Primer Pairs Research Articles

Establishment and application of a rapid visual diagnostic method for Streptococcus agalactiae based on recombinase polymerase amplification and lateral flow strips

This study aims to establish a rapid diagnostic method for Streptococcus agalactiae (GBS) based on recombinase polymerase amplification (RPA) and lateral flow strips (LFS). The best primer pairs designed by SIP gene were screened according to the basic RPA reaction, then the probe was designed. The reaction condition was optimized based on the color development of the LFS detection line. To ascertain the reaction specificity, 10 common clinical pathogens and 10 clinical specimens of GBS were tested. Furthermore, the reaction sensitivity was assessed by utilizing a tenfold gradient dilution of GBS genomic DNA as templates. RPA–LFS method was compared to the qPCR assay and biochemical culture method for the Kappa consistency test. The RPA–LFS technique was able to complete the amplification process within 30 min and the results were observed on lateral flow strips. The method is highly sensitive, with a minimum detection limit of 1.31 ng for GBS. The RPA–LFS method showed consistent accuracy of results compared to qPCR and the culture–biochemical method. The establishment of this method is conducive to the development of on-site immediate detection, which can provide information for the timely development of a reasonable antimicrobial treatment plan, and has a greater potential for clinical application.

A novel fluoro colorimetric Loop mediated isothermal amplification (LAMP) assay for detection of Trichomonasvaginalis

BackgroundTrichomoniasis is a curable, non-viral, sexually transmitted infection. Early diagnosis and treatment of cases can prevent complications and further spread of infection. Rapid diagnostics tests, which can be performed on-site, will help in early diagnosis. The study aims to develop a rapid diagnostic test based on the principle of fluoro-colorimetric LAMP for detecting Trichomonas vaginalis (TV). Materials and methodsT. vaginalis was grown in a modified CPLM medium, and DNA was extracted. Three pairs of LAMP primers targeting the actin gene were designed using the primerexplorer V.5 online tool. The LAMP assay was standardized for temperature and time. To determine the LAMP assay's detection limit, diluted TV DNA and spiked urine samples were used. Conventional PCR was done using previously published primers and compared with LAMP results. The sensitivity and specificity to detect TV from clinical specimens were assessed. ResultsThe optimum performance of the LAMP assay was determined to be 63 °C for 60 min and terminated at 80 °C for 5 min. The LAMP assay could detect 60 fg/μl of diluted TV DNA and up to 1 parasite/ml of spiked samples. The assay was 1000 times more sensitive than PCR. The LAMP assay was 100% sensitive and specific with crude extract, and reactions were visually discernible. Interpretation & conclusionsThe LAMP assay developed in the study is easy to perform and interpret, affordable, rapid, and highly sensitive to detect T. vaginalis. It is ideally suited for the point-of-care test, as it fulfills WHO's recommended ASSURED characteristics.

Association Study of Pleural Mesothelioma and Oncogenic Simian Virus 40 in the Crocidolite-Contaminated Area of Dayao County, Yunnan Province, Southwest China.

Background: In Dayao County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, Southwest China, 5% of the surface is scattered with blue asbestos, which has a high incidence of pleural mesothelioma (PMe). Simian virus 40 (SV40) is a small circular double-stranded DNA polyomavirus that can cause malignant transformation of normal cells of various human and animal tissue types and promote tumor growth. In this study, we investigate whether oncogenic SV40 is associated with the occurrence of PMe in the crocidolite-contaminated area of Dayao County, Yunnan Province, Southwest China. Methods: Tumor tissues from 51 patients with PMe (40 of whom had a history of asbestos exposure) and pleural tissues from 12 non-PMe patients (including diseases such as pulmonary maculopathy and pulmonary tuberculosis) were collected. Three pairs of low-contamination risk primers (SVINT, SVfor2, and SVTA1) were used to detect the gene fragment of SV40 large T antigen (T-Ag) by polymerase chain reaction (PCR). The presence of SV40 T-Ag in PMe tumor tissues and PMe cell lines was detected by Western blotting and immunohistochemical staining with SV40-related antibodies (PAb 101 and PAb 416). Results: PCR, Western blotting, and immunohistochemical staining results showed that the Met5A cell line was positive for SV40 and contained the SV40 T-Ag gene and protein. In contrast, the various PMe cell lines NCI-H28, NCI-H2052, and NCI-H2452 were negative for SV40. PCR was negative for all three sets of low-contamination risk primers in 12 non-PMe tissues and 51 PMe tissues. SV40 T-Ag was not detected in 12 non-PMe tissues or 51 PMe tissues by immunohistochemical staining. Conclusion: Our data suggest that the occurrence of PMe in the crocidolite-contaminated area of Yunnan Province may not be related to SV40 infection and that crocidolite exposure may be the main cause of PMe. The Clinical Trial Registration number: 2020-YXLL20.

One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus.

