Primer Pairs Research Articles

Squash vein yellowing virus from California emerged in the Middle East via intragenic and intergeneric recombination events in the hypervariable potyvirus P1 and ipomovirus P1a genes.

We present the complete sequence of the genomic RNA of an isolate of squash vein yellowing virus (Ipomovirus cucurbitavenaflavi) from California (SqVYV-CA) and show it is a recombinant virus with a highly divergent 5' UTR and proximal P1a gene. The evolution of SqVYV-CA involved an intrageneric event between unknown potyviruses, related to isolates of papaya ringspot virus (Potyvirus papayanuli) from the Old World, and an intergeneric event between this recombinant potyvirus (minor parent) and an isolate of SqVYV from Israel (SqVYV-IL) (major parent). These events occurred in mixed infections and in the potyvirus P1 and ipomovirus P1a recombination hotspots and resulted in SqVYV-CA having a potyvirus 5' UTR and chimeric P1-P1a gene/protein and the remainder of the genome from SqVYV-IL. The SqVYV-CA chimeric P1-P1a gene is under positive selection, and the protein is intrinsically disordered and may localize to the nucleus via nuclear localization signals in the P1 part. The C-terminal SqVYV-IL P1a part also diverged but retained the conserved serine protease motif. Furthermore, substantial divergence in SqVYV isolates from the Middle East was associated with genetic drift and a long evolutionary history in this region. The finding that the host range and symptomatology in cucurbits of SqVYV-CA is similar to those of SqVYV from Florida and SqVYV-IL, indicated that the recombinant part of the genome had no obvious effect on the virus-host interaction. A divergent part of the P1 sequence of the SqVYV-CA P1-P1a gene was used to develop a primer pair and RT-PCR test for specific detection of SqVYV-CA. This test was used to detect spread of SqVYV-CA to a new production area of California in 2021 and 2022. Together, these results demonstrate (i) a high level of genetic diversity exists among isolates of SqVYV and involved intra- and intergeneric recombination and genetic drift (mutation), (ii) evidence that SqVYV originated in the Middle East and that there were independent introductions into the New World and (iii) the remarkable genetic flexibility of the 5' proximal genes of these viruses.

First report of Tomato Zonate Spot Virus infecting Belamcandachinensis in China.

Tomato zonate spot virus (TZSV, Orthotospovirus tomatozonae, genus Orthotospovirus, family Tospoviridae) was first reported to infect tomato (Solanum lycopersicum) in China in 2008 (Dong et al. 2008). Belamcanda chinensis (L.) Redouté is a perennial herbaceous medicinal plant of the family Iridaceae, which is widely distributed in China. Its rhizome contains abundant active components, mainly including flavonoids, and has antibacterial, anticancer, and antioxidative effects. In July 2023, four B. chinensis plants with virus-like symptoms were collected in Fuyuan County, Yunnan Province in China. The diseased leaves showed chlorosis and ringspots (Fig. S1). Spherical virus particles with a diameter of 80-100 nm were observed in the saps of diseased leaves under a transmission electron microscope (Fig. S2). The presence of an orthotospovirus was confirmed by the previously reported method to amplify the partial sequence (312 nt) of L segment (Huang et al. 2018) (Fig. S3). BLASTn analysis showed that the obtained 312-nt sequence was 95.62% nucleotide identity with TZSV tomato-YN isolate (accession no. NC_010491.1). To obtain the complete genome of this isolate, total RNA from symptomatic leaves of two single diseased B. chinensis were extracted using Hipure Universal RNA Mini Kit (Magen Biotech) and subjected to high-throughput sequencing with a NovaPE150 (Illumina, USA) at MAGIGENE (Shenzhen, China). A total of 41,144,571 clean reads were obtained after removing low quality reads. Quality-controlled, qualified reads were assembled into contigs using Megahit v1.1.2 software. Thirteen contigs shared nucleotide identity ranging 86.94%-97.73% with the L, S, and M segments of TZSV using BLASTn searches online (https://blast.ncbi.nlm.nih.gov/Blast.cgi). In addition, no contigs were mapped to other viral (taxid:10239) and viroidal (taxid:12884) sequences in GenBank Databases. The full-length L, M, and S RNA segments of TZSV-Bc isolate was determined tbe 8917 nt (PP314222), 4718 nt (PP314223) and 3213 nt (PP314224), respectively. These segments were validated by RT-PCR, and Sanger sequencing. They shared nucleotide sequence identities of 95.9%, 97.2%, and 93.1% of the L (NC_010491.1), M (NC_010490.1), and S (NC_010489.1) segments, of the TZSV tomato-YN isolate, respectively. Compared to the TZSV tomato-YN isolate, there exists a missing segment with 113 nt in the intergenic region of S RNA and a segment with 199 nt in M RNA. To further confirm the TZSV infection on B. chinensis, three primers pairs Tosp10/ Tosp11， Tosp5/Tosp6, and NSs-F/NSs-R were tested by RT-PCR for TZSV based on the previous report (Dong et al, 2008). The sequences of amplicons shared >99% nucleotide identity with the corresponding TZSV-Bc isolate sequences. Total of 14 B. chinensis samples were detected with the primer pair N-F/N-R (5'-ATGTCTAACGTCCGGAGTTTAACA-3'/ 5'-AAAAGACAGATCATTGCTGCTCTT-3') by One Step RT-PCR, 6 samples (42.85%) showed the positive results. The mechanical inoculation and RT-PCR detection confirmed TZSV-Bc isolate can infect N. bethamiana. So far, tomato zonate spot virus has been detected in different plants including tobacco (N. tabacum) (Huang et al. 2017), sticktight (Bidens pilosa) (Xu et al. 2022), pepper (Capsicum annuum) (Li et al. 2023) in China. To our knowledge, it is the first report of TZSV naturally infecting B. chinensis plants, which enriches information on the host range of TZSV and will be helpful for disease management.

