TGB1 is both necessary and sufficient to remodel host actin and endomembranes and to recruit TGB2/3 to the X-body, thus emerging as the central orchestrator of the X-body. Our results indicate that the actin/endomembrane-reorganizing properties of TGB1 function to compartmentalize the viral gene products of PVX infection.

HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis.

Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.

Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3'UTRs, whereas a distinct pathway degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3'UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.

The 5' cap of tobacco mosaic virus (TMV) is required for virion attachment to the actin/endoplasmic reticulum network during early infection.

Almost nothing is known of the earliest stages of plant virus infections. To address this, we microinjected Cy3 (UTP)-labelled tobacco mosaic virus (TMV) into living tobacco trichome cells. The Cy3-virions were infectious, and the viral genome trafficked from cell to cell. However, neither the fluorescent vRNA pool nor the co-injected green fluorescent protein (GFP) left the injected trichome, indicating that the synthesis of (unlabelled) progeny viral (v)RNA is required to initiate cell-to-cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3-vRNA formed granules that became anchored to the motile cortical actin/endoplasmic reticulum (ER) network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actin-dependent RNA movement. The 5' methylguanosine cap was shown to be required for vRNA anchoring to the actin/ER. TMV vRNA lacking the 5' cap failed to form granules and was degraded in the cytoplasm. Removal of the 3' untranslated region or replicase both inhibited replication but did not prevent granule formation and movement. Dual-labelled TMV virions in which the vRNA and the coat protein were highlighted with different fluorophores showed that both fluorescent signals were initially located on the same ER-bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome.

ADS1 encodes a MATE-transporter that negatively regulates plant disease resistance.

Multidrug and toxic compound extrusion (MATE) proteins comprise the most recently identified family of multidrug transporters. In plants, the numbers of MATE proteins has undergone a remarkable expansion, underscoring the importance of these transporters within this kingdom. Here, we describe the identification and characterization of Activated Disease Susceptibility 1 (ADS1) which encodes a putative MATE transport protein. An activation tagging screen uncovered the ads1-Dominant (ads1-D) mutant, which was subsequently characterized by molecular, genetic and biochemical approaches. The ads1-D mutant was compromised in both basal and nonhost resistance against microbial pathogens. Further, plant defence responses conferred by RPS4 were also disabled in ads1-D plants. By contrast, depletion of ADS1 transcripts by RNA-interference (RNAi) promoted basal disease resistance. Unexpectedly, ads1-D plants were found to constitutively accumulate reactive oxygen intermediates (ROIs). However, analysis of ads1-D Arabidopsis thaliana respiratory burst oxidase (atrboh) double and triple mutants indicated that an increase in ROIs did not impact ads1-D-mediated disease susceptibility. Our findings imply that ADS1 negatively regulates the accumulation of the plant immune activator salicylic acid (SA) and cognate Pathogenesis-Related 1 (PR1) gene expression. Collectively, these data highlight an important role for MATE proteins in the establishment of plant disease resistance.

Live-cell imaging of viral RNA genomes using a Pumilio-based reporter.

We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.

Identification and characterization of barley RNA-directed RNA polymerases.

RNA-directed RNA polymerases (RDRs) play crucial roles in the RNA silencing response of plants by enhancing and maintaining silencing signals. At least two members of the RDR group, namely RDR1 and RDR6, are implicated in defence against plant viruses. RDRs have so far only been characterized in dicot species. In this report, we identified and characterized HvRDR1, HvRDR2 and HvRDR6 genes in the monocot plant barley (Hordeum vulgare). We analysed their expression under various biotic and abiotic stresses including fungal and viral infections, salicylic acid treatment as well as during plant development. The different classes and subclasses of barley RDRs displayed contrasting expression patterns during pathogen challenge and development suggesting their involvement in specific regulatory pathways. Their response to heat and salicylic acid treatment suggests a conserved pattern of expression of these genes between monocot and dicot plant species. The existence of two HvRDR1 and two HvRDR6 genes suggests an evolutionary selection for specialization in response to biotic and abiotic stresses after gene duplication.

BAX INHIBITOR-1 is required for full susceptibility of barley to powdery mildew.