Feline coronavirus (FCoV), feline panleukopenia virus (FPV), and feline leukemia virus (FeLV) are prevalent throughout China and significantly threaten cat health. These viruses cause similar manifestations and pathological damage. Rapid and accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable and rapid method for accurate detection of FCoV, FPV, and FeLV so that a definite diagnosis can be made and effective measures can be taken to prevent and control viral infection. We designed three pairs of specific primers and probes for the detection of FCoV 5' untranslated region, FPV viral protein 2, and FeLV pol genes. Recombinant plasmid constructs were generated for use as standard plasmid constructs. Optimal reaction conditions, including primer and probe concentrations, reaction cycles, and annealing temperatures, were obtained on the basis of optimization tests. One-step triplex real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was successfully established to simultaneously detect FCoV, FPV, and FeLV. The specificity, sensitivity, and repeatability of the assay were analyzed, and its applicability was validated by testing 1175 clinical samples. One-step triplex RT-qPCR had a high degree of specificity only for the detection of FCoV, FPV, and FeLV; it had high sensitivity with limits of detection of 139.904, 143.099, and 152.079 copies/reaction for p-FCoV, p-FPV, and p-FeLV standard plasmid constructs, respectively, and it had reliable repeatability with 0.06%-0.87% intra-assay coefficients of variations. A total of 1175 clinical samples were examined for FCoV, FPV, and FeLV using triplex RT-qPCR, and the FCoV, FPV, and FeLV positivity rates were 18.47%, 19.91%, and 47.57%, respectively. The clinical sensitivity and specificity of one-step triplex RT-qPCR were 93.07% and 97.99%, respectively. We developed a rapid and reliable one-step triplex RT-qPCR method for the detection of FCoV, FPV, and FeLV, which could be used as a diagnostic tool for clinical monitoring and diagnosis.

Construction of a DNA Fingerprint Map and a Core Collection of Platycladus orientalis

Platycladus orientalis is one of the main species used in afforestation projects in the arid mountains of north and northwest China, meaning that the species has high ecological and economic value. Studying its genetic diversity and obtaining a core germplasm base and genetic fingerprint data are important for the screening, development, and utilization of the species. This can provide the core materials for the preservation and evaluation and mining of germplasm resources and can provide superior gene resources for breeding programs. In this study, the genetic diversity among 104 P. orientalis germplasm resources was examined using simple sequence repeat (SSR) markers, and a core germplasm containing 31 accessions was constructed that represents the most genetic diversity of P. orientalis accessions. Each of 20 pairs of primers showed polymorphism, and 117 alleles were identified. The average number of alleles at each locus was six, and the mean effective allele number was 2.607. The average Shannon’s information index was 0.983, and the average polymorphism information content was 0.445. There is thus a significant amount of genetic variation within P. orientalis germplasm, yielding a rich genetic diversity. The constructed core germplasm accounted for 30% of the original germplasm. There was no significant difference in genetic diversity between the core germplasm and the original germplasm resources, indicating that the obtained core germplasm resources could fully represent the original germplasm. Using 17 SSR primers with high polymorphism, the DNA fingerprints of 104 P. orientalis germplasm resources were constructed. The results showed that 98 had specific DNA fingerprints. The results of this study provide a valuable basis for the collection, preservation, and utilization of P. orientalis germplasm resources, and the methods adopted in this study have important reference value for the construction of core germplasm of other perennial woody plants.

Twin Prime Editing Mediated Exon Skipping/Reinsertion for Restored Collagen VII Expression in Recessive Dystrophic Epidermolysis Bullosa

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Novel multiplex-PCR test for Escherichia coli detection.

Detection of E. coli from environmental or clinical samples is important due to the common occurrence of this species of bacteria in all human and animal environments. As commonly known, these bacteria strains can be commensal and pathogenic, causing numerous infections of clinical importance, including infections of the digestive system, urinary, respiratory, and even meninges, particularly dangerous for newborns. The developed multiplex polymerase chain reaction test, confirming the presence of E. coli in samples, can be used in many laboratories. The test provides new opportunities for quick and cheap analyses, detecting E. coli using only three pairs of primers (analysis of the presence of three genes) responsible for metabolism and distinguishing E. coli from other pathogens from the Enterobacteriaceae family. Compared to other tests previously described in the literature, our method is characterized by high specificity and sensitivity.

First Report of Lingonberry Stunted Yellows Disease of Vaccinium vitis-idaea L. associated with 'Candidatus Phytoplasma trifolii'-Related Phytoplasma Strain in Lithuania.

