Polish Isolates Research Articles

Phenotypic Characteristics, Pathogenicity, and Molecular Identification of Hypomyces perniciosus Causing Wet Bubble Disease of Edible Mushrooms

Abstract The research aimed to determine the phenotypic characteristic, pathogenicity and molecular characterization of Hypomyces perniciosus isolates responsible for wet bubble disease of the white button mushroom Agaricus bisporus. Phenotypic characteristics such as colony appearance, mycelium texture, and pigmentation were studied on four different agar media, i.e., potato dextrose agar (PDA), malt extract agar (MEA), Czapek-Dox yeast agar (CYA), and Sabouraud dextrose agar (SDA), after eight days of incubation. Additionally, the growth rate of the tested isolates was studied depending on the pH of the medium. Fungal isolates showed the highest mycelial growth on MEA and SDA at pH 6.0. However, on CYA at pH 7.0, the mycelium exhibited the worst growth. Isolate identification and genetic relationship analysis were carried out using internal transcribed spacer region sequencing and the random amplified polymorphic DNA method. The research confirmed that all Polish isolates belong to the species H. perniciosus, and the genetic diversity is relatively low. Phylogenetic analyses revealed three subgroups of H. perniciosus isolates. The first group included three genetically distinct isolates with a similarity coefficient in the range of 0.76–0.85 to isolates CBS 815.73 and CBS 322.52. The second group was divided into two subgroups and included 16 isolates with a genetic similarity range of 0.91 to 1.0 to CBS 815.73 and CBS 322.52 isolates. Furthermore, the eight genetically similar isolates exhibited the greatest pathogenicity towards A. bisporus.

Genetic Sequence and Pathogenicity of Infectious Bursal Disease Virus in Chickens in Egypt During 2017-2021.

The continued circulation of infectious bursal disease virus (IBDV) in Egypt, despite the use of various vaccines, is a serious problem that requires continuous detection of IBDV. In the current study, real-time reverse transcriptase polymerase chain reaction testing of 100 diseased chicken flocks during 2017-2021 revealed the presence of very virulent IBDV (vvIBDV) in 67% of the flocks, non-vvIBDV in 11%, and a mixture of both vvIBDV and non-vvIBDV in 4%. Twenty-nine IBDV isolates were submitted for partial sequencing of the viral protein 2 hypervariable region (VP2-HVR), and 27 isolates were confirmed to be genogroup A3 (vvIBDV) with 96.3%-98.5% similarity to the global A3 (vvIBDV) and 88.9%-97% similarity to genogroup A1 vaccine strains. The remaining two isolates were non-vvIBDV and showed 91.1% and 100% identity with classical genogroup A1 strains, respectively. Furthermore, the sequence and phylogenetic analysis of VP1 (amino acids 33-254) of two selected isolates of A3, 5/2017 and 98/2021, clustered them as B2, vvIBDV-like, strains with high similarity (99.5%) to four Egyptian, 99% to Chinese and European, and 97.7% to Chinese and Polish vvIBDV isolates. Experimental infection of commercial broiler chickens with two vvIBDV-A3B2 isolates (5/2017 and 98/2021) showed no mortality despite typical tissue lesions, clear histopathological changes, and strong ELISA antibody response. Isolate 98/2021 was more pathogenic, as confirmed by histopathology, whereas isolate 5/2017 induced a stronger serological response. In conclusion, vvIBDV (A3B2) strains with two amino acid (aa) substitutions in VP1 as V141I and V234I as well as VP2 as Y220F and G254S are still circulating in Egypt.

Assessing tropism and genetic traits of carp oedema virus isolates to enhance detection strategies.

