First report of grapevine polerovirus 1 from a grapevine in China

Xinjiang Uygur Autonomous Region is the largest grape-producing area in China, with a grape output of 3.05 million tons in 2020, accounting for nearly 20% of the total grape output in China (National Bureau of Statistics, 2021). Viral disease is a major factor threatening the grape industry and results in large economic losses by affecting the quality of grapes and wines. Actually, nearly 80 different viruses have been recorded in grapevine (Fuchs, 2020). To identify viruses that infect grapevine in Xinjiang, leaves of four vines of cultivar Cabernet Sauvignon with symptoms of chlorotic spots and crinkling were collected from a vineyard at Shihezi University in Shihezi City in May 2019 and pooled for total RNA extraction (Invitrogen™ PureLink ® Plant RNA Reagent, USA). After ribodepletion, a cDNA library was prepared using the Ribo-ZeroTM Kit (Illumina, San Diego, USA) and subjected to high-throughput sequencing (HTS) on the Illumina NovaSeq 6000 platform (Novogene, China). In total, 41,799,420 paired-end reads (150 nt × 2) were obtained after performing quality control using Trimmomatic version 0.39 (Bolger et al., 2014). These reads were de novo assembled into 154,716 contigs using the rnaSPAdes method in SPAdes software with default parameters (Bankevich et al., 2012). BLASTn analysis of these contigs led to the identification of 59 viral-related contigs from 248 -18476 bp. These contigs belonged to six positive-stranded RNA viruses, namely, grapevine polerovirus 1 (GPoV-1; 2 contigs), grapevine berry inner necrosis virus (GBINV; 4 contigs), grapevine leafroll-associated virus 3 (GLRaV-3; 2 contigs), grapevine Pinot gris virus (GPGV; 3 contigs), grapevine rupestris stem pitting-associated virus (GRSPaV; 4 contigs), and grapevine fleck virus (GFkV; 17 contigs); one DNA virus, grapevine geminivirus A (GGVA; 2 contigs); and one viroid, Australian grapevine viroid (AGVd; 1 contig). Among them, GPoV-1 is a newly discovered grape-infecting virus that has recently been reported from Japan and France (Candresse et al., 2020; Chiaki and Ito, 2020). The two contigs of GPoV-1 were assembled manually into a 5627-nt scaffold that covers 99.6% of the genome of the reference GPoV-1 isolate (MT008025). The scaffold shared 98.5% and 98.2% nucleotide (nt) sequence identities with the French GPoV-1 isolate KT (MT008025) and the Japanese GPoV-1 isolate KC (LC505098), respectively. To confirm the GPoV-1 infection of the grapevines used for HTS analysis, we designed a primer pair targeting the coding region of the P1 protein GP30F (5′-CCTCTTTCGCTGCCATAGGC-3′) and GP2180R (5′-CCTGGAGCCTTAAGCTGGTG-3′) and applied them in revere transcription (RT)-PCR using a PrimeScriptTM One Step RT–PCR Kit (Takara, China) to detect GPoV-1. The expected 2151-bp fragment was amplified from one of the four grapevine samples. The amplicon was cloned into the pMD19-T vector (TaKaRa, China) and Sanger sequenced. BLASTn analysis showed that the sequence of the amplicon (GenBank accession no. OK574336) shared 98% identity with the scaffold obtained from HTS and shared 98.5% and 97.4% identity with the GPoV-1 isolates KT and KC, respectively. To determine the occurrence of GPoV-1 in the vineyard, 8 and 20 leaves were randomly collected from grapevines of cvs. Black Monukka and Cabernet Sauvignon, respectively. We designed a primer pair of GPoV4264F (5′-ACTGCACAGACTCTCACACG-3′) and GPoV4657R (5′- TCCTTCGCGCAGTCACTATC-3′), which target the coding region of the P3-P5 fusion protein. An expected 394-bp amplicon was detected in 2 out of the 8 Black Monukka and 7 out of the 20 Cabernet Sauvignon leaf samples. Sanger sequencing confirmed the GPoV-1 identity of the amplicons. Although all the samples used for HTS analysis displayed symptoms, 4 of 9 samples in which GPoV-1 infection was detected were asymptomatic, suggesting that GPoV-1 may be latent, as reported previously (Candresse et al., 2020). To the best of our knowledge, this is the first report of GPoV-1 infection of grapevine in China. Although most members of the genus Polerovirus (family Solemoviridae) are transmitted by aphids, how GPoV is transmitted remains unknown, representing an increased risk for its spread. Therefore, attention should be given to reducing the prevalence of GPoV-1 in grape-producing areas in China, especially in Xinjiang.