Lingonberries (Vaccinium vitis-idaea L.) are low-growing, evergreen shrubs of cooler, northern regions of North America and Europe. These plants produce berries that are unique in flavor, bear high economic significance, and play a vital role in maintaining the diversity of the northern ecosystems (Kowalska, 2021). In October 2023 diseased plants of lingonberry were discovered in Labanoras Forest (55°14'N 25°42'E) (Lithuania). The plants expressed symptoms of stunting, yellowing, little leaf, shortened internodes, and stem distortions. Samples (leaves) were collected and tested from ten asymptomatic and ten symptomatic lingonberry plants. Total genomic DNAs of all samples were extracted by a CTAB protocol. Extracted DNAs were used as a template in direct and nested PCRs using the universal primer pairs P1/P7 and R16F2n/R2, respectively, to amplify phytoplasma 16S rRNA gene 1.2 kb fragments (Lee et al. 1998). The primer pairs SecAFor1/SecARev3 and SecAFor2/SecARev3 were used in direct and semi-nested PCRs, respectively, to amplify phytoplasma secA genes 0.5 kb fragment (Dickinson and Hodgetts, 2013). PCR amplicons of the 16S rRNA and secA genes specific for the phytoplasmas were only obtained from all sampled symptomatic plants. Three R16F2n/R2 and three SecAFor2/SecARev3 amplicons were cloned and submitted for Sanger sequencing (Nature Research Centre, Vilnius, Lithuania by 3500 Genetic Analyser). The three 16S rDNAs as well as the three secA gene fragments were identical. The BLAST analysis (NCBI) of the obtained sequences showed a similarity percentage, ranging from 99.75% to 100% (1247-1250 bp from 1250 bp) for 16SrRNA, and 98.13% to 99,15% (473-478 bp from 482 bp) for secA amplicons, with numerous strains of 'Candidatus (Ca.) Phytoplasma (P.) trifolii' (first hit MT674293 and KR906724, respectively). Additionally, 16S rDNA sequences by using iPhyClassifier were used to create virtual RFLP pattern (Zhao et al. 2009). The generated pattern was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI, subgroup A. The phytoplasma strain detected in lingonberries was designated as lingonberry stunted yellows, LingbSY. Furthermore, the enzymatic RFLP analysis was performed with the 14 restriction enzymes (Lee et al., 1998), and obtained profiles were compared with virtually generated using iPhyClassifier. This yielded the same classification of detected phytoplasma to the 16SrVI-A phytoplasma subgroup. The phylogenetic analysis of both marker gene sequences revealed the same LingbSY phytoplasma classification. Selected sequences were deposited in GenBank (NCBI) with Accession No: PP237769 (16S rRNA gene) and No: PP238489 (secA gene). Phytoplasmas of 16SrI phytoplasma group were identified in lingonberries in Canada (Brochu et al. 2022). Strains of 16SrVI phytoplasma group were reported in Vaccinium myrtillus in Austria (Fernandez et al. 2007). This is the first report of 'Ca. P. trifolii' strain belonging to 16SrVI-A phytoplasma subgroup infecting lingonberry worldwide. Also, this is the first report of 16SrVI phytoplasma group in Lithuania. The presence of this phytoplasma poses a threat to the natural ecosystem and could eventually spread into agricultural settings in our country. Therefore, it's crucial to conduct surveillance for insect vectors, and assess effective control methods. Without proactive action, long term sustainability of lingonberries and their ecosystems may be jeopardized.

Epicoccum sorghinum Causing Leaf Spot on Polygonatum cyrtonema in China.

Polygonatum cyrtonema Hua (family Asparagaceae) is a traditional Chinese medicinal plant that is widely cultivated in various parts of China, including Hunan Province. In summer 2022, a leaf spot disease was observed in 10% of the P. cyrtonema plants (Huang jing) in 18 hectares of this crop in the Hongjiang District (27°18'4″N, 110°11'1″E) of Hunan Province. The initial symptoms of the disease were brown spots on young leaves, and adjacent tissues gradually changed from green to yellow. The entire leaf then became yellow, withered, and eventually exhibited a thn and black appearance. In total, 12 diseased plants from four sampling sites (three plants per site) were collected for laboratory analysis to address the concerns of P. cyrtonema growers. Symptomatic leaf samples were selected, and the leaf fragments containing infected parts of the plants were disinfected with 75% ethanol for 1 min, then immersed in 2.5% hypochlorite for 45 s. After disinfection, symptomatic leaf samples were rinsed three times with sterile water, placed on potato saccharose agar containing 50 μg/ml kanamycin and incubated at 25°C for 2 days. Subsequently, 12 fungal isolates were isolated from various leaf samples through hyphal tip transferring. Ten of the 12 fungal isolates had similar morphological features, and one of them (isolate hjh) was used as the representative isolate for the study. With a growth rate of 6.3 mm per day, its white colonies transformed into red concentric rings in five days; they gradually became black after 10 days of growth. The chlamydospores were round (4.0-9.9 × 3.1-9.3 µm, n = 30), whereas the conidia were ovate (8.0-12.1 × 3.2-6.5 µm, n = 30). The morphological features of the isolate hjh were similar to the features of Epicoccum spp. (Aveskamp et al. 2010). The internal transcribed spacer (ITS) region (including the partial ITS1 sequence and the 5.8S and ITS2 complete sequences), β-tubulin (tub) gene, and large subunit (LSU) rRNA gene, were amplified from the isolate hjh using the primer pairs ITS5/ITS4, Bt2a/Bt2b, and LROR/LR5, respectively (Taguiam et al. 2021). BLASTn analysis showed that the ITS (OR253745), tub (OR253764), and LSU (OR253746) sequences generated from the isolate hjh were 98-99% similar to the sequences of E. sorghinum strains CBS 179.80 and CBS 627.68. Subsequently, the ITS, tub, and LSU sequences were combined using Sequence Matrix software; phylogenetic analysis via Bayesian and maximum likelihood methods (Vaidya et al. 2011; Li et al. 2021) classified the isolate hjh into the E. sorghinum clade. To fulfill Koch's postulates, pathogenicity tests were conducted on healthy (lesion-free and disease-free) 2-year-old P. cyrtonema plants. Three healthy plants were inoculated by spraying whole plant until run-off with a spore suspension of the isolate hjh (1 × 106 conidia/ml); Three other healthy plants were sprayed with sterile water as controls. The inoculated plants were incubated in a growth chamber at 25 ± 2°C with 85% humidity for 28 days(Chen et al. 2021). Leaves from the inoculated plants gradually became brown within 15 days. Finally, the plants died 28 days after inoculation. The control plants showed no symptoms throughout the experimental period. Isolates (isolate hjh1, hjh2 and hjh3) that were reisolated from the inoculated plants exhibited morphologically similar characteristics and molecularly identical to the original isolate hjh. To our knowledge, this is the first report of E. sorghinum causing leaf spot disease on P. cyrtonema. The results of this study may facilitate the production of P. cyrtonema in China.

