Primer Pairs Research Articles

First Report of Rust Disease on Alcea rosea caused by Puccinia modiolae in China.

Alcea rosea, belonging to the Alcea genus in the Malvaceae family, originated from China, but it is now grown worldwide. A. rosea has been widely used in traditional Chinese medicine to alleviate constipation, pain, swelling, and sores. In February 2023, typical symptoms of fungal infection were observed on A. rosea at Guizhou Normal University in Guiyang, Guizhou Province, China. The disease incidence was over 90% (n = 100) for the surveyed A. rosea plants, and the disease severity range from 30% to 90%. The initial symptoms of A. rosea rust were the appearance of chlorotic spots on the leaves. Subsequently, numerous reddish to dark-brown erumpent pustules (telia) were observed. Gradually, the entire plant was covered by rust and the center of each lesion turned brown, necrotic, and ruptured over times, eventually causing defoliation. Voucher specimens of infected A. rosea leaves as representative samples have been deposited at Guizhou Normal University (GNU2023LS008). Telia are round in shape, mostly aggregated in mass, with a diameter of 0.28-0.78 mm (0.46 mm, n = 20). They range in color from reddish-brown to dark brown, and are mainly hypophyllous but occasionally formed on the adaxial leaf surface. The teliospores are fusoid with dimensions of 31.3-93.8 × 10.9-21.3 μm (57.5 × 15.1 µm average, n = 50), hyaline or yellowish to light-brown in color, mostly two-celled, with a smooth wall (1.5-3.0 µm) and a thickened apex (3.0-9.0 µm). However, teliospores which are one-, three-, or four-celled with a notch at the apex, are rarely observed. The morphological characteristics of host symptoms and teliospores were similar to those of Puccinia modiolae (Aime and Abbasi 2018; Albu et al. 2019). For phylogenetic analysis, genomic DNA was extracted from the teliospores of infected leaves. To confirm the species-level identification, PCR was performed on the extracted DNA to amplify the ribosomal DNA internal transcribed spacer (ITS) and large subunit (LSU) regions using primer pairs ITS1/ITS4 (Schoch et al. 2012) and NL1/NL4 (Ziemiecki et al. 1990), respectively. The resulting ITS DNA sequence (GenBank accession no. OR607960) showed 100% identity with P. modiolae sequences (OP369291.1), when the query coverage was 100%. The LSU DNA sequence obtained (OR607961.2) shared 99.85% similarity with P. modiolae (MK458702.1). A phylogenetic tree was constructed using MEGA7.0 and the maximum likelihood method based on the ITS and LSU sequences. The fungal isolates collected in this study and several reference sequences of P. modiolae were grouped within a clade that included the isolates reported on A. rosea in Korea (Ryu et al. 2023), with 100% bootstrap support. Pathogenicity testing was conducted by gently pressing spore powder of naturally diseased leaves onto young leaves of three healthy A. rosea plants, with three noninoculated healthy plants serving as controls. The inoculated and noninoculated plants were kept in a growth chamber at the 26°C with a 12 hour light/dark cycle and 80% humidity. After 2 weeks, all inoculated A. rosea plants showed characteristic disease symptoms of rust infection and telia of P. modiolae, while control plants remained symptomless. The pathogen was identical to that observed on the original diseased leaves. The study results indicate that the causal fungus responsible for the disease is P. modiolae, which has been previously reported on Malvaceae plants (Farr and Rossman 2022). To the best of our knowledge, this is the first report of P. modiolae on A. rosea in China. This study will contribute to an increased understanding of the host range of Puccinia modiolae.

Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen's kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

Molecular authentication and discrimination between banana shrimp and Pacific white shrimp by duplex PCR-lateral flow dipstick (dPCR-LFD) system

Banana shrimp is easily substituted by Pacific white shrimp due to its relatively similar morphology and appearance. The duplex PCR-lateral flow dipstick (dPCR-LFD) system was developed to authenticate these two species based on the mitochondrial cytochrome b (Cytb) gene. Two pairs of species-specific primers were designed for dPCR amplification. The specificity was also tested toward nine marine species. The sensitivity of the method was also evaluated at the DNA range of 0.02 %-60 % of adulterated Pacific white shrimp DNA in DNA from banana shrimp. Several types of cooked samples under various conditions were also tested. The developed dPCR-LFD system could specifically differentiate banana shrimp from Pacific white shrimp. No cross-amplification was observed in the specificity test. The sensitivity test showed the detection limit of 0.02 % (0.01 ng) of Pacific white shrimp DNA. The cooking processes including boiling, frying, and steaming did not show any effect on the efficiency of the dPCR-LFD system. This technique could be therefore implemented for the authentication of banana shrimp and Pacific white shrimp products as well as further use for other seafood frauds.

RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice.

Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

Distribution and Mechanism of Japanese Brome (Bromus japonicus) Resistance to ALS-Inhibiting Herbicides in China.

Bromus japonicus is a common monocot weed that occurs in major winter wheat fields in the Huang-Huai-Hai region of China. Pyroxsulam is a highly efficient and safe acetolactate synthase (ALS)-inhibiting herbicide that is widely used to control common weeds in wheat fields. However, B. japonicus populations in China have evolved resistance to pyroxsulam by different mutations in the ALS gene. To understand the resistance distribution, target-site resistance mechanisms, and cross-resistance patterns, 208 B. japonicus populations were collected from eight provinces. In the resistant population screening experiment, 59 populations from six provinces showed different resistance levels to pyroxsulam compared with the susceptible population, of which 17 B. japonicus populations with moderate or high levels of resistance to pyroxsulam were mainly from the Hebei (4), Shandong (4) and Shanxi (9) Provinces. Some resistant populations were selected to investigate the target site-resistance mechanism to the ALS-inhibiting herbicide pyroxsulam. Three pairs of primers were designed to amplify the ALS sequence, which was assembled into the complete ALS sequence with a length of 1932 bp. DNA sequencing of ALS revealed that four different ALS mutations (Pro-197-Ser, Pro-197-Thr, Pro-197-Phe and Asp-376-Glu) were found in 17 moderately or highly resistant populations. Subsequently, five resistant populations, QM21-41 with Pro-197-Ser, QM20-8 with Pro-197-Thr and Pro-197-Phe, and QM21-72, QM21-76 and QM21-79 with Asp-376-Glu mutations in ALS genes, were selected to characterize their cross-resistance patterns to ALS inhibitors. The QM21-41, QM20-8, QM21-72, QM21-76 and QM21-79 populations showed broad-spectrum cross-resistance to pyroxsulam, mesosulfuron-methyl and flucarbazone-sodium. This study is the first to report evolving cross-resistance to ALS-inhibiting herbicides due to Pro-197-Phe mutations in B. japonicus.

First report of a rust disease of Lycium chinense caused by Puccinia tumidipes in Korea.

Lycium chinense Mill is a deciduous broad-leaved shrub belonging to the Solanaceae family and, is widely distributed throughout Korea. This plant is native to, or cultivated for various oriental medicinal purposes in, multiple regions of Asia, including Korea, China, and Japan (Lee 1982; Kim et al. 1994). Eleven Puccinia species have been reported to infect Lycium species (Otálora et al. 2018). In May and October 2022, symptoms of rust disease caused by Puccinia sp. were observed on almost all the leaves of about 60 sprawling stems of L. chinense plants on the seashore of Jeju island, Korea (33°14'15.0835″N, 126°30'53.40E). Brownish red (uredinium) or blackish brown (telium) pustules were observed on upper and lower surfaces of infected leaves. These symptoms were observed on about 40 L. chinense plants, barely growing between rocks on the seashore of Ulsan Metropolitan City, and on the about 20 L. chinense plants on a small home garden of Jindo-gun, Korea, in June and October 2023, respectively. Uredinia were amphigenous, individually scattered, but sometimes formed groups of two or three on leaves and sepals, ferruginous, pulverulent, and surrounded by a ruptured epidermis, often developing into blackish telia. Urediniospores were either ellipsoid or ovoid, approximately 29.3-34.9 × 17-24.3 μm, with yellowish walls, 1-2 μm thick. The germ pores were bizonate, and each band contained four pores covered by low papillae. Blackish-brown telia were observed on both leaf surfaces. Teliospores were broadly ellipsoidal, and rounded at the apex and towards the base. They were measured approximately 37.1-53.4 × 25-34.5 μm. The walls were light chestnut-brown and 2-3.7 μm thick, apically up to 5-9 μm thick. The swollen pedicel was persistent, basal, hyaline, smooth, and similar in length to the spores (Fig. 1). These morphological characteristics were similar to those of P. tumidipes, as described by Otálora et al. (2018). The representative specimens were preserved at the Animal and Plant Quarantine Agency Herbarium (PQK220531, -230605, and -231026). The fungal internal transcribed spacer (ITS2) and cytochrome oxidase subunit 3(CO3) regions were amplified from the total DNA of the isolates, using the primer pairs ITS5, ITS4, CO3F1, and CO3R1 for phylogenetic analysis (White et al. 1990; Vialle et al. 2009). PCR products were sequenced (Celemics, Seoul, Korea), and deposited in GenBank (Accession numbers are shown in Fig. 2.). The combined ITS2 and CO3 sequences were grouped with those of other isolates of P. tumidipes in the phylogenetic tree (Fig. 2). In November 2022, three pathogenicity tests were conducted using a urediniospore suspension made with the PQK220531 isolate in sterile distilled water. The suspension was smeared onto the upper surface of healthy L. chinense leaves. The three inoculated plants were kept in the dark at saturated moisture levels for 24 hours and placed in an isolated glasshouse together with the three control plants. After two weeks, uredinia of P. tumidipes were observed on the leaves of the inoculated plants, but not on the control plants. Although no spermogonial or aecial stage has been observed in Korea, our study has proven that P. tumidipes is the causal fungus of rust disease in L. chinense. This result is also the first discovery of the New-World P. tumidipes in Asia, showing this fungus is not limitedly distributed in America and suggesting further surveys be done on its exact geographical distribution.