First Report of Powdery Mildew Caused By Golovinomyces monardae on Scarlet Beebalm (Monarda didyma) in China

Scarlet Beebalm (Monarda didyma) is a perennial ornamental flowering plant in the mint family, Labiatae. Due to low-maintenance, and a long blooming period, it is commonly cultivated in gardens as an ornamental plant in China. In May 2021, a disease was observed on the leaves of a capitals beebalm plant in a Ten Mile Flower Sea in Xiayi county (116°13′8″E, 34°14′45″N), Henan province of China. Symptoms first appeared as nearly circular, small, white, powdery mildew-like spots on the leaves which gradually expand, covering the entire leaves. The lesions spread from the lower leaves to the upper canopy, and the stems were also covered by white mycelia. In severe cases, early defoliation occured. About 30% plants were affected. Representative voucher specimens (SQNUMd04, SQNUDn4) were deposited in the herbarium of Shangqiu Normal University (SQNU), Shangqiu, China. Conidiophores (n = 30) were cylindrical, 92.0 to 142.2 µm long and 10.8 to 14.2 µm wide, and produced 5 to 7 immature conidia in a chain. Foot-cells of conidiophores were mostly curved at the base. Conidia (n = 30) were hyaline, ellipsoid, 23.3 to 29.8 μm (avg. 26.6 μm) long, and 11.2 to 16.9 μm (avg. 14.5μm) width, a length/width ratio of 1.5 to 2.1, and germ tubes were produced at the perihilar position. No chasmothecia were observed. Based on morphological comparison using the description by Scholler et al. (2016) description the fungus was tentatively identified as Golovinomyces monardae (G.S. Nagy) M. Scholler, U. Braun & Anke Schmidt. For molecular identification, DNA was extracted from mycelia and conidia, which were collected by scraping symptomatic leaves.The ITS regions and LSU were amplified using primers ITS1/ITS4 (White et al. 1990) and NL1/NL4 (Horisawa et al. 2013). BLASTn analysis of the (MZ303741) and LSU (MZ305434) sequences showed 100% identity with those of G. monardae (AB307667, LC076800, LC076802, LC076808, and AB077691) reported on Monarda species (Matsuda et al. 2003; Takamatsu et al. 2013; Scholler et al. 2016). Pathogenicity tests were carried out by gently dusting conidia from infected leaves onto healthy leaves of five M. didyma plants and five non-inoculated plants used as controls. After 9 days, typical powdery mildew colonies started to appear on the inoculated leaves while control plants remained disease free. All plants were placed in a greenhouse without temperature and humidity control. Based on morphology, fungus was identified as the same as that used for inoculum, fulfilling Koch's postulates. Although G. monardae has been reported on various genera in the Labiatae and Verbenaceae (Farr and Rossman 2021), to our knowledge, this is the first report of G. monardae causing powdery mildew of Scarlet Beebalm(M. didyma) in China.

Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains

Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of vanA and vanB genes using common primers. The system was designed using the Primer3 online software. The specificity of primers and probes was first checked by in silico PCR and by BlastN analysis. The genomic DNA of 255 bacterial isolates, including Enterococcus spp., Gram-negative, and Gram-positive strains, as well as a collection of 50 stool and 50 rectal swab samples, were tested to evaluate the specificity of the new real-time PCR (RT-PCR) system. The results of the designed RT-PCR were 100% specific and 100% positive on tested vancomycin resistant isolates harboring either the vanA or vanB gene. RT-PCR assays were negative for all other bacterial species tested including vancomycin-sensitive Enterococci and Enterococcus strains harboring vanC genes. The limit of detection of vanA and vanB genes by RT-PCR assay was 47 CFU/mL and 32 CFU/mL, respectively. The rapid and accurate detection of vancomycin-resistant Enterococci is the cornerstone for minimizing the risk of nosocomial transmissions and outbreaks. We believe that this assay will strengthen routine diagnostics and surveillance programs.