First Report of Dickeya dadantii Causing Potato Blackleg in Fujian Province China.

Blackleg and soft rot are harmful diseases in potato (Solanum tuberosum) caused by Pectobacterium spp. and Dickeya spp. (Czajkowski et al. 2015). The occurrence of potato blackleg was serious in potato-producing areas around Xiapu County in Fujian Province, China, in 2021 (6 ha) and 2022 (7 ha), with an incidence of approximately 5%, which reached nearly 23%. Three diseased plants were collected to isolate the pathogen. Single colonies from each sampled plant were isolated and streaked onto fresh plates. DNA from three colonies from different plants was PCR amplified with primer pair 27F/1492R (Lane 1991) for the 16S rRNA gene. Since the sequences were identical, we selected strain M2-3 for further analysis. The strain M2-3 was gram-negative, pectolytic on CVP, grew at 37°C and 5% NaCl. The bacterium was positive for phosphatase activity, erythromycin sensitivity, indole production, gelatin liquefaction, malonic utilization, and acid production from, melibiose, raffinose, and arabinose. The bacterium was negative for sucrose, α-methyl glucoside, sorbitol, trehalose, lactose, and sodium citrate (Fujimoto et al. 2018;),although sucrose and lactose did not provide the expected results, there are exception in all species. The genome of strain M2-3 was sequenced and deposited in the NCBI database under accession numbers: CP077422. An Average Nucleotide Identity (ANI) analysis showed that M2-3 clustered with other D. dadantii strains and has a 98.39% identity with D. dadantii strain DSM 18020 (CP023467). The housekeeping genes (recA, dnaX, acnA, gapA, icd, mdh, mtlD and pgi) were amplified with primer pairs designed previously(Fujimoto et al. 2018; Ma et al. 2007) and sequenced. A multilocus sequence analysis (MLSA) was performed by concatenating the 8 gene sequences and constructing a maximum likelihood phylogenetic tree using PhyloSuite version 1.2.1 (Zhang et al. 2020) and IQ-tree version 1.6.8 (Nguyen et al. 2015) software. Strain M2-3 was clustered together with Dickeya dadantii. For the pathogenicity test, three plants per treatment, totaling nine plants, were used. Bacterial suspensions (1×10^8 CFU/mL) were made in a 10mM PBS buffer. 10 μL of M2-3, D. dadantii type strain 18020 (positive control), and buffer (negative control) were injected into the plant stems near the base. Water stains appeared at the site of inoculation after 2 days and they gradually became black and rotten. The leaves became yellow and wilted, and the petiole base rotted within 5 days of inoculation completing the Koch postulate. According to average nucleotide identity and housekeeping gene sequence analysis, strain M2-3 was identified as Dickeya dadantii. Previous studies have reported several pathogens that cause potato blackleg in China, including P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense (Li-ping et al. 2020; Wang et al. 2021). To the best of our knowledge, this study is the first to report potato blackleg disease caused by Dickeya dadantii in Fujian Province, China. This finding suggests that this pathogen may cause a threat to potato production in Fujian Province.

Mixed infections of Tomato yellow leaf curl New Delhi virus and a ‘Candidatus Phytoplasma asteris’ strain in zucchini squash in Italy

A new disease syndrome of zucchini squash was observed in Southern Italy, in 2018 and again in 2020. Affected plants were severely stunted and leaves were bent downwards, small, stiff, thick, leathery, and had interveinal chloroses. In addition, flowers were virescent and fruits were deformed and often cracked. Disease incidence was 20 and 30% in two different zucchini cultivations in Campania region (Southern Italy). Tomato yellow leaf curl New Delhi virus (ToLCNDV) was detected in eight samples, by loop-mediated isothermal amplification–based (LAMP) kit and by PCR and Sanger sequencing of the AV1 gene. Phytoplasmas were detected in the same samples using nested PCR assays with primer pairs P1/P7 and R16F2n/R16R2. Phytoplasma associations in plant samples were confirmed using specific primers for the multilocus genes SecY, tuf and rp. Sequence comparison of multilocus genes and phylogenetic analyses of the 16S rDNA gene confirmed the association of a phytoplasma strain closely related to ‘Candidiatus Phytoplamsa asteris’. This is the first report of mixed infections of ToLCNDV and a putative ‘Ca. Phytoplamsa asteris’ strain in zucchini, associated with a new Squash-Phytoplasma-Begomovirus (SqPB) disease syndrome.

