Sequence Analysis Of Region Research Articles

Molecular Characterization of African Swine Fever Virus From 2019-2020 Outbreaks in Guangxi Province, Southern China.

African swine fever virus (ASFV) causes contagious hemorrhagic disease of pigs with high morbidity and mortality. To identify the molecular characteristics of ASFV strains circulating in Guangxi province, southern China, a total of 336 tissue samples collected from 336 domestic pigs that died as a result of severe hemorrhagic disease during 2019–2020 were tested for ASFV. Furthermore, 66 ASFV strains were genetically characterized by sequence analysis of the C-terminal region of B646L (p72) gene, the complete E183L (p54) gene, the variable region of EP402R (CD2v) gene, the central variable region (CVR) of B602L gene, the full MGF505-2R gene, and the tandem repeat sequence (TRS) within intergenic region (IGR) between the I73R and I329L (I73R/I329L) genes. Phylogenetic analysis revealed that the ASFV strains from Guangxi province belonged to genotypes I and II based on the B646L (p72) and E183L (p54) genes, and there were eight different tetrameric TRS variants based on the CVR of B602L gene. Phylogenetic analysis of the EP402R (CD2v) gene revealed that these ASFV strains belonged to serogroups 4 and 8. Eight of the 66 strains belonged to genotype I and serogroup 4, and showed deletion of whole MGF505-2R gene. The sequence analysis of the IGR between the I73R/I329L genes showed that IGR II and III variants were co-circulating in Guangxi province. The results indicated that ASFV strains circulating in Guangxi province during 2019–2020 outbreaks showed high genetic diversity, of which genotypes I and II, as well as serogroups 4 and 8, were simultaneously circulating in Guangxi province, and there existed wild-type and naturally gene-deleted strains in the field. This is the first detailed report on the molecular characterization of the ASFV strains circulating in southern China, and serogroup 4 in China.

Enhancing the Discovery of Bioactive Secondary Metabolites From Fungal Endophytes Using Chemical Elicitation and Variation of Fermentation Media.

Endophytic microorganisms are an important source of bioactive secondary metabolites. In this study, fungal endophytes obtained from A*STAR’s Natural Product Library (NPL) and previously isolated from different habitats of Singapore were investigated for their diversity, antimicrobial, and cytotoxic activities. A total of 222 fungal strains were identified on the basis of sequence analysis of ITS region of the rDNA gene. The identified fungal strains belong to 59 genera distributed in 20 orders. Majority of the identified strains (99%; 219 strains) belong to the phylum Ascomycota, while two strains belonged to the phylum Basidiomycota, and only one strain was from Mucoromycota phylum. The most dominant genus was Colletotrichum accounting for 27% of all the identified strains. Chemical elicitation using 5-azacytidine and suberoylanilide hydroxamic acid (SAHA) and variation of fermentation media resulted in the discovery of more bioactive strains. Bioassay-guided isolation and structure elucidation of active constituents from three prioritized fungal strains: Lophiotrema sp. F6932, Muyocopron laterale F5912, and Colletotrichum tropicicola F10154, led to the isolation of a known compound; palmarumycin C8 and five novel compounds; palmarumycin CP30, muyocopronol A-C and tropicicolide. Tropicicolide displayed the strongest antifungal activity against Aspergillus fumigatus with an IC50 value of 1.8 μg/ml but with a weaker activity against the Candida albicans presenting an IC50 of 7.1 μg/ml. Palmarumycin C8 revealed the best antiproliferative activity with IC50 values of 1.1 and 2.1 μg/ml against MIA PaCa-2 and PANC-1 cells, respectively.

First report of Podosphaera mors‐uvae causing powdery mildew on Ribes fasciculatum var. chinense in Korea

AbstractIn July 2017, hundreds of Ribes fasciculatum var. chinense plants were found infected with powdery mildew for the first time in Korea. Further field surveys resulted in collecting anamorphs and teleomorphs of the powdery mildew at three additional locations in Korea. Morphological characterization of the holomorph and molecular sequence analyses of the ITS regions and 28S rDNA gene confirmed the identification of this fungus as Podosphaera mors‐uvae, which is already known for a long time on Ribes spp. in Europe and North America. Previous records of P. mors‐uvae on Ribes grossularia in Japan and China are also discussed. This is the first report of P. mors‐uvae from Korea.