Amplicon-Based Analysis of the Fungal Diversity Across Kenyan Soda Lakes

BackgroundMicroorganisms have been able colonize and thrive in environments characterized by low/high pH, temperature, salt or pressure. Examples of extreme environments are the soda lakes and soda deserts. The objective of this study was to explore the fungal diversity across soda lakes Magadi, Elmenteita, Sonachi and Bogoria in Kenya. A new set of primers was designed to amplify a fragment long enough for the 454-pyrosequencing technology. Results Analysis of the amplicons generated showed that the new primers amplified for eukaryotic groups. A total of 153,634 quality-filtered, non-chimeric sequences were used for community diversity analysis. The sequence reads were clustered into 502 operational taxonomic Units (OTUs) at 97% similarity using BLASTn analysis of which 432 were affiliated to known fungal phylotypes and the rest to other eukaryotes. Fungal OTUs were distributed across 107 genera affiliated to the phylum Ascomycota, Basidiomycota, Glomeromycotina and Incertae Sedis. The Phylum Ascomycota was the most abundant phylotype. Overall, fifteen (15) genera (Chaetomium, Monodictys, Arthrinium, Cladosporium, Fusarium, Myrothecium, Phyllosticta, Coniochaeta, Diatrype, Sarocladium, Sclerotinia, Aspergillus, Preussia and Eutypa) accounted for 65.3% of all the reads. The Genus Cladosporium was detected across all the samples at varying percentages with the highest being water from Lake Bogoria (51.4%). Good’s coverage estimator values ranged between 97 and 100%, an indication that the dominant phylotypes were represented in the data. ConclusionThese results provide useful insights that can guide cultivation dependent studies in order to understand the physiology and biochemistry of the as yet uncultured taxa.

First report of Apple rubodvirus 2 infecting pear (Pyrus communis) in South Africa

Apple rubbery wood virus 2 (ARWV-2; Rott et al., 2018) belong to the species Apple rubodvirus 2, a member of the genus Rubodvirus (family Phenuiviridae; Kuhn et al 2020). ARWV-2 was first identified in apples and is associated with apple rubbery wood disease (ARWD) that is characterized by unusual flexibility of stems and branches, reduced growth, shortened internodes and increased cold sensitivity (Jakovljevic et al., 2017, Rott et al., 2018). ARWD was first reported in 1935 in England on apple and has since been found on quince and pear (Jakovljevic et al., 2017; Rott et al., 2018). In January 2021, leaves were collected from a pear tree (Pyrus communis cv. Forelle, F514) in a commercial orchard near Villiersdorp, South Africa. The tree displayed no foliar or tree branch symptoms, except for malformed fruits potentially due to insect feeding damage or pear stony pit disease previously associated with infection of apple stem pitting virus (ASPV) (Paunovic et al. 1999). Leaf petioles (one gram) were used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). A sequencing library was constructed (Illumina TruSeq Stranded Total RNA with plant Ribo-Zero) and sequenced on an Illumina HiseqX instrument (Macrogen, South Korea). A total of 30,709,182 paired-end reads (100 nt) were obtained and trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly, using default parameters of CLC Genomics Workbench 11.0.1 (Qiagen), resulted in 97,294 contigs. BLASTn analysis identified 17 viral contigs, with 14 contigs having high nucleotide identity to ASPV and three to ARWV-2. The latter contigs included all three segments of ARWV-2. The L contig was 7371 nts, M was 1289 nts and S was 1463 nts in length, generated with 7341, 626 and 9161 reads for segment L, M and S, respectively. Segment S had the highest read coverage (524.87x), followed by segment L (88.07x) and M (36.60x). The ARWV-2 GenBank accessions with the highest percentage identity to the contigs were MF062128.1 from United States of America (98.2% to segment L), MN163134.1 from China (97.5% to segment M) and NC_055535.1 from Germany (93.5% to segment S). The contigs spanned 100%, 80.92% and 100% of these accessions of segments L, M and S, respectively and were deposited in GenBank as accessions MZ593725- MZ593727. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the presence of ARWV-2 in sample F514, using primers directed at segments L (con708_178F/con708_666R), M (ARWaV-2S1_38F/ARWaV-2S1_682R) and S (ARWaV-2M567F/ARWaV-2M1342R) (Rott et al., 2018). Amplicon sequences (510 bp (L), 645 bp (M) and 799 bp (S)) were confirmed with bi-directional Sanger sequencing. Fifty-nine additional pear samples were surveyed in 2021 for ARWV-2 using the M segment assay as mentioned above. The survey included the Koue Bokkeveld and Elgin areas, and cultivars Bosc (22 samples), Abate (10 samples), Rosemarie (3 samples), Forelle (9 samples), Packham’s Triumph (12 samples) and Early Bon Chretien (3 samples). A total of 27 samples (11 samples from the Koue Bokkeveld region and 16 samples from the Elgin region) tested positive for ARWV-2, demonstrating the common presence of this virus in pears in South Africa. This is the first report of ARWV-2 infecting pear in South Africa. Although no association with disease symptoms were observed, this study expands the data on the incidence and distribution of this virus in South Africa.