QTL mapping for the flag leaf-related traits using RILs derived from Trititrigia germplasm line SN304 and wheat cultivar Yannong15 in multiple environments

BackgroundDeveloping and enriching genetic resources plays important role in the crop improvement. The flag leaf affects plant architecture and contributes to the grain yield of wheat (Triticum aestivum L.). The genetic improvement of flag leaf traits faces problems such as a limited genetic basis. Among the various genetic resources of wheat, Thinopyrum intermedium has been utilized as a valuable resource in genetic improvement due to its disease resistance, large spikes, large leaves, and multiple flowers. In this study, a recombinant inbred line (RIL) population was derived from common wheat Yannong15 and wheat-Th. intermedium introgression line SN304 was used to identify the quantitative trait loci (QTL) for flag leaf-related traits.ResultsQTL mapping was performed for flag leaf length (FLL), flag leaf width (FLW) and flag leaf area (FLA). A total of 77 QTLs were detected, and among these, 51 QTLs with positive alleles were contributed by SN304. Fourteen major QTLs for flag leaf traits were detected on chromosomes 2B, 3B, 4B, and 2D. Additionally, 28 QTLs and 8 QTLs for flag leaf-related traits were detected in low-phosphorus and drought environments, respectively. Based on major QTLs of positive alleles from SN304, we identified a pair of double-ended anchor primers mapped on chromosome 2B and amplified a specific band of Th. intermedium in SN304. Moreover, there was a major colocated QTL on chromosome 2B, called QFll/Flw/Fla-2B, which was delimited to a physical interval of approximately 2.9 Mb and contained 20 candidate genes. Through gene sequence and expression analysis, four candidate genes associated with flag leaf formation and growth in the QTL interval were identified.ConclusionThese results promote the fine mapping of QFll/Flw/Fla-2B, which have pleiotropic effects, and will facilitate the identification of candidate genes for flag leaf-related traits. Additionally, this work provides a theoretical basis for the application of Th. intermedium in wheat breeding.

Genetic insights into Cyphocharax magdalenae (Characiformes: Curimatidae): Microsatellite loci development and population analysis in the Cauca River, Colombia.

Cyphocharax magdalenae, a Colombian freshwater fish species, plays a vital role in nutrients distribution and serves as a significant food source for other fish species and local fishing communities. Considered a short-distance migratory species, C. magdalenae populations face substantial extinction risk due to human activities impacting their habitats. To address the lack of knowledge on genetic diversity and population structure, this study used next-generation sequencing technology to develop species-specific microsatellite loci and conducted a population genetics analysis of C. magdalenae in the middle and lower sections of the Cauca River, Colombia. Out of 30 pairs of microsatellite primers evaluated in 324 individuals, 14 loci were found to be polymorphic, at linkage equilibrium and, in at least one population, their genotypic frequencies were in Hardy-Weinberg equilibrium. Results showed high genetic diversity levels compared to other neotropical Characiformes, with inbreeding coefficients similar to those reported for phylogenetically related species. Moreover, C. magdalenae exhibits seasonal population structure (rainy-dry) consisting of two genetic stocks showing bottleneck signals and high effective population sizes. This information is essential for understanding the current species genetics and developing future management programs for this fishery resource.

Development of a PCR-based assay for specific and sensitive detection of Fusarium buharicum from infected okra plant.

Fusarium wilt, caused by the fungus Fusarium buharicum, is an emerging disease of okra in Japan. The disease was first reported in Japan in 2015, causing significant damage to okra seedlings. Due to the potential threat in okra cultivation, the development of an accurate detection method for F. buharicum is needed for the surveillance and management of the disease. In this study, we designed a primer set and developed conventional and nested PCR assays for the specific detection of F. buharicum in infected okra plants and contaminated soil, respectively. We compared the diversity of the translation elongation factor 1 alpha (EF-1α) gene of F. buharicum with 103 other fungal species/isolates to design a species-specific primer. This primer pair successfully amplified approximately 400 bp of PCR product that was only detected in the F. buharicum isolate, not in the other fungal isolates. The developed nested PCR method was highly sensitive and could detect the fungus from a 0.01 fg DNA sample. The primer successfully detected the pathogen in artificially infected plants and soil by conventional and nested PCR, respectively. This is the first report of the development of the F. buharicum-specific primer set and detection assays, which can be used for the specific and sensitive detection of F. buharicum in field samples and for taking early control measures.