Enhancing Conventional PCR for Detection of Erwinia amylovora

Polymerase chain reaction (PCR) methods, including conventional PCR (cPCR) and quantitative real-time PCR (qRT-PCR), with both plasmid- and chromosome-targeting primers, are currently the most reliable methods for detecting <i>Erwinia amylovora</i> due to their high sensitivity and specificity. Despite qRT-PCR’s quantitative advantage, cPCR remains an attractive method to detect this bacterium in initial screenings of suspected host plants, as it is cost-effective and does not require skilled personnel in well-equipped laboratories. This study aimed to significantly improve cPCR robustness via application of bovine serum albumin (BSA) as a PCR facilitator, with a modified EaF/R primer pair, as previously reported. Experiments have shown that simple supplementation with BSA (10 mg/ml) enhances cPCR reactions using templates such as genomic DNA, bacterial cells, and infected symptomless host organs, including immature apple fruits and seedlings, with EaF/R primers. The cPCR method described in this study is simple, specific, and reliable, and can be applied in routine assays to diagnose fire blight.

First Report of Uromyces aecidiiformis Causing Rust Disease on Fritillaria unibracteata in China.

Fritillaria unibracteata Hsiao et K. C. Hsia is a recognized source of 'Chuanbeimu' in the 'Chinese Pharmacopoeia'. In China, its bulbs have been used as a traditional herbal cough remedy for about 2,000 years. Surveys for fungal diseases were conducted in Xiaojin and Songpan, Sichuan Province, the primary cultivation region of F. unibracteata, with an area of 150 acres, in May and July 2022. Rust was found in almost all areas and incidence ranged from 5% to 80% in all study areas. Diseased leaves displayed yellow spots on the upper side, and raised buff, golden, or fuscous waxy pustules on the lower side. In severe cases, the infection extended to the stems and petioles, leading to wilting and death of plant. Spermogonia, aecia, and telia were mainly found on the underside of leaves. Spermogonia were scattered among the aecia and exhibited a range of colors from honey-yellow to chestnut-brown. They had a cross-sectional diameter of 94.4 to 214.3 µm height and 94.2 to 197.5 µm in width (n=30). They were nearly spherical, embedded in the host tissue, and had distinct periphysis at the pores. Aecia were hemispherical, initially white, with the peridium later turning yellowish-brown and opening via a central pore. Aeciospores were pale yellow, finely and closely verrucose, measuring 20.6 to 34.1 × 18.4 to 30.1 µm with a cell wall thickness of 1.5 to 2.4 µm (n=51). Prior to plants wilting, elongated telia were observed, gradually exposed, then finally opening through longitudinal cracks in the epidermis. Teliospores were unicellular, dark brown, oblong to oval, and solitary on stems, measuring 24.7 to 38.2 × 19.2 to 27.8 µm (n=130) with a wall thickness of 1.6 to 3.1 µm, with a low hyaline papilla at the apex and were moderately rugose with longitudinal parallel ridges. The characteristics align with previous descriptions of Uromyces aecidiiformi (Rees, 1917, Zhuang, 2005). The primer pair LR0R (Moncalvo et al., 1995)/LR5 (Vilgalys & Hester, 1990) was utilized for amplifying and sequencing the large subunit of the nuclear ribosomal RNA genes from strains IS909-3 and IS1816 (GenBank PQ008482, PQ008483). The obtained sequences showed a high similarity of 99.9% to 100% similarity to strains U1023 and UBC19 of U. aecidiiformis in RustHubb (KR0014142 and PUN23000)( Kaishian et al., 2024). Through examination of morphology, host range, and sequence similarity, we determined the rust species to be U. aecidiiformis. Pathogenicity testing was conducted by spraying a suspension of aeciospores (1×105 spores/mL in 0.05% Tween 20 solution) on six healthy four-year-old F. unibracteata plants indoors in May 2023. The plants were allowed to grow under natural conditions, where the diurnal temperature ranged from 9 to 20℃, with an average temperature of 14℃, which is conducive to the growth of F. unibracteata. Another six seedlings were sprayed with 0.05% Tween 20 solution as controls. After three weeks, all infected plants showed symptoms similar to those seen in the field, while control plants remained symptom-free. Microscopic examination and sequencing confirmed that the pathogen morphology was consistent between the field and the inoculation, meeting Koch's postulates. Although U. aecidiiformis has been previously reported to cause rust of F. pallidiflora and F. ussuriensis(Zhuang, 1989, Zhuang, 2005), this is the first report of U. aecidiiformis causing rust on F. unibracteata in China. This pathogen significantly reduces the yield and quality of Chuanbeimu, highlighting the importance of effectively identifying and controlling it.

Isolation and Preliminary Characterization of Probiotic Isolates from Healthy Thai Adult Feces