Co-Carriage of Metal and Antibiotic Resistance Genes in Sewage Associated Staphylococci

Controlling spread of resistance genes from wastewater to aquatic systems requires more knowledge on how resistance genes are acquired and transmitted. Whole genomic sequences from sewage-associated staphylococcus isolates (20 S. aureus, 2 Staphylococcus warneri, and 2 Staphylococcus delphini) were analyzed for the presence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs). Plasmid sequences were identified in each isolate to investigate co-carriage of ARGs and MRGs within. BLASTN analysis showed that 67% of the isolates carried more than one ARG. The carriage of multiple plasmids was observed more in CC5 than CC8 S. aureus strains. Plasmid exchange was observed in all staphylococcus species except the two S. delphini isolates that carried multiple MRGs, no ARGs, and no plasmids. 85% of S. aureus isolates carried the blaZ gene, 76% co-carried blaZ with cadD and cadX, with 62% of these isolates carrying blaZ, cadD, and cadX on the same plasmid. The co-carriage of ARGs and MRGs in S. warneri isolates, and carriage of MRGs in S. delphini, without plasmids suggests non-conjugative transmission routes for gene acquisition. More studies are required that focus on the transduction and transformation routes of transmission to prevent interspecies exchange of ARGs and MRGs in sewage-associated systems.

Genome Characterization and Phylogenetic Analysis of the First Bovine Rhinitis B Virus Isolate in China

Bovine rhinitis B virus (BRBV) is an emerging viral species in the genus Aphthovirus, family Picornaviridae. Studies suggested that BRBV was considered a potential etiological agent of bovine respiratory disease complex (BRDC). BRBV has been reported in the United States, Sweden, Canada, Japan, and Mexico. However, little information of BRBV was available in China. In this study, we performed viral metagenomic analysis in a calf with respiratory disease. The results showed high abundance (3.85) of BRBV nucleotide and 248 mapped reads in calf samples. Online BLASTn analysis showed that three contigs of those had the highest nucleotide similarity (95%) with one Swedish BRBV isolate (BRBV_SWE1, GenBank accession no. KY432299). To identify the genome characterization of the Chinese BRBV isolate (designated CHN1), six couples of overlapping RT-PCR primers were designed according to genome sequences of BRBV_SWE1. Through gene cloning and splicing, we obtained the genome information of CHN1, possessing 7,465 nucleotides (46.6% G+C). Although CHN1 had the highest nucleotide similarity (95.1%) with BRBV_SWE1, one 11-nucleotide (ACATTTGTTGT) deletion occurred in the 5′ untranslated region compared to SWE1. Phylogenetic analysis showed that CHN1 clustered together with BRBV_SWE1, and far from other BRBV isolates. This study recorded the first discovery of BRBV infection in China. Further investigation should be made in order to evaluate the infection status and epidemiological significance of BRBV in China.

