Identification and Complete Genome Sequence of an Alternavirus From Airborne Spores of the Phytopathogenic Fungus Fusarium Bullatum

A double-stranded RNA (dsRNA) mycovirus was isolated from airborne spores of Fusarium bullatum and was named Fusarium bullatum alternavirus 1 (FbAV1). Sequencing analysis and the rapid amplification of cDNA ends (RACE) of 5’ and 3’-end confirmed three segments: dsRNA1 (3546 nt), dsRNA2 (2511 nt) and dsRNA3 (2484 nt). BLASTN search of sequences showed that FbAV1 has 92-96% identity with Fusarium incarnatum Alternavirus 1 (FiAV1). Phylogenetic analysis of the RdRp amino acid sequences suggested that the dsRNA mycovirus in this study clustered with the newly proposed family “Alternaviridae”. This is the first report of FbAV1 mycovirus from airborne spores of a fungus F. bullatum.

First report of Pectobacterium versatile causing blackleg of potato in Serbia

Potato blackleg is frequently observed on the production fields in the Bačka region of Vojvodina province, which is one of the largest potato-growing areas in Serbia. This disease usually occurs during June and July. In July 2020, blackleg symptoms in the form of stem necrotic lesions, vascular discoloration, hollow stems, and wilting of whole plants were noted on potato cultivar VR808 on a field 28 ha in size located in Maglić village (GPS coordinates 45.349325 N, 19.542768 E). Disease incidence was estimated at 20−25%. Isolations were performed from 12 potato samples on Crystal Violet Pectate medium (CVP). Stem sections consisted of brown lesions and healthy tissue (c.10 cm) were surface sterilized with ethyl alcohol 70% (w/v) and rinsed with sterile distilled water. Small pieces of tissue were taken at the edges of stem lesions (between healthy and diseased tissue) were soaked in phosphate buffer saline for 20 min and plated using a standard procedure (Klement et al. 1990). Single colonies that formed pits after 48 hours at 26 °C were re-streaked onto Nutrient Agar (NA) where creamy white colonies with smooth surfaces were formed. A total of 30 isolates were selected and DNA isolated from the colonies was further analyzed by polymerase chain reaction (PCR) using the partial dnaX gene (DNA polymerase subunit III gamma/tau) with primer pair dnaXf/dnaXr for Pectobacterium and Dickeya species identification (Slawiak et al. 2009). A single characteristic band of 535 bp was amplified in all isolates (Slawiak et al. 2009). DNA sequence alignment showed two distinct groups of isolates (Fig.S1), which were genetically uniform within each group. Using BLASTn search, it was established that the dnaX sequence of the first group (consisting of 19 Serbian potato isolates) had 99.79% identity with NCBI-deposited Pectobacterium versatile strains 14A and 3-2 from potato from Belarus (Acc. No. CP034276 and CP024842, respectively) as well as SCC1 from Finland (Acc. No. CP021894). The remaining 11 dnaX sequences had 100% identity with Pectobacterium carotovorum subsp. carotovorum strain CFBP7081 originating from water in Spain (Acc. No. MK516961). The partial dnaX sequences of three Serbian P. versatile isolates (Pv1320, Pv1520, and Pv1620) and one P. carotovorum subsp. carotovorum (Pcc2520) were deposited in GenBank under Acc. No. MW839571, MW805306, MW839572, and MW805307, respectively. These results, indicating combined infection in the observed field, signify the first identification of P. versatile in Serbia. Multilocus sequence analysis (MLSA) performed with proA (proAF1/ proAR1) and mdh (mdh2/mdh4) genes (Ma et al. 2007; Moleleki et al. 2013) grouped three tested Serbian potato P. versatile isolates together with P. versatile strains from NCBI (Fig.S2). For both tested genes, BLASTn search revealed 100% homology with P. versatile strain SCC1 from Finland. Three Serbian P. versatile potato isolates were deposited under Acc. Nos. MZ682623-25 for proA and MZ682620-22 for mdh genes. According to the routine tests suggested for Pectobacteriaceae (Schaad et al. 2001), Serbian isolates possessed microbiological traits identical to P. versatile description (Portier et al. 2019). Pathogenicity was performed on potato cultivar VR808 with three selected P. versatile isolates (Pv1320, Pv1520, and Pv1620) in the following assays: (i) surface-sterilized tuber slices with holes in the center filled with 100 µL of bacterial suspensions (adjusted to 109 CFU mL-1) to test the isolates’ ability to cause soft rot, and (ii) young, four-week old plants with developed 3rd true leaf (c. 30 cm tall) were inoculated by injecting stems with bacterial suspension adjusted to 107 - 108 CFU mL-1 at a height 5 cm above the soil line. Negative controls were treated with sterile distilled water. Inoculated plants were kept under controlled conditions (25 °C temperature and >70% relative humidity). Each assay was replicated twice. Soft rot appeared on tuber slices 24 h after inoculation. On inoculated stems, initial symptoms manifested as greasy elongated spots at inoculation sites two days after inoculation (DAI), and subsequently extended along the vascular tissue and became necrotic. Whole plant's decay was recorded in five DAI, while negative controls remained healthy. To complete Koch's postulates, bacteria were re-isolated from symptomatic potato plants and confirmed by PCR and sequencing of dnaX. This first report of P. versatile in potato indicates that blackleg currently present in Serbia is caused by a diverse bacterial population. This pathogen was first identified in genome comparison as ‘Candidatus Pectobacterium maceratum’ (Shirshikov et al. 2018) and was later renamed as Pectobacterium versatile sp. nov. (Portier et al. 2019). Thus far, bacterium Pectobacterium carotovorum subsp. brasiliensis has been recognized as dominant pathogen on most of the infected fields in Vojvodina province, and was recently noted on one plot subjected to a combined infection with Dickeya dianthicola (Marković et al. 2021). Findings achieved in this study are highly relevant, as they point to the diversity in potato blackleg pathogens, likely due to the increasingly widespread distribution of imported seed potatoes.