RNA analysis of the GALNS transcript reveals novel pathogenic mechanisms associated with Morquio syndrome A

Morquio syndrome A (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficiency of N-acetyl-galactosamine-6-sulfatase (GALNS) which catabolizes the glycosaminoglycans (GAG), keratan sulfate and chondroitin-6-sulfate. Homozygous or compound heterozygous pathogenic variants in the GALNS result in the deficiency of the enzyme and consequent GAG accumulations. DNA sequence and copy number analysis of the GALNS coding region fails to identify biallelic causative pathogenic variants in up to 15% of patients with Morquio syndrome A. RNA transcript analysis was performed to identify pathogenic alterations in two unrelated families with Morquio syndrome A in whom a single heterozygous or no pathogenic alteration was detected by standard analysis of the GALNS gene. RNA sequencing and quantitative expression analysis identified the overabundance of an aberrant GALNS transcript isoform and a reduction of the clinically relevant isoform (NM_000512.4) in the Morquio syndrome A patients from both families. The aberrant isoform (ENST00000568613.1) was produced by alternative splicing and contained intronic sequence that was likely a cryptic exon predicted to result in a reading frame shift and generation of a premature termination codon. These findings indicated that the aberrant splicing is likely the novel molecular defect in our patients. RNA transcript analysis could be useful to identify pathogenic alterations and increase the yield of molecular diagnosis in patients with Morquio syndrome A whose genetic variants are not found by standard sequencing or gene dosage analysis.

Screening and biocontrol evaluation of indigenous native Trichoderma spp. against early blight disease and their field assessment to alleviate natural infection

BackgroundEarly blight disease of tomato caused by pathogenic fungi Alternaria solani is the most significant and common disease throughout the world as well as in Kingdom of Saudi Arabia. The aim of this study was to isolate and identify native Trichoderma species from the Jeddah region in Saudi Arabia; evaluate their antagonistic potential against A. solani; and study their influence early blight disease severity in greenhouse and in open field.ResultsThe present study focused to explore the biocontrolling potential of native Trichoderma spp. against A. solani strain to compare with a conventional fungicide. Out of 21, 3 Trichoderma isolates showed an antifungal activity and significantly inhibited the mycelial growth of pathogen that were identified as Trichoderma atroviride, T. harzianum and T. longibrachiatum by their ITS region sequence analysis. Strong in vitro mycelial growth suppression (70.66%) was also recorded at 400 ppm Mancozeb (90%WP®) fungicide. Further, these Trichoderma bioagents and fungicide were further evaluated in greenhouse (artificially inoculated) and in field on naturally infected tomato plants. In greenhouse, (13.74%) disease severity after T. harzianum treatment was recorded, followed by T. longibrachiatum (25.83%) and T. atroviride (21.67%). The disease severity after fungicide (50 mg/L; 10 ml per plant) application was (7.91%). Further, positive impact on the plant biomarkers was demonstrated by all selected Trichoderma isolates in greenhouse. Under natural infection in season I, the disease severity (%) after T. longibrachiatum, T. atroviride and T. harzianum treatments was 11.5, 13.26 and 16.81%, respectively, followed by control (32.12%), whereas 7.18% disease severity was recorded after fungicide application.ConclusionsThe results revealed that native Trichoderma of this region had potential to mitigate the early blight disease intensity in field.

Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment.

In many parts of the world, human-mediated environmental change is depleting biodiversity faster than it can be characterized, while invasive species cause agricultural damage, threaten human health and disrupt native habitats. Consequently, the application of effective approaches for rapid surveillance and identification of biological specimens is increasingly important to inform conservation and biosurveillance efforts. Taxonomic assignments have been greatly advanced using sequence-based applications, such as DNA barcoding, a diagnostic technique that utilizes PCR and DNA sequence analysis of standardized genetic regions. However, in many biodiversity hotspots, endeavors are often hindered by a lack of laboratory infrastructure, funding for biodiversity research and restrictions on the transport of biological samples. A promising development is the advent of low-cost, miniaturized scientific equipment. Such tools can be assembled into functional laboratories to carry out genetic analyses in situ, at local institutions, field stations or classrooms. Here, we outline the steps required to perform amplicon sequencing applications, from DNA isolation to nanopore sequencing and downstream data analysis, all of which can be conducted outside of a conventional laboratory environment using miniaturized scientific equipment, without reliance on Internet connectivity. Depending on sample type, the protocol (from DNA extraction to full bioinformatic analyses) can be completed within 10 h, and with appropriate quality controls can be used for diagnostic identification of samples independent of core genomic facilities that are required for alternative methods.