Probiotics are known for health-promoting microorganisms, for examples, assisting gastrointestinal digestion and short chain fatty acid producers. Several current oral human probiotics were derived from human samples; yet, of various ethnic (genetics) and dietary patterns. As probiotics from similar ethnic (genetics) and dietary pattern might confer certain advantages (e.g., more compatibility with hosts), this study isolated and characterized probiotics from healthy Thai adults. To screen for potential probiotic isolates from healthy Thai adult feces, and preliminary characterize these probiotic isolates by biochemical tests and 16S rRNA gene sequencing, following the established Thailand’s Food and Drug Administration (FDA) and National Center for Genetic Engineering and Biotechnology (BIOTEC) guidelines. Twenty fecal samples with clinically verified healthy Thai males and females aged 22-42 years were cultivated on MRS (or M17) media along the broad probiotic screenings (pH 4.0 and 0.1% oxgall) for bacterial probiotic propagation. All derived colonies of different morphologies were continued colony PCR using universal probiotic primers (specific for genera Bifidobacterium, Enterococcus, Lactobacillus, Pediococcus and Streptococcus thermophilus), and only PCR-positive colonies from any of these probiotic primer pair were further cultivation for isolates. Then, these isolates were screened and characterized general probiotic properties that included acid tolerance (pH 2.0), bile salt tolerance (0.3% bile salt), hydrophobicity, antibiotic (ampicillin, ciprofloxacin, erythromycin and vancomycin) susceptibility, no hemolysis activity, catalase, and potassium hydroxide (KOH). The final passing isolates represented potential probiotic isolates that could perhaps maintain in gastrointestinal tract with safety, and were full-length 16S rRNA gene sequenced to identify the genus and species by BLASTN against non-redundant GenBank database. A total of 295 bacterial isolates were obtained from the cultivated on MRS (or M17) media along the broad probiotic screenings from 20 fecal samples. Then, following the general probiotic property characterizations and the full-length 16S rRNA gene sequence analysis, 7 potential isolates were finally derived: 2 Bifidobacterium animalis subsp. lactis, 1 Lactobacillus (new genus name as Limosilactobacillus) reuteri, 1 L. reuteri or L. vaginalis, and 3 Pediococcus pentosaceus. These isolates exhibited strong acid and bile salt tolerances, and relatively high cell surface hydrophobicity. These isolates were susceptible to ampicillin and erythromycin, similar to a reference probiotic control Lactobacillus casei strain Shirota; and the CU13-450 and CU13-451 were also susceptible to vancomycin, similar to a reference control Bifidobacterium breve. Catalase-negative consistent with these bacteria in intestinal tract as anaerobes or facultative anaerobes, and KOH-negative result inferred the Gram-positive bacteria consistent with the full-length 16S rRNA gene sequence identification (Bifidobacterium, Lactobacillus, and Pediococcus are Gram-positive bacteria). No hemolysis activity was observed for these isolates. These 7 probiotic isolates had general probiotic properties and with confirmed the 16S rRNA gene sequences, that follow the Thailand’s FDA and BIOTEC guidelines. However, additional beneficial function investigations, such as in vitro epithelial cell adhesion and in vivo animal model, and whole genome sequencing, should be performed to better validate their possible human health benefits and safety.

Occurrence of Phytoplasmas Belonging to 16SrII Group Associated with Cyanthillium cinereum Witches'-Broom Diseases in China.

Cyanthillium cinereum, which belongs to the family of Asteraceae, is an annual or perennial herbaceous plant with significant medicinal uses for treating colds and fever. During September to November of 2020, C. cinereum showing symptoms of witches'-broom were found in economic forests distributed in Ding'an, Hainan Province of China, with 20% incidence. The symptoms of the plant were consistent with infections by 'Candidatus Phytoplasma' species. To identify the pathogen, five symptomatic and three asymptomatic C. cinereum samples were collected. Total DNAs were extracted using 0.10 g fresh leaf tissues of symptomatic and asymptomatic C. cinereum through a CTAB DNA extraction method according to Doyle and Doyle (1990). PCR amplification were performed employing the primer pairs of R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008) specific for the conserved gene fragments of 16S rRNA and secA from phytoplasma. The PCR products were purified and sequenced through Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China), and the obtained sequences were deposited in GenBank. The phytoplasmal 16S rRNA and secA gene fragments obtained in the study were all identical with the length of 1325 bp (GenBank accession: PP098738) and 741 bp (PP072217), respectively. The phytoplasma strain was described as CcWB-hnda. A BLAST search based on 16S rRNA genes indicated that CcWB-hnda strain was identical to phytoplasmas belonging to 16SrII group like peanut witches'-broom phytoplasma strain T48 (OR239773) and 'Ca. Phytoplasma aurantifolia' strain TB2022 (CP120449). Virtual RFLP profiles based on 16S rRNA gene fragments obtained by iPhyClassifier (Zhao et al., 2009) showed that CcWB-hnda strain was a member of 16SrII-A subgroup with 1.00 similarity coefficient to the reference phytoplasma strain (L33765). A BLAST search based on secA genes indicated that CcWB-hnda had 100% sequence identity with phytoplasmas belonging to 16SrII group such as 'Ca. Phytoplasma aurantifolia' isolate TB2022 (CP120449), Vigna unguiculata witches'-broom phytoplasma (OR661282) and Emilia sonchifolia witches'-broom phytoplasma (MW353710). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing Neighbor-Joining method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) demonstrated that CcWB-hnda was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value respectively. To our knowledge, this is the first report of C. cinereum infected by phytoplasmas belonging to 16SrII-A subgroup in China. Identification of the vector insects of the pathogens is necessary in future, revealing the epidemiology of the related diseases. Phytoplasmas belonging to same 16Sr group or subgroup can infect different plants and spread through them in nature. The finding in this study will be beneficial to epidemic monitoring and early warning of C. cinereum witches'-broom disease and the related plant diseases caused by the phytoplasmas belonging to 16SrII group.

Spore PCR and qPCR Methods for Rapid Detection of Five Colletotrichum Species Responsible for Pepper Anthracnose in Korea

Pepper anthracnose, caused by <i>Colletotrichum</i> spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of <i>Colletotrichum</i> spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.

Molecular Detection by Rolling Circle Amplification Combined with Deep Sequencing of Mixed Infection by Bovine Papillomaviruses 2 and 4 in Carcinoma In Situ of the Bovine Esophageal Mucosa.

Papillomaviruses (PVs) are oncogenic and infect the skin and mucosa of various host species. Considering the recent advances in research on PVs using rolling circle amplification (RCA) followed by high-throughput sequencing (HTS), in this study, we aimed to investigate the bovine papillomavirus (BPV) types associated with proliferative lesions in the upper alimentary tract of an affected bull and characterize the viral strains through complete genome sequencing using this strategy. We analyzed the PV strains associated with two hyperplastic esophageal lesions through PCR using degenerate primer pairs and RCA, followed by HTS. HTS of the libraries generated using RCA products provided the whole genome sequence of BPV4 present in squamous papilloma, whereas the complete genome sequence of BPV2 and subgenomic fragments of BPV4 were identified in carcinoma in situ (CIS). For the first time, we have sequenced BPV2 identified from the CIS of the bovine upper alimentary canal. Additionally, RCA followed by HTS allowed characterization of the mixed infection by BPV2 and BPV4 in this lesion. These data reveal that BPV4 is not the only BPV type present in CIS of the esophageal mucous membrane; moreover, a mixed infection caused by BPV2 and BPV4 at the tested anatomical site was demonstrated.