Carp oedema virus (CEV) is a relatively understudied poxvirus. It exhibits an affinity for gill and skin epithelial cells. Investigations were conducted into selected aspects of CEV biology, with a focus on determining cell and tissue tropism of CEV, acquiring gene sequences and updating CEV tests in fish tissues. A total of 238 common carp tissue samples from nine aquaculture farms were analysed. The study evaluated the efficacy of intermediate detection of CEV by real-time PCR and in situ hybridisation. The genes encoding protein P4a were sequenced, analysed and aligned in a phylogenetic tree using a molecular evolution model. In situ hybridisation revealed the necessity to validate the Centre for Environment, Fisheries and Aquaculture Science protocols for sampling for CEV detection and to use the tissues for which the virus has the highest tropism, namely the skin and kidneys, rather than solely the gills. The level of genetic variability was determined, and it was shown that CEV mutates systematically. The creation of two distinct phylogenetic clades confirms certain strains' description as Polish isolates. Determining the localisation of CEV genetic material in organs and tissues is pivotal for shaping the World Organisation for Animal Health guidelines. The utility of molecular diagnostics has been demonstrated in the skin and kidney of carp, in addition to the gills, impelling their inclusion in diagnostic protocols. The clusters identified in the phylogenetic tree offer valuable insights for developing the current PCR primers. The prevalence of CEV infection in aquaculture, juxtaposed with its notably lower detection in wild fish, underscores the significance of mandatory molecular diagnostic testing for CEV in carp farming.

Variability of <i>Puccinia komarovii</i> var. <i>komarovii</i> isolates from <i>Impatiens parviflora</i> DC. and its occurrence in the Drawa and Wigry National Parks in Poland

Small balsam (<i>Impatiens parviflora</i> DC.), an invasive plant native to central Asia, is currently one of the most extensive invasive plants in Central Europe, including Poland. Its expansion may affect populations of native species, such as touch-me-not balsam (Impatiens noli-tangere). It is particularly dangerous in legally protected areas, where the possibilities of eradicating it are very limited. There is some hope for stopping its expansion by using pathogens as a biological weapon. In this case, useful may be host-specific rust fungus <i>Puccinia komarovii</i> var. <i>komarovii</i>. Our three-year observations of I. parviflora infection severity by <i>P. komarovii</i> var. <i>komarovii</i> carried out in Poland in Drawa National Park (DNP) and Wigry National Park (WNP) showed the higher incidence of rust on leaves in DNP, where the median blade infestation for two years was 51% of leaf coverage. In contrast, for three years in WNP, the median blade infestation amounted to only 25%. The most severe symptoms occurred during the summer months. However the percentage of leaf coverage by pathogen exhibited fluctuations depending on the year, implying an influence of environmental conditions that stabilized the infection each season. Molecular analyses, in addition to confirming the infection of the tested plants by <i>P. komarovii</i> var.<i> komarovii</i>, enabled the characterization of its population based on comparative analysis of ITS and LSU sequences. Clear differences were found between the studied isolates and <i>P. komarovii</i> from <i>I. glanduliferae</i> and <i>I. brachycentra</i>. There was no diversity among our isolates, but they differed from the sequences of Polish isolates originating from <i>I. parviflora</i> available in GenBank, which indicates some diversity of rust in a relatively small area. It probably results from sexual reproduction and may enable rapid adaptation of the pathogen to the region or host plant, or it may indicate certain “entry points” to Poland, where the isolates arrived from their native place of occurrence.

Detection and Molecular Characterization of Peach Latent Mosaic Viroid Isolates Infecting Stone Fruit Trees in Poland