First Report of Little cherry virus 1 Infecting Sweet Cherry in Ontario, Canada

The Niagara fruit belt is one of the richest fruit-producing areas in Canada, contributing to 90% of Ontario's tender fruits such as peach, plum and sweet cherry. Little cherry virus 1 (LCV1) of the genus Velarivirus is a causal agent of little cherry disease which has devastated cherry crops in many regions (Eastwell and Bernardy 1998, Jelkmann and Eastwell, 2011). From 2013 to 2018, foliar symptoms indicative of viral infection such as leaf deformation, ringspot, mottling, vein clearing, and reddening were found on sweet cherry trees grown in the Niagara region. To determine if these trees were infected by a virus, small RNAs (sRNAs) were isolated from separately pooled asymptomatic and symptomatic leaves using the mirPremier microRNA isolation kit (Sigma Aldrich Canada, Oakville, ON). The sRNAs were used to create two libraries (four leaves per library) with the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA). The sRNA libraries were separately sequenced with the MiSeq Desktop Sequencer (Illumina, San Diego, CA). In total, 5,380,196 reads were obtained and Trimmomatic (Bolger et al. 2014) was used to remove adaptors. The remaining 4,733,804 clean reads were assembled into contigs using Velvet 0.7.31 (Zerbino and Birney, 2008) and Oases 0.2.09 (Schulz et al. 2012) with minimum length of 75 nt (Supplementary Table 1). A BLASTn search (Altschul et al. 1997) of the contigs identified the presence of Cherry virus A (genus: Capillovirus), two members of the Ilarvirus genus (Prunus necrotic ringspot virus and Prune dwarf virus) in both libraries. LCV1 was only found in contigs derived from the symptomatic library. Of the clean reads, 22,016 were assembled into six contigs (with lengths ranging from 86 to 116 nt, Supplementary Table 1) mapping to LCV1, covering 7.07% of the viral genome. To confirm LCV1 infection, primers were designed from the assembled contigs and used for reverse transcription polymerase chain reaction (RT-PCR). Amplicons were sequenced and the terminal sequences were determined using 5’ and 3’ RACE Systems (Invitrogen, Burlington, ON). Degenerate primers were designed from multiple sequence alignments of published LCV1 genomes for amplification and primer walking to obtain the sequence of LCV1 (Table S2). The complete genome sequence of LCV1 has a length of 16,934 nt and was deposited in GenBank (accession no. MN508820). A BLASTn search showed that this isolate is nearly identical (99.6% sequence identity) to an isolate from California (accession no. MN131067). To determine the incidence of infection, a field survey was performed at the same location during spring months of 2014 to 2018 using RT-PCR with primers specific to the viral coat protein gene (Supplementary Tale 2). Among 46 cherry trees sampled, two (4.3%) trees were infected with LCV1 and showed negative results with CVA, PNRSV and PDV. Both trees displayed mild suturing of primary and secondary veins (Supplementary Figure 1). LCV1 has been identified in Western stone fruit producing regions (British Columbia in Canada, and Washington, California, and Oregon in the United States of America). To the best of our knowledge, this is the first report of LCV1 in any eastern region of Canada. The low incidence of LCV1 suggests that this virus is not widespread in this region. Routine monitoring and detection of LCV1 is required to prevent this devastating cherry disease from spreading in this region.