BAX INHIBITOR-1 (BI-1) is one of the few proteins known to have cross-kingdom conserved functions in negative control of programmed cell death. Additionally, barley BI-1 (HvBI-1) suppresses defense responses and basal resistance to the powdery mildew fungus Blumeria graminis f. sp. hordei and enhances resistance to cell death-provoking fungi when overexpressed in barley. Downregulation of HvBI-1 by transient-induced gene silencing or virus-induced gene silencing limited susceptibility to B. graminis f. sp. hordei, suggesting that HvBI-1 is a susceptibility factor toward powdery mildew. Transient silencing of BI-1 did not limit supersusceptibility induced by overexpression of MLO. Transgenic barley plants harboring an HvBI-1 RNA interference (RNAi) construct displayed lower levels of HvBI-1 transcripts and were less susceptible to powdery mildew than wild-type plants. At the cellular level, HvBI-1 RNAi plants had enhanced resistance to penetration by B. graminis f. sp. hordei. These data support a function of BI-1 in modulating cell-wall-associated defense and in establishing full compatibility of B. graminis f. sp. hordei with barley.

The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence.

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.

Combining bacteriophage engineering and linear dichroism spectroscopy to produce a DNA hybridisation assay.

Nucleic acid detection is an important part of our bio-detection arsenal, with the COVID-19 pandemic clearly demonstrating the importance to healthcare of rapid and efficient detection of specific pathogenic sequences. As part of the drive to establish new DNA detection methodologies and signal read-outs, here we show how linear dichroism (LD) spectroscopy can be used to produce a rapid and modular detection system for detecting quantities of DNA from both bacterial and viral pathogens. The LD sensing method exploits changes in fluid alignment of bionanoparticles (bacteriophage M13) engineered with DNA stands covalently attached to their surfaces, with the read-out signal induced by the formation of complementary duplexes between DNA targets and two M13 bionanoparticles. This new sandwich assay can detect pathogenic material down to picomolar levels in under 1 minute without amplification, as demonstrated by the successful sensing of DNA sequences from a plant virus (Potato virus Y) and an ampicillin resistance gene, ampR.

List of non-EU viruses and viroids infecting potato (Solanum tuberosum) and other tuber-forming Solanum species.

The European Commission requested a pest categorisation of the non-EU viruses and viroids of potato (hereafter referred to as viruses). As a first step, a systematic literature and database search was carried out to identify the viruses reported to naturally infect Solanum tuberosum and other tuber-forming Solanum spp (hereafter referred to as potato). Based on the global distribution and on the prevalence inside the European Union (EU), the Panel identified 40 non-EU viruses known to occur only outside the EU or with only a limited presence in the EU (reported in only one or few Member States (MSs) and/or with restricted distribution, outbreaks). Twenty-seven viruses were identified as having a significant presence in the EU (known to occur in several MSs, frequently reported in the EU, widespread in several MSs) or reported only from the EU so far, and will be excluded from further categorisation in the frame of the present mandate. Five viruses remained with an undetermined standing because the available information did not allow their allocation to one of the above groups. The viruses considered non-EU and those with undetermined standing will be further categorised if not addressed by EFSA in previous scientific opinions. Seven viruses for which non-European isolates are specifically regulated in Annex I of directive 2000/29/EC will be categorised separately. The main knowledge gaps and uncertainties of this grouping concern the natural host status of potato, the taxonomy, and/or information on the geographical distribution and prevalence of some of the analysed viruses.

Pest categorisation of non-EU viruses and viroids of potato.

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of those viruses and viroids (hereafter referred to as viruses) of Solanum tuberosum and other tuber-forming Solanum spp. (hereafter referred to as potato) which are considered to be either non-EU or of undetermined standing based on a previous EFSA opinion. These viruses belong to different families and genera and either have an established identity or produce consistent symptoms. Plants for planting is the main pathway for entry for all categorised viruses as they can all be transmitted by vegetative propagation. Several categorised viruses have a relatively wide host range and/or are vector-transmitted, increasing the potential for entry. The information currently available on geographical distribution, biology, epidemiology, impact and potential entry pathways has been evaluated with regard to the criteria to qualify as potential Union quarantine pest or as Union regulated non-quarantine pest (RNQP). Since this opinion addresses specifically the non-EU potato viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non-quarantine pests. The following viruses meet the criteria to qualify as potential Union quarantine pest: APLV, APMMV, APMoV, ChiLCV, CYSDV, PAMV, PBRSV, PVH, PVP, PVT, PYDV, PYMV, PYV, PYVV, RCVMV, SALCV, SB26/29, ToCV, ToLCNDV, ToMHaV, ToMoTV, ToSRV and ToYVSV. With the exception of the criterion regarding the potential for consequences in the EU territory, for which the Panel is unable to conclude because of lack of information, AVB, CPSbV, PaLCrV, PapMV, PVB, PVU, SB41 and TVBMV meet all the other criteria to qualify as potential Union quarantine pest. PotLV and WPMV do not qualify as potential Union quarantine pest, since they are not reported to have any impact. For most of the categorised viruses, the conclusions of the Panel have inherent uncertainties, due to the lack of quantitative data on their impact and/or absence or limited availability of information on the biology, epidemiology and geographical distribution.