First Report of Enterobacter mori Causing Bacterial Wilt on Tomato in China.

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in China. In October 2023, a new bacterial disease was discovered on tomato plants in a 0.3-acre farm's greenhouse (35.514806N, 118.996106E) in Longshan Town, Shandong Province, China. Over 50% of the tomato plants showed symptoms of stem rot, leaf wilt, or plant death. Three diseased tomato plants were collected for pathogen isolation and purification. Two leaf samples, each about 1 cm2, were cut from the junction area of healthy and diseased parts and disinfected with 75% alcohol for 60 s, followed by 0.1% HgCl2 for 90 s, and then washed three times with sterilized H2O. The samples were subsequently ground with 1.0 mL sterilized H2O. The ground samples were diluted to 10-4, 10-5, and 10-6 and then were plated on a potato dextrose agar (PDA) plate, respectively. White mucous bacterial colonies appeared at 28℃ for 24~48 h, no fungal colony was observed. Six bacterial colonies were randomly selected for gram staining and found to be gram-negative. To further determine their species classification, fragments of the 16SrDNA, hsp60, gyrB, and rpoB genes were separately amplified using previously reported PCR conditions and with primer pairs, including 27F/1492R (Wu et al., 2023), HSP60-F /HSP60-R (Gül et al., 2023), gyrB UP-1 / gyrB UP-2r (Yamamoto et al., 1995) and rpoB CM81-F / rpoB CM32b-R (Brady et al., 2008). Sequence analysis showed that the obtained sequences of the 16SrDNA, hsp60, gyrB, and rpoB genes among these six colonies were identical and 100%, 100%, 99.31%, and 99.36% similar to those of Enterobacter mori accessions OP601841 (with a coverage of 100%), MT199160 (83%), OP676246 (100%), and MN594495 (100%), at nucleotide level, respectively. Sequences of the above four genes of 23LSFQ were submitted to GenBank under the accession numbers PP461247, PP474090, PP136037, and PP136038, respectively. We selected one of these six colonies, 23LSFQ, for further analysis. The phylogenetic tree based on the concatenated sequences of the above four genes using the maximum likelihood method with MEGAX software showed that 23LSFQ is grouped with E. mori LMG25706 (NCBI: txid980518). To determine the pathogenicity of 23LSFQ , we sprayed 23LSFQ (OD600=0.8) onto five 30-day-old healthy plants of the tomato cultivars Alisa Craig, Jinpeng NO.1, and Chaobei, respectively. These seedlings were incubated in a chamber at 28°C with a 16 h light/ 8h dark photoperiod and 60% relative humidity. The leaves of the inoculated plants became curled and wilted at two days post inoculation (dpi) and appeared necrotic at 10 dpi. The symptoms were similar to those observed in field-infected tomato plants. No symptoms were observed on the plants inoculated with water. We further sequenced the re-isolated bacteria from the symptomatic inoculated seedlings. Results showed that they belong to E. mori. The experiment was repeated three times. E. mori has been found to cause diseases on peaches (Ahmad et al., 2021), watermelons (Wu et al., 2023), Canna indica, (Zhang et al., 2023), and strawberries (Ji et al., 2023). E. cloacae has been found to cause diseases on tomatoes in Heilongjiang province (Jin et al., 2023). This is the first report of E. mori causing leaf yellowing and wilting on tomatoes in China. These results are significant for the safe production and disease control of greenhouse tomatoes.

Analysis of the Genetic Diversity and Mating System of the Endangered Plant Keteleeria davidiana var. calcarea

Keteleeria davidiana var. calcarea is an endangered plant endemic to China. It is mainly distributed in the karst areas of the Guangxi and Guizhou Provinces. It is characterized by small populations and intermittent distribution. This study aimed to explore the genetic diversity and mating system of wild populations of Keteleeria davidiana var. calcarea in fragmented habitats. To achieve this, we genotyped 46 maternal trees and 214 progenies from four fragmented populations of Keteleeria davidiana var. calcarea using nine pairs of microsatellite primers. The genetic diversity of Keteleeria davidiana var. calcarea (Ho = 0.68, He = 0.63) was lower than that of the species overall but higher than that of other Keteleeria plants. The incidence of unbiased expected heterozygosity (uHe) and allelic richness (Ar) was higher in the maternal generation than in the progeny. This suggests that the genetic diversity of the progeny was lower than that of the maternal generation. Keteleeria davidiana var. calcarea is divided into four populations, but there is significant genetic exchange between the populations according to STRUCTURE and gene flow analyses. The multilocus mating system analysis (MLTR) results indicate that the multilocus outcrossing rate (tm) was 0.902, the single-locus outcrossing rate (ts) was 0.606, the bimaternal inbreeding coefficient (tm-ts) was 0.295, and the coefficient of inbreeding depression(δ) was 0.904. These results suggest a certain degree of selfing and inbreeding in Keteleeria davidiana var. calcarea. To prevent problems associated with inbreeding and conserve the genetic diversity of Keteleeria davidiana var. calcarea, we recommend establishing seed gardens, using artificial pollination, and employing asexual propagation techniques for conservation intervention.