DETECTION OF ALFACORONAVIRUSES, BETACORONAVIRUSES AND ASTROVIRUSES IN BAT FECAL SAMPLES FROM MOSCOW REGION

In our study we analyzed viral RNA in bat fecal samples from Moscow region (Zvenigorod district) collected in 2015. To detect various virus families and genera in bat fecal samples we used PCR amplification of viral genome fragments, followed by high-throughput sequencing. Blastn search of unassembled reads revealed the presence of viruses from families Astroviridae, Coronaviridae and Herpesviridae. Assembly using SPAdes 3.14 yields contigs of length 460–530 b.p. which correspond to genome fragments of Coronaviridae and Astroviridae. The taxonomy of coronaviruses has been determined to the genus level. We also showed that one bat can be a reservoir of several virus genuses. Thus, the bats in the Moscow region were confirmed as reservoir hosts for potentially zoonotic viruses.

DETECTION OF ALFACORONAVIRUSES, BETACORONAVIRUSES AND ASTROVIRUSES IN BAT FECAL SAMPLES FROM MOSCOW REGION

In our study we analyzed viral RNA in bat fecal samples from Moscow region (Zvenigorod district) collected in 2015. To detect various virus families and genera in bat fecal samples we used PCR amplification of viral genome fragments, followed by high-throughput sequencing. Blastn search of unassembled reads revealed the presence of viruses from families Astroviridae, Coronaviridae and Herpesviridae. Assembly using SPAdes 3.14 yields contigs of length 460–530 b.p. which correspond to genome fragments of Coronaviridae and Astroviridae. The taxonomy of coronaviruses has been determined to the genus level. We also showed that one bat can be a reservoir of several virus genuses. Thus, the bats in the Moscow region were confirmed as reservoir hosts for potentially zoonotic viruses

BIoInfoRmatIonal analySIS of YersiniapseudotuberculosisIP32953 CRISPR/CaSSyStem

The results of this study include Yersinia pseudotuberculosis CRISPR/Cas system structure analysis. CRISPR/Cas system is a specific adaptive protection against heterogeneous genetic elements. The object of research was the complete genome of Y. pseudotuberculosis IP32953 (NC_006155). CRISPR/Cas system screening was performed by program modelling methods MacSyFinder ver. 1.0.2. CRISPR loci screening and analyzing were carried out by program package: CRISPR Recognition tool (CRT), CRISPI: a CRISPR Interactive database, CRISPRFinder, and PilerCR. Spacer sequences were used in order to find protospacers in ACLAME, GenBank-Phage and RefSeq-Plasmid databases by BLASTn search algorithm. Protospacer sequences could be found in genomes of phages, plasmids and bacteria. In last case complete genomes of bacteria were analyzed by online-tool PHAST: PHAge Search Tool. Y. pseudotuberculosis IP329353 has CRISPR/Cas system that consists of one sequence of cas-genes and three loci. These loci are far away from each other. Locus YP1 is situated in close proximity to cas-genes. Protospacers were found in genomes of Y. pseudotuberculosis PB1/+, Y. intermedia Y228, Y. similis str. 228, Salmonella phage, Enterobacteria phage, Y. pseudotuberculosis IP32953 plasmid pYV and plasmid of Y. pseudotuberculosis IP31758. Thus, the combination of four program methods allows finding CRISPR/Cas system more precisely. Spacer sequences could be used for protospacer screening.