Pest categorisation of potato virus M (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus M (PVM). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact compared to the current situation in the EU and availability of control measures of non-EU isolates of PVM has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PVM are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. Populations of PVM can be subdivided into two strains: the ordinary strain (PVM-O) is present in the EU, while the divergent strain (PVM-D) is absent from the EU or considered to have at most a limited distribution in the EU. Non-EU isolates of PVM-O are not expected to have an additional impact in the EU compared to EU isolates and therefore do not meet the corresponding criterion to qualify as a potential Union quarantine pest. The Panel is unable to conclude on the potential impact of non-EU PVM-D isolates in the EU territory, but PVM-D isolates meet all the other criteria to qualify as a potential Union quarantine pest.

Pest categorisation of potato virus S (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus S (PVS). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact compared to the current situation in the EU, and availability of control measures of non-EU isolates of PVS has been evaluated with regard to the criteria to qualify as potential Union quarantine pest. Because non-EU isolates of PVS are absent from the EU, they do not meet one of the requirements to be regulated as an RNQP (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. Populations of PVS can be subdivided into two strains: the ordinary strain (PVS-O) with a worldwide distribution (including the EU), and the Andean strain (PVS-A) which is absent from the EU or considered to have at most a limited distribution in the EU. Two additional divergent isolates (PVS-A/PVS-O recombinants and PVS-arracacha) have also been categorised. Non-EU isolates of PVS-A are expected to have an additional impact as compared to the PVS isolates currently present in the EU, and therefore meet all the criteria to qualify as potential Union quarantine pests; the magnitude of the additional impact is, however, unknown. Non-EU isolates of PVS-A/PVS-O recombinants and of PVS-arracacha also meet these criteria, with the exception of the criterion regarding the potential additional consequences in the EU territory for which the Panel was unable to conclude. Non-EU PVS-O isolates are not expected to have an additional impact in the EU as compared to EU isolates and therefore do not meet the corresponding criterion.

Pest categorisation of potato virus A (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus A (PVA). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact over the current situation and availability of control measures of non-EU isolates of PVA has been evaluated with regard to the criteria to qualify as potential Union quarantine pest. Because non-EU isolates of PVA are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. This categorisation was performed considering two groups of isolates: those reported in Solanum betaceum (PVA-TamMV, not reported from the EU) and all other isolates (hereafter referred to as PVA, worldwide distribution). Non-EU isolates of PVA and of PVA-TamMV do not meet one of the criteria evaluated by EFSA to be regarded as a potential Union quarantine pest, since they are not expected to have an additional impact in the EU.

Pest categorisation of potato virus V (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus V (PVV). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact and availability of control measures of non-EU isolates of PVV has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PVV are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. This categorisation was performed considering two lineages, PVV-I (present in and outside the EU) and PVV-II (not reported in the EU), and isolate PVV-PA4 (unknown distribution). Non-EU isolates of PVV-I and PVV-PA4 do not meet one of the criteria evaluated by EFSA to be regarded as a potential Union quarantine pest, since they are not expected to have an additional impact in the EU. With the exception of the criterion regarding the potential consequences in the EU territory, for which the Panel is unable to conclude, non-EU isolates of PVV-II meet all the other criteria to qualify as a potential Union quarantine pest.

Pest categorisation of potato virus X (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus X (PVX). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact and availability of control measures of non-EU isolates of PVX has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PVX are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. On the basis of their ability to overcome potato resistance genes, PVX isolates can be divided into several pathotypes. PVX isolates that are not able to overcome resistance genes and PVX isolates that are able to overcome the Nb and/or Nx resistance genes are already present in the EU. Isolates able to overcome the Rx resistance gene have only been reported from South America. These Rx breaking isolates could potentially have an additional impact over the current situation in the EU and therefore meet all the criteria to qualify as a potential Union quarantine pest. All other non-EU isolates, should they be introduced, are not expected to have additional impact and therefore do not meet this criterion to qualify as a potential Union quarantine pest.