Development and preliminary application of a quadruplex real-time PCR assay for differential detection of porcine circovirus 1-4 in Chengdu, China.

Porcine circovirus (PCV) typically causes severe immune suppression in pigs, leading to mixed clinical infections with various pathogens that can cause significant harm to the pig industry. PCV has four subgenotypes, with PCV4 being an emerging virus that requires investigation due to its potential for epidemic outbreaks. Therefore, there is a need to develop a method that can detect all four PCV strains simultaneously. In this study, four pairs of specific primers and TaqMan probes were designed based on the conserved sequence of the PCV1-4 ORF2 gene to establish a PCV1-4 TaqMan multiplex real-time quantitative PCR method. The novel method was compared to six commercial testing kits for its efficacy. Then, a total of 595 mixed samples of spleen and lymph node collected from 12 districts in Chengdu from July to December 2021 were tested using the novel method. The results showed that the novel PCV1-4 TaqMan multiplex real-time quantitative PCR detection method has satisfied specificity, sensitivity, and repeatability. The positive rates of PCV1, PCV2, and PCV3 in Chengdu were 2.18%, 31.60%, and 15.29%, respectively, while no positive PCV4 was detected. The mixed infection rate of PCV2 and PCV3 was 5.21%. Our novel method may be as a potential method for PCV1-4 detection. Currently, PCV2 is the main epidemic PCV subtype in Chengdu, while the potential threat of PCV4 should also be considered.

On the Universal Presence of Mathematics for Incompletely Predictable Problems in Rigorous Proofs for Riemann Hypothesis, Modified Polignac’s and Twin Prime Conjectures

We validly ignore even prime number 2. Based on all arbitrarily large number of even Prime gaps 2, 4, 6, 8, 10...; the complete set and its derived subsets of Odd Primes fully comply with the Prime number theorem for Arithmetic Progressions, and our derived Generic Squeeze theorem and Theorem of Divergent-to-Convergent series conversion for Prime numbers. With these conditions being satisfied by all Odd Primes, we argue Polignac's and Twin prime conjectures are proven to be true when they are usefully treated as Incompletely Predictable Problems. In so doing [and with Riemann hypothesis being a special case], this action also support the generalized Riemann hypothesis formulated for Dirichlet L-function. By broadly applying Hodge conjecture, Grothendieck period conjecture and Pi-Circle conjecture to Dirichlet eta function (proxy function for Riemann zeta function), Riemann hypothesis is separately proven to be true when it is usefully treated as Incompletely Predictable Problem. Crucial connections exist between these Incompletely Predictable Problems and Quantum field theory whereby eligible (sub-)functions and (sub-)algorithms are treated as infinite series.

Optimization of Amplicon-Based Sequencing for Large-Scale Diagnostics of Known Pome Viruses and Viroids

The implementation of reliable methods for the simultaneous detection of multiple viruses is challenging in plant viral diagnosis. In the present study, we report the development and effectiveness of an optimized version of a robust multiplexed PCR (HiPlex) followed by simultaneous targeted amplicon sequencing to detect 14 viruses and 5 viroids of pome fruit trees. We used amplicon sequencing data from 1,400 pome fruit tree samples previously diagnosed with the HiPlex v.1.0, which included 290 primer pairs for pome viruses. The analysis enabled the identification of highly efficient and specific primer pairs to be assembled in a new HiPlex (dubbed HiPlex v.2.0). Additionally, newly designed primers were implemented for 30% of targeted viruses and viroids. Overall, 116 primer pairs were assembled into the HiPlex v.2.0, resulting in a reduction of the number of primers and multiplex PCR reactions needed by 60 and 50%, respectively. The new HiPlex also includes two nepoviruses, tomato ringspot virus and tobacco ringspot virus, showing flexibility at augmenting and expanding the number of targeted diagnosed viruses using this approach. This optimized set of primers increased the cost-effectiveness and sensitivity while reducing background amplification. Comparable results for virus and viroid detection were obtained using the HiPlex v.2.0 and individual RT-qPCRs, even at a 10,000-fold dilution, demonstrating similar sensitivity to standard PCR-based diagnostics. This new HiPlex-based amplicon sequencing represents a powerful, sensitive, and low-cost tool for large-scale screening of pome fruit trees for all reported viruses and viroids infecting these crops. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Development and evaluation of genomics informed real-time PCR assays for the detection and strain typing of Mycobacterium avium subsp. paratuberculosis.