Pichia bovicola sp. nov., a yeast species associated with small-intestinal content of cattle.

Three yeast strains, DMKU-MP6-4T, DMKU-MP2-6 and DMKU-MP5-1, were isolated from the small-intestinal content or Pia of cattle in Thailand during the investigation of yeast diversity in this habitat. According to the D1/D2 domain of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) region sequence analysis, these strains represent a novel yeast species in the genus Pichia. The species produced one to four ascospores per ascus with spherical to ellipsoidal shape and heterogenous in terms of size. These three strains were identical and differed from their closely related species, Pichia exigua NRRL Y-10920T by 2% (six nucleotide substitutions and five gaps) in the D1/D2 domain of the large subunit rRNA gene, while ITS region sequences differed by 3.1% (16 nucleotide substitutions and 27 gaps), 3.7% (19 nucleotide substitutions and 28 gaps) and 3.1% (16 nucleotide substitutions and 27 gaps) for DMKU-MP6-4T, DMKU-MP2-6 and DMKU-MP5-1, respectively. The name, Pichia bovicola, is proposed to accommodate these species. The holotype is DMKU-MP6-4T (TBRC 15616T=PYCC 8905T).

Identification and phylogenetic analysis of the pathogen of jute anthracnose in China

<p id="C3">Anthracnose is a major fungal disease that seriously affects the yield and quality in jute fiber. Identifying the types of anthracnose pathogens and determining their pathogenicity will help jute disease-resistant breeding and develop elite disease-resistant gene resources. The 92 strains were isolated and purified from samples of diseased jute, which were collected from 7 jute production areas including Fuzhou and Zhangzhou in Fujian Province, Xinyang City in Henan Province, Liu’an City in Anhui Province, Hangzhou City in Zhejiang Province, Nanning City in Guangxi Province, and Changsha City in Hunan Province. The sequence analysis of the rDNA-ITS region showed that 11 out of the 92 strains were typical anthracnose pathogens. Further identification of the phylogenetic tree of the LSU region and morphological characteristics revealed that 10 strains, including ZZ4 and GX19, were <italic>Colletotrichum gloeosporioides</italic>, while the strain of CS3 was <italic>Colletotrichum dematium</italic>. The pathogenicity test of artificial inoculation indicated that there was a significant difference in pathogenicity between <italic>Colletotrichum gloeosporioides</italic> and <italic>Colletotrichum dematium</italic>. These results laid a basis for identification of resistance gene loci and effective controlling of anthracnose in jute.

Two new species of Fusicolla (Hypocreales) from China

Two new Fusicolla species are described and illustrated based on strains isolated from soil and rotten twig. Morphological features and DNA sequence analyses of combined ITS and LSU regions support their placements in Fusicolla and recognition of new species. Fusicolla gigas sp. nov. is characterized by C-shaped, 0−4(−5)-septate microconidia, falcate, (1–)4–9-septate macroconidia up to 80 µm long, and pinkish orange colonies on PDA. Fusicolla guangxiensis sp. nov. is distinguished by long-fusiform, falcate, (0–)1(–3)-septate macroconidia with acute ends, and orange colonies on PDA. Their macroconidia produced on OA are obvious larger than those on PDA. Distinctions between the new species and their close relatives are compared in detail.

Isolation, identification, and application of yeast strains from the local ecosystem of Summer Black vineyard

Several indigenous yeast strains originating from the local ecosystems of Summer Black vineyard in Jingjiang, Jiangsu, China, were isolated and identified using Wallenstein Laboratory (WL) medium combined with 26S rDNA D1/D2 region sequence analysis. The 18 isolated colonies were classified into four different culture types. Two yeast strains were selected for vinification of Summer Black wines, and the volatile compounds were analyzed by headspace solid-phase microextraction–gas chromatography-mass spectrometry (HS-SPME/GC-MS). 2,3-butanediol, ethyl octanoate, ethyl hexanoate, and ethyl decanoate were the key odor-active compounds. The wines vinificated using the two selected yeast strains had higher quantities of identified compounds than that vinificated by commercial yeast. The two yeast strains isolated from the local ecosystem of Summer Black vineyard were more effective in producing aroma in Summer Black wines than commercial yeast was. This study will give some guidance or fundamental data toward wine making and yeast selection and fermentation. Novelty impact statement Saccharomyces cerevisiae and Brettanomyces bruxellensis were two main strains isolated from local “Summer Black” grape yard located in Jingjiang, Jiangsu Province, China. 2,3-butanediol, ethyl octanoate, ethyl hexanoate, and ethyl decanoate were major contributors to the flavor of the grape wine. The two yeast strains were much more effective in producing aroma in wine than commercial yeast strain BV818.