Three new species of Calocybella from India based on morphology and molecular phylogeny

Three new species of Calocybella, C. lohitha, C. babruka and C. swetha are described from Kerala State, India based on morphology and molecular phylogeny. Comprehensive descriptions of these species are provided along with photo-illustrations and comparisons with phenetically similar and phylogenetically related species. Nuclear ribosomal internal transcribed spacer (ITS) region of the three species were sequenced and analyzed. In addition, nuclear ribosomal large subunit (nrLSU) region of Calocybella swetha was sequenced and used in BLASTn search to find similar sequences. ITS-based phylogenetic analysis using Maximum likelihood (ML) method supported both the novelty of these species and their placement within the genus. The discovery of these three species forms the first record of Calocybella outside Europe.

Three new species of Russulaceae from India based on morphology and molecular phylogeny

Three new species of Russulaceae, Lactifluus umbonatus and Lactifluus indicus belonging to Lf. subg. Gerardii and Lactarius keralensis belonging to L. subg. Piperites, are described from Kerala State, India. Comprehensive descriptions, photographs, line drawings and comparisons with phenetically similar and phylogenetically related species are given. Nuclear ribosomal internal transcribed spacer regions (ITS) of the three species were sequenced and analyzed. In addition, nuclear ribosomal large subunit (nLSU) gene of Lactifluus indicus was sequenced and the sequence was used in BLASTn search to find similar sequences. Maximum likelihood (ML) analysis based on the ITS sequences confirmed both the novelty of these species and their placement within their respective genera. The discovery of Lactifluus umbonatus and Lf. indicus represent the first report of novel species belonging to Lf. subg. Gerardii from India. Lactifluus indicus represents the second record of a pleurotoid Lactifluus species from India.

Identification and Characterization of Panama Wilt Causing Fungal Isolates to Musa Paradisiaca cv. Puttabale

Musa paradisiaca L. (Musaceae) cultivar ‘Puttabale’ is an indigenous banana commonly cultivated in the farmyards of Chitradurga,Davanagere, Chikmagalur and Shivamogga district of Karnataka. The fruits are valued for delicious taste. This banana cultivar is highlysusceptible to Fusarium infection and the yield loss has estimated to 30-40% at standing stage. The corm tissue of Musa paradisiaca cv.Puttabale showing typical panama wilt symptoms were collected from pathogen infected banana farmyard of Bhadravati, Sagara, Shikaripura,Holehonnur, Soraba, Thirthahalli, Gonibeedu and Lakkavalli regions of Shivamogga district of Karnataka. These infected corms were cultureon PDA medium and examined carefully by morphological and molecular identification. Out of the eight fungal isolates, the colonies of fungalisolates collected from Bhadravati, Sagara, Holehonnur, Thirthahalli, Gonibeedu and Lakkavalli regions produced a dense white aerialmycelium that evenly spread on the growth medium similar to the colony texture of Fusarium species. Among these fungal isolates, thepathogen collected from Gonibeedu region showed the presence of sickle or rod shaped, four-celled macro conidiophores, round or ovalshaped chlamydospores and the BLASTn search result showed 99 % similarity with the sequences of Fusarium oxysporum f. sp. cubense(Foc). The only one fungal isolate showed the presence of actual panama wilt causing fungi.DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11135 Int J Appl Sci Biotechnol, Vol. 2(4): 420-425