Abstract Field surveys were carried out to assess the incidence of peach latent mosaic viroid in nine orchards of stone fruit trees in four growing regions of Poland. Forty of 119 peaches, six of 37 apricots, five of eight nectarines and one of four plums trees tested positive by RT-PCR for this viroid. Most of the infected stone fruit trees did not show any symptoms. Leaf yellow mosaic was only noticed on ‘Maycrest’ peach and ‘Saturn’ nectarine trees, and additional symptoms of cracked fruits with corky sutures and enlarged pits were observed on one ‘Maycrest’ tree. One or two isolates from each orchard and each species of infected tree were selected, and together, 11 isolates of peach latent mosaic viroid were used for genetic analyses. The nucleotide sequence similarity of selected isolates found in Poland was 93.6–100%. They shared 82.0–98.8% similarity with the viroid strains from different hosts and geographical regions. The phylogenetic analysis led to the clustering of all isolates detected during this study in the most numerous represented subgroup II-A, composed of peach latent mosaic viroid strains originating from different geographic regions and host plants. Although Polish isolates were clustered in the same subgroup, some differed in length and nucleotide sequences. It was shown that the genetic variability of isolates found in Poland was unrelated to the regions of their origin. A positive signal was obtained in dot-blot hybridization of all analyzed 11 isolates.

Viruses infecting Capsicum crops in Poland and molecular characterization of newly detected bell pepper alphaendornavirus (BPEV)

Viral diseases are one of the main factors limiting the cultivation of Capsicum annuum L. worldwide, causing yield losses of up to 80%. Cucumber mosaic virus (CMV) is a prevalent virus affecting the quality and yield of pepper; however, the presence of other viruses has been also noticed. In this study, pepper fruits originated from retail chains, home gardens and greenhouses were analysed to determine the occurrence of viruses infecting sweet pepper crops in Poland. A total of 32 symptomatic and asymptomatic fruit samples were tested for the presence of: CMV, pepper mild mottle virus (PMMoV), tomato spotted wilt virus (TSWV), tomato mosaic virus (ToMV), tobacco mosaic virus (TMV) and potato virus Y (PVY). The presence of viruses in single and mixed infections, with a predominance of CMV, was confirmed. In addition, two symptomatic pepper samples that were negative in RT-PCR for common viruses were subjected to high-throughput sequencing and the presence of new viruses in Poland: bell pepper alphaendornavirus (BPEV) and pepper cryptic virus 2 (PCV2) in a mixed infection with other viruses was demonstrated. The presence of new viruses was verified in all tested samples and BPEV was the most prevalent. The phylogenetic analysis of the BPEV population showed that the Polish isolates are diverse and grouped within two clusters. Although BPEV does not pose a direct threat to pepper crops, its presence may cause changes in the physiology of the host, as well as may interact with other viruses in mixed infections.

Characterization of three-nucleate Rhizoctonia AG-E based on their morphology and phylogeny

The genus Rhizoctonia has been classified into two main groups according to the number of nuclei. Binucleate Rhizoctonia strains have two nuclei in each cell, whereas multinucleate Rhizoctonia fungi were observed to have a variable number of nuclei ranging from 4 to 16 in each cell. In the study, twelve Polish isolates were tested. According to ITS1-5,8S-ITS2 rDNA sequences, the isolates were classified in the AG-E. Their affiliation to AG was confirmed by anastomosis reactions with tester isolates. The number of nuclei was counted with DAPI staining under a fluorescent microscope, and the diameter of the hyphae was also measured. Not all AG-E isolates had the same number of nuclei in their cells: one group among these fungi produced cells with a diverse number of nuclei, usually 3; however, this number ranged from 2 to 4, making the average number of nuclei close to 3. It can be assumed that all isolates with three nuclei belong to this group, which may greatly facilitate the preliminary identification of trinucleate isolates of Rhizoctonia spp. belonging to AG-E. Based on these characters, we call these isolates AG-E-3n isolates. The thiamine requirement is not helpful in classifying and describing the AG-E strains.

Zucchini Yellow Mosaic Virus (ZYMV) as a Serious Biotic Stress to Cucurbits: Prevalence, Diversity, and Its Implications for Crop Sustainability.