Expression of Regucalcin, a calcium-binding protein is regulated by hypoxia-inducible factor-1α

Regucalcin (RGN) regulates intracellular Ca2+ homeostasis and the activity of several proteins involved in intracellular signaling pathways, which highlights its importance in cell biology. Regucalcin has cytoprotective effects reducing intracellular levels of oxidative stress, also playing a crucial role in the control of cell survival and apoptosis. In an effort to assess its gene regulation, we initially identified the expression of Regucalcin in rat lungs treated with hypoxia at various time points. Previously, HIF-1α expression was also reported to be upregulated in hypoxia. Interestingly hypoxic induced Regucalcin expression in a fashion similar to that of HIF-1α expression in rat lungs. Sequence analysis of the Regucalcin promoter region revealed the presence of putative HRE binding motifs. Further analysis of the 1 kb Regucalcin promoter region with 5′ deletion and point mutants of HRE binding motif showed that the HRE binding site was critical for high promoter activity. In addition, HIF-1α protein binds directly to the HRE binding motifs within the Regucalcin promoter in-vivo, and regulates Regucalcin gene expression. All together, these findings suggest that Regucalcin is the novel target gene of HIF-1α and that Regucalcin gene expression in hypoxia may be regulated by the control of HIF-1α expression.

Genetic diversity in enhancer II region of HBV genotype D and its association with advanced liver diseases.

BackgroundHepatitis B Virus (HBV) is one of the most common human infectious agents, and the mutations in its genome may cause chronic hepatitis (CH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). This study was designed to characterize the enhancer-II (Enh-II) region of X gene in HBV positive patients to assess the association of such mutations with CH, LC, and HCC.MethodsHBV positive samples (N = 200) with patients’ demographic and clinical data were collected from different regions of Khyber Pakhtunkhwa (KP), Pakistan. The Enh-II region of the HBx gene was sequenced and zanalyzed for polymorphism associated with advanced liver disease. Univariate and logistic regression analyses were performed to evaluate potent mutations associated with a risk for LC and HCC.ResultsHBV Enh-II region sequences analysis revealed 25 different mutations. The highest frequency of mutations S101F (62.2%), A102V/R/G/I (56.25%), M103L/A (68.75%)were found in HCC, followed in LC and CH patients as 57.1%, 42.8%, 28.52% 16%, 15.2% and 18.4% respectively. H94 deletion in the α-box of the Enh-II region, associated with a high risk of HCC was found in half of the HCC patients. This deletion was present in 28.5% of LC and 6.5% of CH patients. Importantly, the high frequency of some notable mutations such as E109A/Y, A110S/K, Y111D/E, and F112L was first time reported in the entire study population. The frequencies of these mutations were high in HCC (43.75%, 37.5%, 50% and 43.75% respectively) as compared to LC (14.28%, 14.28%, 28.2% and 42.8%) and CH patients (12.8%, 15.2%, 16.8% and 16% respectively).ConclusionMutations associated with LC and HCC are prevalent in the Enh-II region in Pakistani HBV isolates. The mutations found are alarming in CH patients as these may progress to LC and HCC in a large number of patients.

Molecular detection of aster yellows phytoplasmas in ashwagandha plants in South India

Ashwagandha is a native medicinal plant of Indian sub-continent grown in different parts of the world for its medicinal value. The little leaf symptoms were observed in ashwagandha plants in Tamil Nadu, India. The nested PCR with the primer pair fU5/rU3, amplified 890 bp fragment of the 16S rRNA region of phytoplasma from five symptomatic leaf samples of ashwagandha plants. The nucleotide sequence analysis of 16S rRNA region revealed that the sequences of the five samples were identical and confirmed 99% nucleotide sequence identity with 16S ribosomal RNA gene sequences of ‘Candidatus Phytoplasma asteris’. This result indicate the presence of aster yellows phytoplasmas in this plants from South India.