RGeasy: a reference gene analysis tool for gene expression studies via RT-qPCR

Gene expression through RT-qPCR can be performed by the relative quantification method, which requires the expression normalization through reference genes. Therefore, it is essential to validate, experimentally, the candidate reference genes. Thus, although there are several studies that are performed to identify the most stable reference genes, most them validate genes for very specific conditions, not exploring the whole potential of the research since not all possible combinations of treatments and/or conditions of the study are explored. For this reason, new experiments must be conducted by researchers that have interest in analyzing gene expression of treatments and/or conditions present, but not explored, in these studies. Here, we present the RGeasy tool, which aims to facilitate the selection of reference genes, allowing the user to choose genes for a greater number of combinations of treatments/conditions, compared to the ones present in the original articles, through just a few clicks. RGeasy was validated with RT-qPCR data from gene expression studies performed in two coffee species, Coffea arabica and Coffea canephora, and it can be used for any animal, plant or microorganism species. In addition to displaying a rank of the most stable reference genes for each condition or treatment, the user also has access to the primer pairs for the selected reference genes.

A molecular genetic toolkit for the differential expression analysis of soybean β-conglycinin subunit genes

The majority of proteins in soybean seeds are storage ones, including β-conglycinin and glycinin, which are necessary for seed germination. At the same time, they are the most valuable soy proteins used in the food industry, since their subunit composition and proportion of total protein can affect the quality of the resulting food product. β-conglycinins are trimers with different composition of subunits which are designated as α', α, β and encoded by the CG-1, CG-3, and CG-4 genes, respectively. The PCR analysis employed a model soybean cultivar ‘Sentyabrinka’. A complementary DNA synthesized from the RNA isolated from seeds of the studied cultivar served as a template. The in silico created pairs of primers for CG-1, CG-3, and CG-4 gene transcripts were used. As the result of PCR and the analysis of the obtained electrophoregrams, optimal annealing temperatures of primers for the CG-1, CG-3 and CG-4 genes were selected, at which only the characteristic fragment was observed. Thus, a molecular genetic toolkit has been developed for a comprehensive study of the qualitative and quantitative composition of soybean protein and can be used for further analysis of differential expression of genes responsible for the synthesis of β-conglycinin subunits.

First report of Cotton leaf curl Multan virus of Spinach in China.

Cotton leaf curl Multan virus(CLCuMuV; Begomovirus gossypimultanese, family Geminiviridea) is a single-stranded circular DNA virus, with a genome size of about 2.7 kb, CLCuMuV, which is commonly associated with its satellite DNA, Cotton leaf curl Multan betasatellite (CLCuMuB) (Mansoor et al., 2003), is a serious threat in cotton production causing cotton leaf curl disease (CLCuD) (Briddon et al., 2000). The spread of CLCuMuV is closely linked to its insect vector, whitefly (Bemisia tabaci), which is the exclusive vector species for CLCuMuV transmission (Pan et al., 2018). In May 2019, two spinach (Spinacia oleracea L) samples (XJBC01, XJBC02) showing upward curling of the leaf margins, vein thickening, and enation, symptoms were collected in Shihezi City, Xinjiang, China (Fig. 1B). A 570 bp fragment was amplified from the two symptomatic spinach samples using Begomovirus universal primer pair AV494 (5'-GCCYATRTAYAGRAAGCCMAG-3') and COPR (5'-GANGSATGHTRCADGCCAT ATA-3'), Sequences generated from these amplicons shared 99% nucleotide sequence identities with CLCuMuV DNA-A sequences, suggesting CLCuMuV infection in spinach. To our knowledge CLCuMuV has not been reported in spinach previously. The complete sequences of CLCuMuV and CLCuMuB were then sequenced using CLCuMuV-specific primers GD37-F (5'-GGATCCATTGTTAAACGAATTTCC-3') and GD37-R (5'-GGATCCCACATGTTTGAATTTGA-3') (Gu et al., 2015), as well as betasatellite universal primers β01 (5'-GGTACCACTACGCTACGCAGCAGCC-3') and β02 (5'-GGTACCTACCCTCCCAGGGGTACAC-3') (Zhou et al.,2003). The full length CLCuMuV DNA-A in spinach spans 2737 nt (GenBank accession number: MW561346), while CLCuMuB in spinach covers 1343 nt (GenBank accession number: MW561347). The 2737 nt full length CLCuMuV DNA-A and the associated 1343 nt CLCuMuB genome sequences generated from spinach samples were deposited in the GenBank with accession numbers MW561346 and MW561347. The MW561346 shared 99.5% sequence identity with CLCuMV GD37 from Hibiscus rosasinensis. Whereas the MW561347 shared 98.4% sequence identity with CLCuMuB GD37β. Therefore, we used infectious clones of CLCuMuV (GD37) and CLCuMuB (GD37β), provided by Xueping Zhou (Gu et al., 2015), to inoculate healthy spinach via Agrobacterium. Infected plants showed typical symptoms 14 days post-inoculation, including leaf edge curling, shrinkage, and vein enlargement, which is consistent with symptoms observed in infected spinach plants in the field (Fig. 1C). The expected 570 bp fragments were amplified in the uninoculated upper leaves of spinach showing symptoms, while not detected in the control spinach, indicating that the symptoms on spinach plants were caused by CLCuMuV associated with CLCuMuB. The transmission efficiency of CLCuMuV to spinach was assessed using two whitefly species, MEAM1 and MED, which were fed on h. rosasinensis infected with CLCuMuV. To compare the transmission efficiency between the two species, 14 spinach plants were inoculated with MEAM1, and 11 spinach plants were inoculated with MED. Each spinach plant was inoculated by releasing 10 whiteflies. After 30 days, MEAM1 transmitted CLCuMuV to spinach inducing typical symptoms (Fig. 1D), with a 78.57% (11/14) transmission efficiency. Similarly, MED also transmitted CLCuMuV to spinach but with a lower efficiency of 54.54% (6/11). These results suggested both MEAM1 and MED could transmit CLCuMuV to spinach, with MEAM1 demonstrating higher efficiency than MED. To the best of our knowledge, this study marks the first report of CLCuMuV infecting spinach, indicating an expanded host range for the virus.