Zucchini yellow mosaic virus (ZYMV) is a severe threat to cucurbit crops worldwide, including Pakistan. This study was pursued to evaluate the prevalence, geographic distribution, and molecular diversity of ZYMV isolates infecting cucurbits in Pakistan's Pothwar region. Almost all the plant viruses act as a biotic stress on the host plants, which results in a yield loss. These viruses cause losses in single-infection or in mixed-infection cucurbit crops, and we have found a number of mixed-infected samples belonging to the Curubitaceae family. Serological detection of the tested potyviruses in the collected cucurbit samples revealed that ZYMV was the most prevalent virus, with a disease incidence (DI) at 35.2%, followed by Papaya ringspot virus (PRSV) with an incidence of 2.2%, and Watermelon mosaic virus (WMV) having an incidence as little as 0.5% in 2016. In the year 2017, a relatively higher disease incidence of 39.7%, 2.4%, and 0.3% for ZYMV, WMV, and PRSV, respectively, was recorded. ZYMV was the most prevalent virus with the highest incidence in Attock, Rawalpindi, and Islamabad, while PRSV was observed to be the highest in Islamabad and Jhelum. WMV infection was observed only in Rawalpindi and Chakwal. Newly detected Pakistani ZYMV isolates shared 95.8-97.0% nucleotide identities among themselves and 77.1-97.8% with other isolates retrieved from GenBank. Phylogenetic relationships obtained using different ZYMV isolates retrieved from GenBank and validated by in silico restriction analysis revealed that four Pakistani isolates clustered with other ZYMV isolates in group IIb with Chinese, Italian, Polish, and French isolates, while another isolate (MK848239) formed a separate minor clade within IIb. The isolate MK8482490, reported to infect bitter gourd in Pakistan, shared a minor clade with a Chinese isolate (KX884570). Recombination analysis revealed that the recently found ZYMV isolate (MK848239) is most likely a recombinant of Pakistani (MK848237) and Italian (MK956829) isolates, with a recombinant breakpoint between 266 and 814 nucleotide positions. Local isolate comparison and recombination detection may aid in the development of a breeding program that identifies resistant sources against recombinant isolates because the ZYMV is prevalent in a few cucurbit species grown in the surveyed areas and causes heavy losses and economic damage to the agricultural community.

Molecular Characteristics of Barley Yellow Dwarf Virus-PAS-The Main Causal Agent of Barley Yellow Dwarf Disease in Poland.

Barley yellow dwarf is a threat to cereal crops worldwide. Barley yellow dwarf virus-PAS (BYDV-PAS) was detected for the first time in Poland in 2015, then in 2019. In the spring of 2021, in several locations in Poland, winter wheat and barley plants with dwarfism and leaf yellowing were collected. Reverse transcription-polymerase chain reaction results revealed BYDV presence in 47 samples and excluded wheat streak mosaic virus infections. Next, immuno-captured polymerase chain reactions confirmed only one case of co-infection caused by BYDV and wheat dwarf virus. Moreover, restriction fragment length polymorphism analysis showed that BYDV-PAS was predominant. The preliminary results were confirmed using sequencing. Infected cereal plants originated mainly from northwestern Poland. The complete coding sequence of coat protein (CP) and a fragment of RNA-dependent RNA polymerase (RdRp) genes of 14 Polish isolates were determined and deposited in the GenBank database. The nucleotide and deduced amino acid sequences of local isolates were compared with others reported to date, indicating their high similarity, from 75.4% to 99.5% and from 81.1% to 100% nucleotide sequence identity, in RdRp and CP, respectively. Phylogenetic analysis, based on the CP gene, revealed the presence of 3 main groups. The Polish isolates clustered together within the Ia group.

Molecular analysis Polish isolates of goose hemorrhagic polyomavirus from geese and free-living birds

Goose haemorrhagic polyomavirus (GHPV) is the viral agent of hemorrhagic nephritis and enteritis of geese (HNEG), a lethal disease of goose. The study describes the results of a molecular analysis Polish isolates of GHPV from geese and free-living birds based on complete VP1 gene and VP2 gene sequences. The sequences were analyzed and aligned with different GHPV isolates sequences accessible in the GenBank database. This study indicates affiliation GHPV isolates from fee-living birds and GHPV isolates circulating in Polish goose flocks and around the world to the same genetic groups, which proves their evolutionary relationship and indicates the potential role of free-living birds as a source of infections for poultry.

Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Isolates in Terms of Efficacy against Cereal Ground Beetle Zabrus tenebrioides (Coleoptera: Carabidae): Morphometry and Principal Component Analysis.

One of the most dangerous pests of cereals is Zabrus tenebrioides and, in Poland, it is becoming a serious pest. Entomopathogenic nematodes (EPNs) seem to be a very promising, biological control agent for this pest. Native EPN populations are well adapted to local environmental conditions. The current study characterized three Polish isolates of the EPN Steinernema feltiae, which differed in their effectiveness against Z. tenebrioides. In the field, isolate iso1Lon reduced the pest population by 37%, compared with 30% by isolate iso1Dan and 0% by the iso1Obl isolate; the number of plants damaged by Z. tenebrioides in the presence of the different isolates reflected the results in terms of the decrease in pest population size. After incubation in the soil for 60 days, recovered EPN juveniles of all three isolates were able to infect 93-100% of the test insects, with isolate iso1Obl again showing the lowest effectiveness. The juveniles of isolate iso1Obl were also morphometrically distinct from the other two isolates, as revealed by principal component analysis (PCA), which helped to distinguish the EPN isolates. These findings showed the value of using locally adapted isolates of EPNs; two of the three isolates randomly selected from Polish soil outperformed a commercial population of S. feltiae.

Genetic Characterization of the Poultry Red Mite (Dermanyssus gallinae) in Poland and a Comparison with European and Asian Isolates.

(1) Background: The blood-feeding mite Dermanyssus gallinae (De Geer 1778) continues to attract wide interest from researchers and bird breeders. The aim of this study was to evaluate the genetic diversity of D. gallinae populations in five commercial laying hen farms in Poland and to determine their similarity with isolates from other countries. The study involved an analysis of a fragment of the cytochrome c oxidase subunit I gene (COI). A total of 38 isolates obtained from Polish farms and 338 sequences deposited in GenBank were analyzed. (2) Results: Haplotype No. 46 was dominant (90%) in Polish isolates and was homologous with the isolates from Great Britain, the Netherlands, Belgium, Japan, and South Korea. These results are indicative of high genetic homogeneity and common ancestry of the poultry red mite and point to a common source of infestation in the examined farms. (3) Conclusions: The genetic diversity of D. gallinae should be further studied to promote a better understanding of how this parasite is disseminated within and between countries.

Characterisation of ORF3, M, N and E Gene Sequences of Porcine Epidemic Diarrhoea Virus from Domestic Pigs in Poland.

Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen causing porcine epidemic diarrhoea and acute gastroenteritis in pigs of all ages. Previous analysis of the viral genome of PEDV in Poland was only based on the spike protein (S) gene sequences and no analysis of other genes has been performed. The aim of this study was to analyse the envelope (E), membrane (M) and nucleocapsid (N) protein and open reading frame 3 (ORF3) gene sequences. Viral RNA from 18 Polish pig faecal samples that were quantitative reverse transcription PCR-positive for PEDV was analysed in four genomic regions (E, M, N and ORF3). Phylogenetic analysis based on these regions' sequences revealed that Polish PEDV isolates were highly related and were clustered into group G2a across the four genes compared. Moreover, the Polish strains were located in distinct subclusters on the phylogenetic trees, which suggests the presence of at least three independently evolving PEDV genetic lines circulating in Poland. The occurrence of unique mutations in the sequences of Polish PEDV strains suggests that PEDV continues to undergo evolutionary processes, accumulating the mutations necessary for viral fitness in its natural hosts. The Polish PEDV strains differed genetically from the CV777 vaccine strain, suggesting the risk of relatively low vaccine efficacy if this strain is used. Our results promote a better understanding of the genetic diversity of PEDV field isolates in Poland and highlight the importance of molecular characterisation of PEDV field strains for the development of an effective vaccine against PEDV.