Preliminary stock structure assessment of the ladyfish Elops machnata (Forsskal, 1775) in south-east and south-west coasts of India by mitochondrial DNA control region sequence analysis

Preliminary stock structure assessment of the ladyfish Elops machnata (Forsskal, 1775) in south-east and south-west coasts of India by mitochondrial DNA control region sequence analysis

Identification and Molecular Investigation of Diplostomum in Capoeta umbla Caught from Freshwater Sources, Turkey

In this study, identification of the cytochrome c oxidase subunit I (COI) gene region in mitochondrial DNA of the Diplostomum specimens, which are parasitic in the eyes of Capoeta umbla fish naturally distributed in Karakaya Dam Lake, Keban Dam Lake and Lake Hazar, (Elazığ province) was performed. A total of 8 haplotypes were determined using sequence analysis of the mtCOI gene region and phylogenetic trees were constructed by determining the genetic distance based on these haplotypes by the Maximum Likelihood (ML), Neighbor Joining (NJ), Minimum Evolution (ME) and Bayes methods. As a result, the lowest genetic distance was 0,00209 and the highest genetic distance was 0,01264. Phylogenetic analysis revealed a closer relationship between haplotype 3 and haplotype 4. It has been evaluated that all the samples studied belong to Diplostomum spathaceum and that the parasite may have been spread by the main hosts between study sites.

16S rDNA and ITS Sequence Diversity of Burkholderia mallei Isolated from Glanders-Affected Horses and Mules in India (2013-2019).

Glanders is a highly contagious and fatal infection of equids caused by the bacteria known as Burkholderia mallei. It is one of the notifiable equine diseases and is still present in Asia, South America and Africa. In India, glanders re-emerged in 2006, and thereafter, increasing numbers of cases were reported in different regions of the country. Between 2013 and 2019, 39 B. mallei were isolated from glanders-affected horses (n = 30) and mules (n = 9) from seven states of India such as Uttar Pradesh, Haryana, Delhi, Himachal Pradesh, Gujarat, Maharashtra and Tamil Nadu. In this study, the phylogenetic relationships of these isolates were assessed by sequence analysis of 16S rDNA gene and ITS region. Purified PCR-amplified products of 16S rDNA gene and ITS region were sequenced, aligned and phylogenetic trees were constructed using MEGA 11 software. Additionally, B. mallei 16S rDNA (n = 36) and ITS (n = 18) sequences available in the GenBank were also included for analysis to determine the diversity of older B. mallei isolates with recent Indian isolates. Both the phylogeny showed that the majority of the recent isolates from India are closely related to each other, but are genetically diverse from older isolates that originated from India. Nucleotide substitutions were also observed in a single and double position in 12 recent and two old Indian isolates. The study also indicates that similar B. mallei strains were responsible for glanders outbreaks in different states (Uttar Pradesh- Himachal Pradesh and Uttar Pradesh- Haryana) and this is due to the migration of infected animals from one state to another state. This study implies that 16S rDNA and ITS region may be used for molecular characterization of B. mallei associated with glanders in resource-limited settings.

Screening and evaluation of acaropathogenic fungi against the bulb mite Rhizoglyphus robini

• Fungal isolates effective in controlling bulb mites ( Rhizoglyphus robini ) were screened. • The selected fungal isolates have high virulence and thermotolerance. • Three Metarhizium anisopliae were determined to be the most suitable for bulb mite control. Bulb mites ( Rhizoglyphus robini ) damage the bulbs, corns, and tubers of garlic, shallots, and onions. Bulb mites have recently become a serious problem because of the continuous use of acaricides, which has resulted in resistance among bulb mite populations. Thus, there is a need to find alternative control methods to suppress bulb mite populations. Here, we report the results of screening pathogenic fungi for the control of R. robini . The initial screen was performed using 342 isolates of entomopathogenic fungi from soils from South Korea. As a result, 57 isolates of putative acaropathogenic fungi were selected from cadavers of bulb mites supporting fungal sporulation. However, 11 isolates were finally selected for further study through a re-evaluation of the pathogenicity of the isolates. These isolates were identified as two isolates of Metarhizium pemphigi , two isolates of Metarhizium pingshaense , and seven isolates of Metarhizium anisopliae by a microscopic examination and sequence analysis of the ITS region and EF-1α gene. The virulence, thermotolerance and UV-B tolerance of the 11 isolates were further evaluated and compared. Eight isolates showed more than 80% mortality and three isolates showed 100% mortality at 7 days after treatment. The thermotolerance of conidia showed a large difference depending on the fungal isolate, and five isolates showed a conidial germination rate of approximately 60% or more even after 2 h of heat treatment. On the other hand, the UV-B tolerance was very low in all the isolates, and only one isolate showed more than approximately 80% tolerance to 0.1 J cm −2 , but the other isolates showed a conidial germination rate of less than 30%. Based on all of the above results, three isolates, M. anisopliae 4–3-2, 4–8-1, and 4–31-2, were the most effective isolates in controlling bulb mites and could be considered promising biological control agents against bulb mites.