Strawberry, a new natural host of Brassica yellows virus in China

Brassica yellows virus (BrYV; genus Polerovirus, family Solemoviridae) has an icosahedral spherical virion with a positive-sense single-stranded RNA genome and it is distinguished from turnip yellows virus (TuYV) based on differences in ORF0 and ORF5 (Xiang et al., 2011). To investigate the occurrence and distribution of viruses infecting strawberry (Fragaria ananassa) in the main production areas in China, a survey of nine greenhouses (667 m2 each) was conducted in the cities of Yantai and Beijing, China in August 2020. About 1% of strawberry plants in each greenhouse showed virus-like symptoms of chlorotic spots; 89 symptomatic leaf samples were randomly collected for virus testing. Total RNA was extracted from a pool of eight samples of four different cultivars (Hokowase: 2, Mibao: 2, Sagahonoka: 2, Monterey: 2) from Yantai using RNAprep Pure Plant Plus Kit (TianGen, China). A cDNA library was constructed by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) after ribosomal RNA-depletion using an Epicentre Ribo-Zero™ rRNA Removal Kit (Epicentre, USA). High-throughput sequencing was done on Illumina Hiseq 4000, generating 70,931,850 high-quality 150 bp paired-end reads. Clean reads were de novo assembled by Trinity (v2.2.0) and the resulting contigs were screened by BLASTn and BLASTx against GenBank database as described previously (Grabherr et al., 2013). A total of 1,432,164 high-quality reads unmapped to the strawberry genome were obtained and assembled into 93 contigs (ranging from 33 to 8,031 nt). Seven of these contigs (277 to 1,254 nt) shared 98.2 to 100% nt identities with BrYV-A (accession no. HQ388348) and covered 89.5% of the genome of BrYV-A. Subsequent analyses indicated the presence of Strawberry pallidosis-associated virus and Strawberry mottle virus in the analyzed sample, both have been reported in strawberry in China (Shi et al., 2018; Fan et al., 2021). To confirm BrYV infection, total RNA was isolated from the eight samples used for HTS and reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers (CP and rtp, Supplementary Table 1) designed based on the assembled contigs. PCR products with expected sizes (587 and 609 bp) were observed in one sample (cv. Mibao). BLASTn analysis indicated that the amplicons (accession no. MW548437 and MW548438) shared 98.6% and 99.3% nt identity with BrYV-A, respectively. To obtain the complete sequence of the putative BrYV isolate, the gaps were bridged and the terminal sequences were determined using 5ʹ and 3ʹ RACE kits (Clontech, China) based on the assembled contigs. The complete genome sequence of the putative BrYV isolate has a length of 5,666 nt (accession no. MZ666129) and shares more than 94.3% nt identities with other BrYV isolates. Phylogenetic analysis indicated that the isolate grouped closely with BrYV and further from TuYV (Figure S1). In addition, 11 samples (cv. Benihoppe) of the remaining 81 symptomatic strawberry samples tested positive for BrYV by RT-PCR with the two pairs of primers mentioned above. The sequences (accession no. MZ407232 and MZ407233) revealed 99.5% and 99.3% nt identities with MW548437 and MW548438. To the best of our knowledge, this is the first report of natural infection of BrYV in strawberry plants. Our findings expand the host range of BrYV, but disease association is difficult to establish due to presence of mixed infection and non-fulfillment of Koch's postulates.

Identification of Plectosphaerella melonis from cucumber plants in Ukraine

A fungus was isolated from diseased roots of Cucumis sativus grown in greenhouses. The morphological and cultural characteristics of the isolate allowed it to be classified as Plectosphaerella melonis. BLASTn analysis revealed 99% homology of the ITS sequence from the isolate with 14 Acremonium cucurbitacearum and P. melonis isolates, allowing attribution of the isolate to P. melonis (syn. A. cucurbitacearum). Koch’s hypothesis requirements were fulfilled for the isolate. Symptoms on host roots developed after 14 d of growing cucumber plants on infested soil. Plants of the cucumber variety Nizhynskyi 12 were very susceptible at the two leaf growth stage (2 weeks after sowing). Above-ground disease symptoms were absent after 14 d, even with severely diseased roots. This is the first report of P. melonis on C. sativus in Ukraine.

Biological Characteristics And Genomic Analysis of A Novel Bacteriophage BUCT609 Infecting Stenotrophomonas Maltophilia

Stenotrophomonas maltophilia is widely distributed in nature and has a high isolation rate in nosocomial infections. Due to its potential application and important role in clinical practice, the relevant studies of S.maltophilia have received much attention. S.maltophilia phage BUCT609 (GenBank: MW960043) was isolated from hospital sewage with S.maltophilia strain No. 3015 as a host. Morphologically, it can be inferred as Podoviridae phage from the result of transmission electron microscopy (TEM). The electron microscopy also shows that the phage has an isometric capsid ~50 nm in diameter. The one-step growth curve demonstrated that the incubation period of 10 min and the burst size is 382 pfu/cell when its optimal multiplicity of infection (MOI) is 0.01. Phage BUCT609 had a high survival rate at pH 3 to 10 and tolerant temperature from 4 ℃ to 55 ℃. Next-Generation Sequencing (NGS) results demonstrated that its complete genome is linear double-stranded DNA of 43145 bp in length, and the GC content is 58 %. It has very little resemblance to other phages. The BlastN analysis shows that the genome of phage BUCT609 shares 22 % homology with S.maltophilia phage Ponderosa (GenBank: MK903280.1), and it encodes 56 putative proteins, of which only 25 have annotated function. Phage BUCT609 with a relatively large burst size and excellent survival ability in a wide pH and temperature range, suggests BUCT609 is a potential alternative for multi-drug resistance S.maltophilia therapy.