This study aimed to identify specific genomic targets for the detection and strain typing of Map and analyse their sensitivity and specificity, and detect Map directly from faeces. A comparative genomics approach was used to identify specific genomic targets for the detection and strain typing of Map. A Map specific qPCR using the primer pair 7132 that targets a DNA segregation ATPase protein was able to detect all strains of Map and is more sensitive than the current Johne's disease PCR assays with a sensitivity of 0.0002 fg µl-1. A strain specific qPCR using the Atsa primer pair that targets the arylsulfase gene was able to differentiate between Type S and Type C strains of Map and was more sensitive than the IS1311 PCR and REA with a sensitivity of 40 fg µl-1 and was specific for Type S Map. Both assays successfully detected Map directly from faeces. This study developed and validated two genomics informed qPCR assays, 7132B Map and Atsa Type S and found both assays to be highly specific and sensitive for the detection of Map from culture and directly from faeces. This is the first time that a probe-based qPCR has been designed and developed for Map strain typing, which will greatly improve the response time during outbreak investigations.

First Report of Leaf Blight Caused by Botryosphaeria dothidea on Aucuba japonica in Zhejiang Province, China.

Aucuba japonica var. variegata Dombrain is a common evergreen cultivated ornamental in China (Li et al. 2016). In December 2022, severe leaf blight on A. japonica was observed next to the Meishiyuan of Zhejiang Normal University (29°8'4″N, 119°37'54″E) in Jinhua City, Zhejiang Province, China. There were seven plants in the surveyed area, and over 50% of leaves were affected. The early symptoms were small gray spot parts with brown borders on the tip of the leaves. Then the grey parts gradually expanded and became brownish black. In severe cases, the whole leaves became black and blighted. To identify the pathogen, 5 symptomatic leaves were randomly collected from 5 plants and cut into small pieces (5 mm × 5 mm), surface disinfected in 1% sodium hypochlorite solution for 3 min, followed by 75% alcohol for 30 s, then rinsed in sterile distilled water thrice. Tissues were cultured on potato dextrose agar (PDA) and incubated at 28°C for 7 days. Pure cultures were obtained by the single-spore method. Thirteen strains were isolates from the tissues, and nine of them showed similar morphological characteristics. Colonies were white initially, then became gray. The undersides of the colonies became black gradually. Hyaline, fusiform conidia (n = 30) were 17.1 to 24.76 µm (average 20.39 ± 1.906 µm) in length and 5.4 to 6.61 µm (average 6.19 ± 0.434 µm) in width. The DNA of nine isolates were extracted by Ezup Column Bacteria Genomic DNA Purification Kit, and their sequences were identical, so they were named QM1. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1), and β-tubulin (TUB2) genes were amplified with primer pairs ITS1/ITS4, TEF1-728F/TEF1-986R and βt2a/βt2b (Slippers et al. 2004), respectively. The BLAST analysis indicated that ITS (OR215464), TEF1 (OR243689), and TUB2 (OR243688) of the isolate QM1 were 99 to 100% identical to those of Botryosphaeria dothidea (GenBank accession nos. MH329646 for ITS sequences; OL891702 for TEF1 sequences; MK511445 for TUB2 sequences). In addition, the phylogenetic tree based on sequences from ITS, TEF1 and TUB2 was constructed with MEGA 11 by use of the maximum likelihood method with 1,000 bootstrapping iterations. Based on the multi-locus phylogeny and morphological features, the isolate QM1 was identified as B. dothidea. To test the Koch's postulates, ten leaves from three healthy two- to three-year-old A. japonica plants were surface disinfested with 75% ethanol for 30 s, rinsed with ddH2O three times. The leaves were wounded with a sterile needle and inoculated with 2ml drop of the isolate QM1 conidial suspension (106 spores/mL), with sterile distilled water as a control. All plants were placed in a greenhouse at 28°C, >70% relative humidity and 12 h light/day. The experiment was repeated three times. After 7 days, leaves of the inoculated group showed symptoms similar to those observed on the naturally infected leaves, while leaves of the control group remained asymptomatic. The pathogen was reisolated from inoculated leaves and was confirmed as B. dothidea based on morphological and molecular analyses. It has been reported B. dothidea cause leaf disease in a wide range of hosts in China, such as Camellia oleifera (Hao et al. 2023), Kadsura coccinea (Su et al. 2021). To our knowledge, this is the first report of Botryosphaeria dothidea causing leaf blight on Aucuba japonica in Zhejiang Province of China. B. dothidea are usually secondary invaders and are known to cause diseases in stressed plants. The results further expand the host-range of B. dothidea, and would help to establish control strategy against the disease.

First Report of Colletotrichum siamense Causing Anthracnose on Bixa orellana in China.