Neurodegenerative disease in association with sexual transmission of human T-cell lymphotropic virus type 2 subtype b in Argentina.

The aim of this study is to show that human T-cell lymphotropic virus type 2 (HTLV-2) infection produces symptoms resembling those described for HTLV-1-associated myeloneuropathy and to highlight the role of sexual transmission in the silent dissemination of HTLV-2. Patient samples were tested by particle agglutination and indirect immunofluorescence assay. The HTLV type was defined by molecular techniques. Nucleotide sequence analysis of HTLV-2 long terminal repeat region, T cell CD3/CD4 and T cell CD3/CD8 counts and typing of human leucocyte antigen (HLA) alleles A, B, C and DRB1 were also performed. HTLV-2 subtype b infection was confirmed in two blood donors and their sexual partners. Two patients exhibited distinctive signs and symptoms of progressive neurological disease. Three infected patients carried HLA-C*04. Both patients with neurological disease also carried HLA-A*31 and HLA-DRB1*07 alleles. Herein we describe for the first time sexual transmission of HTLV-2 in a non-endemic region of Argentina, highlighting the relevance of this transmission route in HTLV-2 silent dissemination out of the clusters of endemicity. We also provide evidence that HTLV-2 infection causes symptoms resembling those described for HTLV-1-associated myeloneuropathy. The evidence presented herein points to the critical need for public health strategies to reduce the spread of this neglected infection.

Zoonotic Genotypes of Enterocytozoon bieneusi in Wild Living Invasive and Native Carnivores in Poland.

Wild carnivores, both introduced and native species, are able to adapt well to peri-urban environments, facilitating cross-species pathogen transmission with domestic animals, and potentially humans. The role of wild living reservoir hosts cannot be ignored because of their known carriage of E. bieneusi zoonotic genotypes. In the past decades, populations of wild living carnivores, i.e., native, such as red foxes, and invasive, such as raccoon dogs and raccoons, have increased and adapted to synanthropic environments across Europe, including Poland. The knowledge concerning E. bieneusi genotype identification and distribution in wild carnivores is limited worldwide. A total of 322 individual fecal samples from six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine and beech martens, were collected and then analysed for the presence of E. bieneusi using the nested PCR method. Overall prevalence of the pathogen was estimated to be as high as 27.3%. The infection rates for E. bieneusi varied between the carnivore species, from 13.7% in beech martens to 40.4% in raccoon dogs. Based on sequence analysis of the ITS region of the rRNA gene marker, we detected five known genotypes of E. bieneusi in examined animals. In the invasive species, E. bieneusi NCF2 and D genotypes have been identified, whereas in the native ones, E. bieneusi NCF2, D, C, EbCar2 and Type IV genotypes were identified. All E. bieneusi genotypes recorded in this survey clustered in Group 1, showing their zoonotic potential. Our results provide the first description of the occurrence and genotypes of the microsporidian E. bieneusi in wild living population of raccoon dogs in Europe. Our findings are important for the study of pathogen epidemiology and emphasize the fact that the invasive and the native wild living carnivores, both widely distributed, should be considered more seriously as significant sources of zoonotic pathogens hazardous to domestic and farmed animals and humans.

Occurrence of collar rot on berseem (Trifolium alexandrinum L.) caused by Sclerotium rolfsii and its molecular characterization in India

Berseem (Trifolium alexandrinum L.) is one of the important legume fodder crops. It is widely grown in Central and Northern parts of India during winter season. Disease symptoms resembling to collar rot was observed in the breeder seed production and research fields of berseem at ICAR-IGFRI, Jhansi, Uttar Pradesh, India in March 2019 and April 2020. Disease incidence was ranged from 15–20% and 20-25% in 2019 and 2020, respectively. Disease symptom appeared as water soaked lesions on surface of collar region followed by the development of rotten areas with white mycelial mass and finally plants were dried. The associated pathogen was isolated and identified as Sclerotium rolfsii (Teleomorph: Athelia rolfsii) on the basis of morphological characters and sequence analysis of ITS, LSU and SSU regions of rDNA. Artificial inoculation of pathogen revealed cent per cent disease incidence and wilting of plants. To best of our knowledge, this is first complete report on etiology of collar rot disease caused by Sclerotium rolfsii on berseem in India.