Molecular Characterization of a Novel Budgerigar Fledgling Disease Virus Strain From Budgerigars in China.

Budgerigar fledgling disease virus (BFDV) is the causative polyomavirus of budgerigar fledgling disease, an important avian immunosuppressive disease in budgerigars (Melopsittacus undulatus). In the current study, we explored the etiological role and molecular characteristics of BFDV. We identified a novel BFDV strain, designated as SC-YB19, belonging to a unique cluster with three other domestic strains (WF-GM01, SD18, and APV-P) and closely related to Polish isolates based on complete sequences. Sequence analysis showed that SC-YB19 had an 18-nucleotide (nt) deletion in the enhancer region, corresponding to the sequence position 164–181 nt, which differed significantly from all other BFDV strains. Based on sequence alignment, three unique nucleotide substitutions were found in VP4 (position 821), VP1 (position 2,383), and T-antigen (position 3,517) of SC-YB19, compared with SD18, WF-GM01, QDJM01, HBYM02, APV7, and BFDV1. Phylogenetic analyses based on complete sequences suggested that SC-YB19, along with the domestic WF-GM01, SD18, and APV-P strains, formed a single branch and were closely related to Polish, Japanese, and American isolates. These results demonstrate that BFDV genotype variations are co-circulating in China, thus providing important insight into BFDV evolution.

Diversity of the Polish isolates of bean yellow mosaic virus (BYMV) isolated from broad bean

Bean yellow mosaic virus (BYMV) is one of the most widespread and damaging viruses of cultivated legumes in the world. In order to characterise BYMV isolates detected in ten broad bean plants in Poland, total nucleic acids were isolated, a fragment of the gene encoding the polyprotein was amplified by RT-PCR and then sequenced. The sequences of the coat protein (CP) gene of the three Polish isolates (E1, E2, E3) were compared with 33 sequences retrieved from the GenBank database. The sequences of the tested isolates showed 81,8–99,8% identity at the nucleotide level and 84,9–99,6% at the amino acid level with other BYMV isolates. The analysis performer based on CP sequences confirmed the genetic diversity of the BYMV population and revealed the presence of three phylogenetic groups. The Polish isolates were classified into I (E1 and E2) and III (E3) group.

Pathogenicity of Phytophthora× alni Isolates Obtained from Symptomatic Trees, Soil and Water against Alder

Phytophthora alni complex (P. × alni, P. × multiformis, P. uniformis) are pathogens attacking alder seedlings and trees, causing significant losses in nurseries and natural tree stands. Decay of alder trees has been observed in Poland for over a dozen years. Overall, 25 Polish isolates of P. × alni obtained from symptomatic alder trunks, rhizosphere soil surrounding infected trees, and nearby natural streams were compared with isolates from symptomatic trunks obtained in France, Belgium and Hungary. Morphologic characterization of mycelium, vegetative and generative organs, temperature effect on mycelium growth, and their pathogenicity were studied. The mycelium growth rate of isolates from symptomatic plants was fastest on Carrot Agar (CA) medium, and from soil and water on Vegetable Agar (V8A) medium. The sizes of zoosporangia varied depending on their origin. The isolates that originated from the soil had the largest zoosporangia. The diameter of the oogonia and antheridia did not differ regardless of their origin. The results of pathogenicity tests of P. × alni isolates obtained from different sources showed that the soil isolates were the most aggressive in each test, followed by the isolates from the trunks and water. A simple test of leaf colonization can give an idea of the aggressiveness of the isolate towards the shoots and roots. No morphological or physiological markers of aggressiveness have been found.

Complete Genome Sequence of a Polish Enterococcus faecalis vanA-Positive Hospital Isolate.