In-silico identification of simple sequence repeat (SSR) markers and phylogenetic analysis from chloroplast genomes of the genus Bambusa

Microsatellites or simple sequence repeats (SSRs), are short DNA sequences composed of repetitive units typically 1 to 6 base pairs long. These highly variable sequences are found in both protein-coding and noncoding DNA regions and exhibit a high degree of polymorphism. In the present study, chloroplast genomes of five species of Bambusa viz. B. bambos, B. multiplex, B. teres, B. vulgaris, and B. tulda, along with three outgroup species viz. Sorghum timorense, Hordeum vulgare, and Triticum aestivum were retrieved from NCBI in FASTA and GenBank formats and screened for chloroplast SSRs (cpSSRs) using MISA Perl script. The sequence analysis of the chloroplast genomes of different species of Bambusa confirmed the presence of 144 cpSSRs. The number of cpSSRs varied among the five bamboo species, with B. vulgaris exhibiting the highest count (31) and B. multiplex showing the lowest (27). Tetranucleotides were the most observed repeats in Bambusa, followed by mono-, di- and trinucleotides. Of the 144 SSR markers identified, only 16 (11.11 %) were suitable for primer design since the remaining sequences failed to produce acceptable primer pairs. An unweighted pair-group method with an arithmetic mean (UPGMA) dendrogram was constructed to analyze the genetic relationships among the genotypes. This analysis revealed three distinct clusters among the genotypes. SSR markers were found to be informative in differentiating the species of Bambusa and were likely to find potential applications in genetic fingerprinting, population genetics, conservation, and phylogenetics.

First Report of Leaf Blight of Syringa oblata Caused by Botryosphaeria dothidea in China.

As an important landscaping plant, Syringa oblata is widely planted in northern China with high ornamental, medicinal, and edible value (Men et al. 2023). In September 2023, a new leaf blight disease on S. oblata was observed in Tianjin (39.0916°N, 117.1019°E), China. The onset of foliar symptoms was marked by the appearance of yellow-brown spots that originated from the tip and margin, subsequently evolving into irregularly shaped brown lesions. Finally, the lesions are distributed throughout the leaf surface, causing the leaf to wilt and seriously affecting photosynthesis. To identify the pathogen responsible for leaf blight of S. oblata, symptomatic leaves were collected and cut into square leaf blocks with a size of 0.3 cm², which were sterilized by immersion in 75% ethanol for 60 s and 5% NaClO for 30 s, and rinsed three times with sterile distilled water. The sterilized leaf pieces were then placed on potato dextrose agar (PDA) and incubated at 25 °C for 3-5 days. The peripheral hyphae of the fungal colony which developed from the infected tissues were isolated onto PDA plates. The fungal cultures on PDA plates were used for morphological observation and identification of the fungus. Colonies of B. dothidea on PDA medium were initially off-white, batting-shaped and gradually grayish-black. Aerial hyphae were well developed and could reach the tip of the petri dish. To induce sporulation, the hyphae were picked into medium containing sterilized pine needles. Conidia were found on pine needles after 30 d of incubation at 25°C. Conidia were hyaline, unicellular, oblong to spindle-shaped, and 17.5-23.0 µm in size × 6.1-8.5 µm(n=50). Based on these characteristics, the isolates were preliminarily identified as B. dothidea (Phillips et al. 2005). To provide additional evidence for the classification of the isolate, genomic DNA was extracted from the isolates of B. dothidea and used for Polymerase Chain Reaction (PCR). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPDH), and actin (ACT) were amplified with the primer pairs ITS1/ITS4, GDP1/GDP2, and ACT-512F/ACT-783R, respectively (Jiang et al. 2022). A BLAST search of sequences showed the ITS, GPDH, and ACT sequences had >99% identity with homologous sequences from B. dothidea isolates Bb158-4(HQ392696.1), PPO-46523(MG761771.1), and CMW7779 (AY972117.1), respectively. Phylogenetic analysis determined that the isolate was in the same clade position as B. dothidea, which confirmed the above morphological identification. To assess pathogenicity, the fungal cakes (6 mm diameter) were obtained from the edge of a fresh colony (cultured on a PDA plate for 7 days) using a sterile perforator. Ten surface-sterilized leaves of healthy S. oblata with uniform growth condition were collected and inoculated with fungal cakes after wounding. Ten leaves were inoculated with sterile PDA medium blocks as control. The test was repeated three times. All leaves were kept at 25°C and sterile H2O was sprayed daily to keep leaves surface moist. After five days, all vaccination sites showed lesions similar to those of the S. oblata diseased leaves in the field, while the controls were asymptomatic. B. dothidea was reisolated from symptomatic tissues, thus fulfilling Koch's postulates. B. dothidea is a member of Botryosphaeriaceae. Currently, B. dothidea infection of Syringa spp. plants has not been reported. However, in China, it has been reported to cause stem rot on Forsythia suspense (He et al. 2022) and leaf dieback on sweet osmanthus (Ling et al. 2010), demonstrating that B. dothidea can infect Oleaceae species. This study found that S. oblata could be infected by B. dothidea. To the best of our knowledge, this is the first report of B. dothidea causing leaf blight on S. oblata in China. Identifying the pathogen of S. oblata leaf blight is essential for the prevention and management of disease associated with S. oblata.