First Record of Trichodorus primitivus and Morphological and Molecular Identification of Longidorus elongatus from Canada

During July 2008, sandy loam soil samples were collected near the rhizosphere of Kentucky bluegrass (Poa pratensis L.) and tall fescue (Festuca arundinacea Schreb.) at a depth of 15 to 20 cm from Caledonia Park, Toronto, Canada. Samples were suspended in water and nematodes were collected on sieves with 250- and 74-μm openings. Among the nematodes recovered were one species of Longidorus Micoletzky, 1922 (4) and one species of Trichodorus Cobb, 1913. The Trichodorus species (2), was identified as T. primitivus (de Man, 1880) Micoletzky, 1922, in which females have rod-like sclerotized pieces parallel to the vagina lumen, each with dimensions of 3.1 × 1.2 μm and at a distance of 2 to 3 μm from each other; and males have spicules with a wide nonoffset capitulum and very narrow blade, three ventromedian cervical papillae (CP), and three precloacal supplements with the anterior one at the level of the capitulum of retracted spicules. Males showed unusual variation in the position of CP2 (i.e., posterior to the onchiostyle region, except for one specimen with CP2 located within the posterior onchiostyle region, which is typical for the species). The morphometric data for nine males are: L, 665 to 805 μm; a, 23.0 to 27.5; b, 3.7 to 5.2; c, 46 to 56; onchiostyle, 47 to 52 μm; and spicules, 32.5 to 40.5 μm. Measurements for 13 females are: L, 630 to 775 μm; a, 20.5 to 24.8; b, 3.5 to 5.5; c, 75.5 to 158.5; and V, 54 to 61%. To our knowledge, this is the first report of T. primitivus in Canada. According to Chen et al. (1), the Longidorus species was identified as L. elongatus (de Man, 1876) Micoletzky, 1922 (3). The morphometric data for females (n = 13) are: L, 5.1 to 6.0 mm; a, 78.5 to 106.5; b, 11.5 to 13.5; c, 86.0 to 120.5; V, 45.5 to 53.0%; odontostyle, 79 to 91 μm; odontophore, 57.5 to 66.5 μm; and tail length, 44.5 to 59.0 μm. Males were not found. Four juvenile stages were identified. J1 (n = 2) with a body length of 1.1, 1.2 mm and replacement/functional odontostyle 59, 59/53.5, 54.5 μm, J2 (n = 2) with L: 1.7, 2.0 mm and replacement/functional odontostyle 62.5, 68.0/58.7 μm, 59.0, J3 (n = 2): L: 2.8, 3.0 mm and replacement/functional odontostyle 76.5, 77/66.5, 67.0 μm and J4 (n = 2) with L: 3.6, 3.8 mm and replacement/functional odontostyle 87.0, 90.5/75.0, 77.5 μm. Due to large morphometric overlap in Longidorus species identification, 2,472 bp of the near full-length 18S and the internal transcribed spacer 1 region of rDNA (Accession No. GU199044) were sequenced. The Blastn search of the partial 18S revealed 100% identity with a population of L. elongatus from Scotland (GenBank No. AY687992, 1,707 bp compared), 99% identity (3 bp difference, 1,707 bp compared) with a population of L. elongatus from Iran (EU503141) and 99% identity (4 bp difference, 1,707 bp compared) with a population of L. elongatus (AF036594, sample location unknown). A Blastn search of the 18S and ITS region revealed only 1 to 3 bp differences with two populations of L. elongatus from Switzerland (AJ549986 and AJ549987) and a population of L. elongatus (AF511417) from Scotland. These molecular data further confirmed the identity of the population from Canada to be L. elongatus. References: (1) Q. Chen et al. Fundam. Appl. Nematol. 20:15, 1997. (2) W. Decraemer. Kluwer Academic Publishers, Dordrecht, the Netherlands, 1995. (3) J. G. de Man. Tijdschr. Ned. Dierk. Ver. 2:78, 1876. (4) H. Micoletzky. Archiv. Naturgesch. 87:1, 1922.