ABSTRACTEnterococcus faecalis is an important human pathogen involved in health care-associated infections, and its increasing resistance to vancomycin is worrisome. Here, we report the complete genome sequence of a Polish hospital vanA-positive isolate of E. faecalis, consisting of a 3,264,821-bp chromosome and six plasmids.

First report of Tomato black ring virus infecting raspberry and blackberry in Poland.

Raspberry (Rubus idaeus L.) and blackberry (Rubus fruticosus L.) are infected by at least 29 viruses, including the Tomato black ring virus (TBRV) (Martin et al. 2013). TBRV belongs to the genus Nepovirus (subgroup B) of the family Secoviridae and is listed as a plant pathogen in over 40 countries. TBRV infects a wide range of herbaceous and woody plants. In Poland, TBRV has been described on the plants of the following species: Tagetes patula, T. erecta, Cucumis sativus, Cucurbita pepo, Lactuca sativa, Solanum tuberosum, S. lycopersicum, Sambucus nigra, and Robinia pseudoacacia (Jończyk et al. 2004, Hasiów-Jaroszewska et al. 2015). To this date, there is no information on the incidence of TBRV in raspberry and blackberry in Poland. In the spring of 2019, 52 blackberry leaf samples and 408 raspberry leaf samples were collected from 4 plantations located in central Poland. None of the raspberry plants (cvs. Glen Ample, Polka, Sokolica), nor the blackberry plants (cvs. Thornfree, Polar, Gaj, Kotata) exhibited viral symptoms. Enzyme-linked immunosorbent assay (ELISA) was carried out for extracts from the 460 collected leaf samples to detect TBRV using commercial antisera (Loewe Biochemica GmbH, Germany). The results indicated that 9 samples (4 blackberry, 5 raspberry) were infected with TBRV. The isolates of the virus were transferred by sap inoculation and maintained in Nicotiana tabacum cv. Xanthi. Systemic ringspot, necrosis and patterned lines were observed on tobacco leaves. The presence of the virus in tobacco leaf samples was confirmed by reverse transcription PCR (RT-PCR). Total RNA was extracted from all 9 samples using the silica capture (SC) method described originally by Boom et al. (1990) and adapted to the detection of plant viruses by Malinowski (1997). Part of the CP gene was amplified with the CPF (5'-GCCTGTCTCTCTCGCAATG-3') and CPR (5'-AAGGAGCCAAACTGAAATGT-3') primer pair (Hasiów-Jaroszewska et al. 2015). Amplicons of the expected size (763 bp) were obtained for each sample. The amplified products were purified, sequenced in both directions, deposited in GenBank and assigned accession numbers: MT507387 to MT507390 and MT507394 for the isolates from Rubus idaeus and MT507391 to MT507393 and MN954654 for the isolates from Rubus fruticosus, respectively. The 9 newly obtained TBRV CP gene sequences, together with the 25 isolates deposited in GenBank, were aligned by ClustalW. The isolates obtained in this study showed a 99.0-100% nucleotides (nt) and a 98.7-100% amino acids (aa) identity in the part of the CP, respectively. Comparison of the part of the CP of the 4 blackberry and the 5 raspberry TBRV isolates with 25 TBRV isolates available in GenBank showed a 80.6-97.8% nt and a 87.9-99.5% aa identity, respectively. The results of the phylogenetic analysis have revealed that the TBRV isolates obtained in this study are closely related to 3 Polish isolates (AY157994, KR139941, KR139951) and 1 Bioreba ctrl Switzerland isolate (KT923164). These findings are of epidemiological significance due to the fact that TBRV was detected on symptomless Rubus plants, which therefore represent a reservoir of the virus and a threat in case of a symptomatic infection of sensitive cultivars. Accordingly, the results will assist in using appropriate strategies for reducing TBRV incidence in Rubus-growing areas. Moreover, this is, to the best of our knowledge, the first report of TBRV in raspberry and blackberry in Poland.