Development of microsatellite markers and evaluation of the genetic diversity of the edible sea anemone Paracondylactissinensis (Cnidaria, Anthozoa) in China.

Paracondylactissinensis Carlgren, 1934 is a sea anemone with economic value in China. The wild population of P.sinensis has been shrinking due to overfishing and environmental pollution, which have caused price instability. In winter, the price of P.sinensis can reach 25 USD per kilogram. Up to now, there are no genetic markers developed for P.sinensis, preventing a further exploration of their population genetic diversity. In this study, the full-length transcriptome of P.sinensis was sequenced and microsatellite DNA markers (simple sequence repeats [SSRs]) were developed from those transcripts. A total of 52 primer pairs, which can amplify specific polymorphic bands in PCR experiments, were designed for the SSR markers. Genetic diversity and population genetics were analysed for P.sinensis populations collected from the coasts of Taizhou and Rizhao using six microsatellite DNA loci. While inbreeding was detected in both populations (Fis > 0), the overall number of alleles (Na = 11.3) and bottleneck analysis suggested that the genetic diversity of P.sinensis has not been greatly impacted. Clustering analyses using STRUCTURE, principal coordinate analysis and unweighted pair group method with arithmetic mean tree revealed that the Taizhou population diverged from the Rizhao population; however, the genetic differentiation between the populations was moderate. Human-mediated commercial activities may be the principal reasons for the gene flow between the populations. Our study provides the first evaluation of the genetic resources of wild P.sinensis populations in China, which can serve as a useful reference for future comparative studies on population genetics and may guide policy-makers in initiating strategies for germplasm conservation and artificial breeding.

First Report of Citrus Leaf Blotch Virus on Nagami Kumquat in Turkey.

Kumquat, known as the smallest of citrus fruits, has seen an increase in the number of commercial orchards in recent years. In May of 2022, a PCR-based survey for citrus virus diseases was conducted in commercial Nagami kumquat (Fortunella margarita (Lour.) Swingle) orchards in the Cukurova region of Turkey. During the survey, five trees with bud union disorder symptoms were found in two different kumquat orchards in the Erdemli district of Mersin province. Young leaf samples from the five trees showing bud union disorder symptoms and two symptomless trees were collected from 6-year-old kumquat. Common citrus viruses and viroids, such as citrus psorosis virus, citrus exocortis viroid, and hop stunt viroid, were not detected in PCR tests performed on all collected samples. The samples were also tested for citrus leaf blotch virus (CLBV; genus Citrivirus, family Betaflexiviridae), a virus associated with a bud union crease in kumquat (Vives et al.2001). Total RNA was isolated from collected leaf samples using the RNeasy Plant Mini Kit (Qiagen, USA). In the RT-PCR assay, two separate specific primer pairs were used to determine the presence of CLBV. The coat protein gene (CP), was amplified with primers KU-18 (5'-TTAAGATTACAGACACGAAGG- 3') and KU-19 (5' CTGTTTTTGAATTTTGCTCG-3'), and the RNA-dependent RNA polymerase gene (RdRp) was amplified with KU-27(5'-GATGCAAGCCAGGATGAATAC-3') and KU-15 (5'-CAGACACTCCAAGACCTTTCC-3') primers (Vives et al. 2002). All of symptomatic samples yielded amplicons of the expected size (456-bp RdRp and 438-bp CP). No amplicons were obtained from symptomless samples. Two PCR products were sequenced to confirm their identity by direct sequencing (GenBank accession no: OQ718432, OQ718433, PP726107, PP726108). Consensus sequences of these two genes showed 96% and 97% nt identity with CP and RdRP genes of the CLBV sequence in GenBank (MT038390, EU857539), respectively. For biological indexing, four RT-PCR-positive samples were used as inoculum sources. Four of Etrog citron (C. medica) and four of Dweet tangor (C. tangerina) seedlings were graft-inoculated for each source, and two seedlings for each cultivar were used as a negative control. Two months later, chlorotic blotching symptoms were observed on Dweet tangor leaves. Six months later, Etrog citron exhibited stem pitting symptoms. None of the negative control seedlings developed any symptoms. RT-PCR was performed to confirm the presence of CLBV in the virus-inoculated seedlings, and amplicons of the expected sizes (456-bp RdRp and 438-bp CP) were obtained. To the best of our knowledge, this is the first report of the CLBV infection in citrus in Turkey. CLBV is thought to have originated from infected grafting material in the country, although it is not known where it may have originated. Further research is needed to determine the distribution of CLBV and its potential to impact commercial citrus production in Turkey.

First Report of Rhizome Rot Caused by Pectobacterium aroidearum on Ginger in China.