Annatto (Bixa orellana L.) is widely cultivated in China. Its seed is used as medicine and as an astringent antipyretic. Since 2019, anthracnose-type lesions have been observed on the annatto leaves in the field (about 30 hectares) in Zhanjiang (21˚18'12''N, 110˚17'22''E), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 3 ha). The early symptoms were yellow spots on the edge or tip of leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions (Supplemental Figure 1-A). Ten symptomatic leaves from 10 plants were sampled. The margins of the lesions were cut into 2 × 2 mm pieces and the surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec. After that, pieces were rinsed thrice in sterile water, placed on potato dextrose agar(PDA) medium, and incubated at 28 ℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (BOC-1, BOC-2, and BOC-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. Besides, the three isolates were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University. Colonies on PDA were white to gray with cottony mycelia after incubating in the dark for 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 9.6 to 18.5 µm × 3.5 to 5.5 µm (n = 50). Appressoria were oval to irregular in shape and dark brown, and they measured 6 to 9 µm × 4.5 to 8 µm (n = 30) (Supplemental Figure 1-D, E and F). These morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009;Sharma et al. 2013). For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates were amplified using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences were deposited in GenBank under nos. MZ047377-MZ047379 (ITS), MZ126934-MZ1269346 (GAPDH), MZ126904-MZ1269046 (CHS-1), and MZ126844-MZ1268446 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from ITS, GAPDH, CHS-1, and ACT sequences that clustered the three isolates with C. siamense (the type strain MFLU 090230), (Supplemental Figure 2). The pathogenicity of the three isolates was tested respectively in a greenhouse maintained at 25 to 29℃ and 80% relative humidity. Annatto seeding ( n =5, 2-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water.. The experient was repeated three times. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy (Supplemental Figure 1-G, and H). The same pathogen was re-isolated from all the inoculated leaves based on morphology and ITS analysis. C. siamense has been reported to cause anthracnose in a broad range of hosts (Weir et al. 2012; Wang et al. 2017; Liu et al. 2017; Zhuo et al. 2017 ), but not in B. orellana. To our knowledge, this is the first report of C. siamense causing anthracnose on B. orellana in China. Our study provides important reference information for controlling this disease.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Youngia japonica subsp. elstonii in Hainan Province, China.

Youngia japonica (L.) DC. is a polymorphic annual herb of the Asteraceae family. Although this plant originated in Asia, it is now world-widely distributed. In China, Y. japonica is used for edible or folk medicine to treat viral infections and various kinds of inflammation (Yu et al. 2021). As a traditional Chinese medicinal herb, Y. japonica used for the treatment of inflammatory diseases, such as angina, leucorrhea, mastitis, conjunctivitis, and rheumatoid arthritis (Chen et al. 2006). During the spring of 2023, powdery mildew symptoms were observed on 60% of Y. japonica subsp. elstonii plants in a greenhouse on the Hainan Medical University campus (19° 58' 53″ N; 110° 19' 47″ E) in Haikou, Hainan Province, China. Powdery mildew colonies covered the leaf surfaces and stems of affected plants, causing discoloration and defoliation. Mycelia were superficial and hyphal appressoria were nipple-shaped. Conidiophores (n =30) were unbranched, cylindrical, 99 to 166 × 11 to 16 µm, and produced three to five immature conidia in chains with a crenate outline. Foot cells (n =30) were cylindrical, straight or sometimes curved at the base, and 35 to 61 µm long. Conidia (n =100) were ellipsoid-ovoid to doliiform, 21 to 40 ×13 to 21 µm (length/width ratio = 1.4 to 2.3), with well-developed fibrosin bodies, and produced germ tubes from the lateral position. Based on these morphological characteristics, the pathogen was provisionally identified as Podosphaera xanthii (Braun and Cook 2012). The teleomorph was not observed. A specimen was deposited in the Hainan Medical University Plant Pathology Herbarium as HMYJ-23. To confirm the genus identification and ascertain a putative species, genomic DNA was extracted from mycelium, conidiophores, and conidia using a fungal DNA kit (Omega Bio-Tek, USA). The rDNA internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 (White et al. 1990) and sequenced directly. The resulting 575-bp sequence was deposited in GenBank (accession no. OR229712). A BLASTn search in GenBank of this sequence showed 99% similarity with the ITS sequences of P. xanthii isolates from China (MT260063, OP765400, MW422608, and MT739423), Thailand (LC270778, LC270779, and LC270780), and Argentina (AB525914). Additionally, the 613-bp 28S rDNA region was amplified using the primer pairs NL1 and NL4 (O'Donnell 1993; accession no. OR240257). This region shared 100% similarity with P. xanthii isolates (MK357436, LC371333, LC270780, OP765401, and AB936277) as well. To confirm pathogenicity, five healthy potted plants of Y. japonica subsp. elstonii were inoculated by gently pressing a powdery mildew-infected leaf onto the young leaves. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 24 to 30°C, 70% relative humidity, with a 16-h photoperiod. After 7 days, inoculated leaves showed powdery mildew symptoms whereas no symptoms were observed on control plants. The fungal colonies observed on inoculated plants were morphologically identical to those found on the originally infected leaves collected from Hainan Province. Based on the morphological characteristics and molecular identification, the fungus was identified as P. xanthii. To our knowledge, this is the first record of P. xanthii infecting Y. japonica subsp. elstonii in Hainan province, China. We are concerned that the pathogen could become a threat to the widespread planting of Y. japonica subsp. elstonii in the future.