Dissemination of Klebsiella pneumoniae ST147 NDM-1 in Poland, 2015-19.

To assess the spread of New Delhi metallo-β-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae ST147 organisms in Poland since an introduction from Tunisia in March 2015, including their phylogenetic position in the global population of the high-risk clone. Out of 8925 unique NDM-positive K. pneumoniae isolates identified in Poland from April 2015 till December 2019, 126 isolates, including the Tunisian imports, were related by PFGE and blaNDM gene-carrying Tn125 transposon derivatives. Forty-seven representative isolates were sequenced by Illumina MiSeq. The phylogeny, resistome, virulome and plasmid replicons were analysed and compared with the international ST147 strains. Plasmids of six isolates were studied by the MinION sequencing. A high homogeneity of the 47 isolates was observed, with minor variations in their resistomes and plasmid replicon profiles. However, the detailed SNP comparison discerned a strict outbreak cluster of 40 isolates. All of the organisms were grouped within the ST147 phylogenetic international lineage, and four NDM-1 producers from Tunisia, Egypt and France were the closest relatives of the Polish isolates. Yersiniabactin genes (YbST280 type) were located within the ICEKpn12-like element in most of the outbreak isolates, characterized by O2v1 and KL64 antigen loci. The blaNDM-1 genes were located in double-replicon IncFIIK2+IncFIBK plasmids. The continuous spread of K. pneumoniae ST147 NDM-1 in Poland since 2015, largely in the Warsaw area, is demonstrated by this genomic analysis. The isolates showed a high degree of homogeneity, and close relatedness to organisms spreading in the Mediterranean region.

The first genotype II African swine fever virus isolated in Africa provides insight into the current Eurasian pandemic

African swine fever (ASF) caused by the African swine fever virus (ASFV) is ranked by OIE as the most important source of mortality in domestic pigs globally and is indigenous to African wild suids and soft ticks. Despite two ASFV genotypes causing economically devastating epidemics outside the continent since 1961, there have been no genome-level analyses of virus evolution in Africa. The virus was recently transported from south-eastern Africa to Georgia in 2007 and has subsequently spread to Russia, eastern Europe, China, and south-east Asia with devastating socioeconomic consequences. To date, two of the 24 currently described ASFV genotypes defined by sequencing of the p72 gene, namely genotype I and II, have been reported outside Africa, with genotype II being responsible for the ongoing pig pandemic. Multiple complete genotype II genome sequences have been reported from European, Russian and Chinese virus isolates but no complete genome sequences have yet been reported from Africa. We report herein the complete genome of a Tanzanian genotype II isolate, Tanzania/Rukwa/2017/1, collected in 2017 and determined using an Illumina short read strategy. The Tanzania/Rukwa/2017/1 sequence is 183,186 bp in length (in a single contig) and contains 188 open reading frames. Considering only un-gapped sites in the pairwise alignments, the new sequence has 99.961% identity with the updated Georgia 2007/1 reference isolate (FR682468.2), 99.960% identity with Polish isolate Pol16_29413_o23 (MG939586) and 99.957% identity with Chinese isolate ASFV-wbBS01 (MK645909.1). This represents 73 single nucleotide polymorphisms (SNPs) relative to the Polish isolate and 78 SNPs with the Chinese genome. Phylogenetic analysis indicated that Tanzania/Rukwa/2017/1 clusters most closely with Georgia 2007/1. The majority of the differences between Tanzania/Rukwa/2017/1 and Georgia 2007/1 genotype II genomes are insertions/deletions (indels) as is typical for ASFV. The indels included differences in the length and copy number of the terminal multicopy gene families, MGF 360 and 110. The Rukwa2017/1 sequence is the first complete genotype II genome from a precisely mapped locality in Africa, since the exact origin of Georgia2007/1 is unknown. It therefore provides baseline information for future analyses of the diversity and phylogeography of this globally important genetic sub-group of ASF viruses.