Ginger (Zingiber officinale) is a vital commercial crop in China, valued for its medicinal and nutritional properties. From June to September 2023, soft rot symptoms were observed on ginger rhizomes in fields in Shucheng County, Lu'an City, Anhui Province, China (31°24'N, 116°53'E). Brown, water-soaked lesions emerged at the stem base, accompanied by leaf yellowing and wilting. Approximately 20% of the ginger fields in this region (about 530 hectares) displayed soft rot symptoms. Rhizomes from ten randomly diseased ginger plants from three different locations were surface disinfected (Liu et al. 2020). The disinfected rhizomes were ground in sterile water, and serial dilutions of the supernatant were then placed on Luria-Bertani (LB) agar, resulting in the isolation and purification of 432 colonies. Using the primer set 27F/1492R (Weisburg et al. 1991), the 16S rRNA gene was amplified and sequenced. BLASTn analysis indicated that Pectobacterium species were isolated from all three locations, accounting for one-fifth of the total isolates. Three isolates, PA03, PA05, and PA08, were randomly chosen for further analysis. These colonies appeared milky white, round, with a smooth surface and smooth margins. All three isolates were Gram-negative, tested negative for methyl red and catalase, but positive for urease. The 16S rRNA sequences obtained (GenBank accession No. PP935462, PP935465 and PP935466) were 100% identical to Pectobacterium aroidearum strains KNUB-05-21 (LC777355, 1,375/1,375 bp), 3898C (KY446053, 1,418/1,418 bp), and MY11 (MT834965, 1,373/1,373 bp). The icdA, mdh, and proA genes of the isolates were amplified by PCR using specific primer pairs icdA-F/icdA-R, mdh-F/mdh-R, and proA-F/proA-R, respectively (Ma et al. 2007). BLASTn searches showed that the icdA (PP922142), mdh (PP922148), and proA (PP922145) sequences of PA03 had similarities of 99.25%, 98.26%, and 98.01%, respectively, with those of P. aroidearum strain UNEB3 (MH500095, 563/536 bp; MH507329, 522/394 bp; MH507331, 655/702 bp). Similarly, the icdA (PP922143), mdh (PP922149), and proA (PP922146) sequences of PA05 were 99.05%, 99.09%, and 97.18% identical to those of P. aroidearum strain MTL1911110204 (MT013063, 529/552 bp; MT013079, 498/399 bp; MT013095, 717/714 bp). The icdA (PP922144), mdh (PP922150), and proA (PP922147) sequences of PA08 shared 99.25%, 98.27%, and 97.94% similarity with those of P. aroidearum strain CCRM35 (MH500096, 537/537 bp; MH507330, 513/396 bp; MH507332, 630/705 bp). Phylogenetic trees were generated using MEGA11.0 software based on 16S rRNA and concatenated sequences of icdA-mdh-proA (Tamura et al. 2021). The results of phylogentic analysis revealed that the isolates, PA03, PA05, and PA08, clustered with P. aroidearum strains. To test pathogenicity, 10 µl of bacterial suspensions (1×108 CFU/ml) of isolates PA03, PA05, and PA08 were inoculated into the stem base of ginger plants using a sterile syringe, with sterile water used as a control. Each isolate was inoculated into five pots, with three plants per pot. The plants were maintained in a greenhouse at 28°C with 75% relative humidity. Rhizomes inoculated with bacterial suspensions displayed soft rot symptoms resembling those observed in the field seven days after inoculation, while the control group remained free of symptoms. Bacteria were re-isolated from diseased tissues and identified by PCR with specific primer pairs icdA-F/icdA-R, mdh-F/mdh-R, and proA-F/proA-R. The re-isolated strains had the identical sequences as PA03, PA05, and PA08, confirming their pathogenicity and fulfilling Koch's postulates. P. aroidearum is known to cause soft rot in various crops (Barroso et al. 2019; Morase et al. 2020; Tang et al. 2021). To our knowledge, this is the first report of P. aroidearum causing soft rot disease of ginger rhizomes in China. In 2023, soft rot disease led to a 15 to 20% reduction in ginger yield, posing a substantial risk to ginger cultivation in Lu'an city. Identifying this pathogen is crucial for preventing disease outbreaks and developing effective disease management strategies.

Integrated characterization and in vitro biocontrol of Fusarium sp. using Trichoderma asperellum

Pepper is a crop of great economic importance in Mexico. However, it is affected by multiple diseases. The aim of this research was to identify through morphological and molecular methods the causal agent of both foliar and root wilting in jalapeño pepper plants (Capsicum annuum L.), and the ability of Trichoderma asperellum to control these diseases in vitro. The causal agent of the disease was isolated from soil and foliar samples of jalapeño pepper fields in Delicias, Chihuahua, Mexico. The fungus was cultured on solid PDA medium to study its morphology. The CTAB method was used to extract DNA from Fusarium sp. The ITS1 and ITS4 primer pair was used to amplify a 850 bp DNA fragment by PCR. The sequence was deposited in Genbank (acc. no. OR801070) and used for phylogenetic analysis using Bayesian inference. The analysis revealed five clades and the isolate was placed in clade 4, which showed high genetic similarity with F. oxysporum (99.28% identity). Based on these molecular, morphological and phylogenetic data, the isolate was identified as Fusarium oxysporum Schltdl. The biocontrol potential of T. asperellum (accession No. MN95047) against F. oxysporum was tested in vitro and it showed 40% to 87.5% inhibition of the pathogen. These findings are important for managing this pathogen and reducing the use of chemical fungicides.

Development of SSR markers for genetic diversity analysis and species identification in Polygonatum odoratum (Mill.) Druce based on transcriptome sequences.

Polygonatum odoratum (Mill.) Druce is a well-known traditional Chinese herb belonging to the Polygonatum. However, the understanding of the genetic diversity of this species at the molecular level is limited due to the lack of transcriptomic and genomic information. In this study, 37,387 unigenes were assembled based on the transcriptome sequencing of the rhizome of Polygonatum odoratum (Mill.) Druce., and 11,021 single- sequence repeats (SSR) motifs, mainly consisting of single-nucleotide repeats (44.44%), dinucleotides (31.06%), and trinucleotides (22.59%), were identified. Based on these SSR motifs, 9,987 primer pairs of SSR markers were designed and 68 SSR markers were randomly selected for verification, of which 21 SSR markers showed polymorphisms among the 24 Polygonatum odoratum germplasms. Ninety-four alleles were detected: the observed alleles ranged from 2 to 11, the effective alleles varied from 1.086 8 to 4.916 8, the Shannon diversity index was 0.173 2~1.749 7, and the polymorphism information content PIC ranged from 0.076 7 to 0.803 9. Based on our analysis of genetic diversity (SSR genotypes) and population structure, we divided the 24 germplasm resources into two groups, indicating that the germplasm with similar geographical origins can be grouped together. In addition, the primers 'YZ14' and 'YZ47' could effectively distinguished the related species: Polygonatum kingianum Coll.et Hemsl., Polygonatum sibiricum Red., Polygonatum cyrtonema Hua, Polygonatum zanlanscianense Pamp. and Polygonatum odoratum (Mill.) Druce. This is the first study in which a dataset of expressed sequence tag (EST)-SSR markers is constructed for the Polygonatum odoratum (Mill.) Druce, and these newly developed EST-SSR markers provided a very efficient tool for genetic relationship analysis, species identification and marker-assisted selection breeding of Polygonatum odoratum (Mill.) Druce.

Exploring Sampling Strategies and Genetic Diversity Analysis of Red Beet Germplasm Resources Using SSR Markers

By using 14 SSR primer pairs, we here analyzed and compared the amplification results of 534 DNA samples from six red sugar beet germplasm resources under three treatments. These data were used to explore the sampling strategy for the aforementioned resources. Based on the sampling strategy results, 21 SSR primer pairs were used to evaluate the genetic diversity of 47 red sugar beet germplasm resources. The six population genetic parameters used for exploring the sampling strategy unveiled that individual plants within the population had a relatively large genetic distance. The genetic parameters Ne, I, and Nei’s of the randomly mixed sampling samples increased rapidly between 10 and 30 plants before decreasing. Therefore, when SSR technology was used to analyze the genetic diversity of the red sugar beet germplasm resources, the optimal sampling gradient for each population was the adoption of a random single-plant mixed sampling sample of no less than 10 plants and no more than 30 plants. The 21 SSR primer pairs were used to detect genetic diversity in 30 random mixed samples of 47 resources. The average polymorphic information content (PIC) was 0.5738, the average number of observed alleles (Na) was 4.1905, the average number of effective alleles (Ne) was 2.8962, the average Shannon’s information index (I) was 1.1299, the average expected heterozygosity (Nei’s) was 0.6127, and the average expected heterozygosity (He) was 0.6127. The genetic distance of the 47 germplasm resources ranged from 0.0225 to 0.551 (average: 0.316). According to the population structure analysis, the most suitable K value was six, which indicated the presence of six populations. Based on the clustering analysis results, the 47 germplasm resources were segregated into six groups, with obvious clustering and some germplasm resources noted for having groups with close genetic relationships. We here established a more accurate and scientific sampling strategy for analyzing the genetic diversity of red sugar beet germplasm resources by using SSR molecular markers. The findings provide a reference for collecting and preserving red sugar beet germplasms and protecting their genetic diversity.

First Report of Powdery mildew Caused by Golovinomyces ambrosiae on Verbena × hybrida in the U.S.

Verbena × hybrida, also known as common garden verbena, has an important ornamental value for their wide range of flower colors and for attracting hummingbirds and butterflies. During the winter of 2021-2022 (December through February), more than 50% pot-grown verbena plants showed symptoms of powdery mildew in a field trial at a Syngenta Crop Protection research facility in Vero Beach, FL. Symptoms were characterized by the development of white, superficial mycelium on the adaxial side of leaves which, eventually, progressed to covering the whole surface of leaves, causing leaf discoloration, shoot distortion, and eventual plant death. Morphological characterization was carried out by observing powdery mildew colonies under the microscope. This powdery mildew forms dense patches of white mycelia, mainly on the adaxial leaf surfaces. The mycelium was a mat of hyphae with septa. Conidiophores were erect. The foot cells were straight, followed by one to three short cells bearing short chains of up to four conidia. The conidia were hyaline and ellipsoidal to doliiform in shape. Conidial germination is of the Eudoidium type. The conidia ranged from 25 to 32 μm long by 12 to 16 μm wide. The length to width ratio ranged between 1.6 and 2.3, but most were between 2.0 and 2.2. This is further verification of its identity as Golovinomyces ambrosiae and not Golovinomyces latisporus, because the length to width ratio of the latter species is consistently less than 2.0 (Qiu et al. 2020). Chasmothecia were not observed. Additionally, the ITS, GAPDH, and IGS regions were sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), PMGAPDH1/PMGAPDH3R (Bradshaw et al. 2022a), and IGS-12a/NS1R (Carbone and Kohn 1999), respectively. The ITS region (GenBank number=PP924119) cannot distinguish between G. latisporus and G. ambrosiae and as such aligned 100% with both species on GenBank. However, the GAPDH and IGS regions can be used to distinguish G. ambrosiae from G. latisporus (Bradshaw et al. 2022b). The GAPDH (GenBank number=PP931995) and IGS (GenBank number=PP931996) regions aligned 100% with multiple G. ambrosiae sequences from GenBank including ON360708 and MK452567, respectively. The specimen was deposited in the Larry F. Grand Mycological Herbarium (NCSLG 24479). To confirm pathogenicity, 'Tuscany® Pink Picotee' and 'Quartz XP Violet with Eye' plugs were transplanted to 10-cm diameter pots containing ProMix potting mix and maintained in a greenhouse (± 26 °C). Inoculation was carried out 21 days after transplanting by touching infected leaves onto healthy leaves of 15 disease-free plants of each variety. Fifteen non-inoculated plants of each variety were used as controls. Typical powdery mildew symptoms and signs were first observed ten days after inoculation and the pathogen was more aggressive on 'Tuscany® Pink Picotee'. Symptoms were not observed on non-inoculated plants. The fungus was morphologically identical to the one originally recovered from infected plants in the field. There have been many reports of Golovinomyces spp. affecting Verbena spp. worldwide; however, this is the first report of G. ambrosiae causing powdery mildew on Verbena × hybrida in the U.S. (Braun and Cook, 2012, Choi et al., 2021; Bradshaw et al. 2024). Powdery mildews reduce plant quality and decreases the aesthetics value of infected plants, causing great losses to the ornamental industry. Correct identification of the causal agent is crucial to recommend appropriate control methods, as they may differ according to the pathogen species.