Outbreak of Septoria leaf spot caused by Sphaerulina musiva on Populus × euramericana in China.

In June 2023, severe leaf spots were noted in Populus × euramericana cv 'Nanlin95' plantations located in the Nanjing Baguazhou Wetland Park (32°09'16.97″N, 118°48'16.74″E) of Jiangsu Province and Populus × canadensis cv 'Sacrau 79' and Populus × canadensis cv 'Guariento' in the Liyuan Village in Nanyang City (32°53'43.70″N, 112°17'29.12″E) of Henan Province, respectively. The disease incidence in both locations could reach 97.9% (556 out of 568 trees) and 98.9% (2409 out of 2436 trees), respectively. The initial symptoms appear as numerous small and circular spots (1.59 to 3.18 mm in diameter) with gray or tan centers and dark-brown margins on the leaves. As the spots age, they sometimes enlarge, often coalesce, and may extend down the petioles. Diseased leaves and petioles were both surface sterilized with 75% ethanol for 30 seconds. With the aid of a hand lens, pycnidia (brown to black, spherical in profile, 90 to 250 µm diam) were easily picked out in the center of the spots and subsequently transferred into 1 mL sterilized water for preparing the spore suspension plated on KV8 medium amended with 100 mg/liter streptomycin sulfate and 50 mg/liter chloramphenicol. After 12 days of incubation, 86 single-spore isolates were obtained and identified as typical Septoria-like fungi according to morphological features, including slow-growing, gray or black colonies with pink mucilaginous matrix and hyaline, straight or curved conidia (size = 25 to 59 × 3.5 to 4 μm; septa = 1 to 6). Species identification was further validated by PCR amplification and sequencing of the internal transcribed spacer (ITS) region with ITS1/ITS4 primer pairs. Multiple sequence alignments with ClustalW revealed that the obtained ITS sequences of 86 isolates were 100% identical to each other. A BLAST search in GenBank indicated that the selfsame sequences of two representative isolates (isolate BGZ11 of Jiangsu Province, accession no. OR660379; isolate KZB22 of Henan Province, accession no. OR711499) shared 99.8% identity (494 of 495 bp) and 100% identity (504 of 504 bp) with related sequences of Sphaerulina musiva (Peck) Quaedvlieg, Verkley, and Crous (syn. = Septoria musiva Peck) in GenBank (MN275187; KF251619), respectively. Furthermore, we used a S. musiva-specific PCR assay (Abraham et al. 2018) on symptomatic leaf samples collected from the plantation. Each sample consisted of 20 cut-out leaf spots per leaf. Eight of the 10 samples were positive for S. musiva DNA. To confirm pathogenicity, six sterile tissue culture of poplar plants (Populus trichocarpa and Populus × euramericana cv 'Nanlin895') were respectively transplanted into pots and grown in a greenhouse for a week and for a month with an 18-h photoperiod augmented with sodium lamps and a 20°C (day)/16°C (night) temperature regime. Inoculations were conducted by spraying the plants with conidia suspension (106 conidia/mL) (LeBoldus et al. 2010). Control plants were sprayed with distilled water. Leaf spots were developed on the inoculated P. trichocarpa leaves at one week and P. × euramericana cv 'Nanlin895' leaves at 10 days after inoculation while no symptoms were observed on the control plants. The fungus S. musiva was successfully reisolated from all symptomatic leaves fulfilling Koch's postulates. Sphaerulina musiva only causes an endemic leaf spot disease on its natural North American host Populus. deltoides (Feau et al. 2010; Ostry 1987). However, on susceptible Populus species (e.g., P. balsamifera, P. trichocarpa, P. maximowiczii) and hybrids, S. musiva causes not only leaf spots but also severely damaging stem and branch cankers (Jeger et al. 2018; LeBoldus et al. 2009; Sondreli et al. 2020). To our knowledge, this is the first report of S. musiva causing leaf spots on poplar in China. Large-scale timber imports (e.g., cut branches, isolated bark, wood with and without bark) potentially lead to anthropogenic-facilitated transport of this pathogen. This outbreak of Septoria leaf spot underscores the potential threat of this pathogen to P. × euramericana in China, where it is widely planted as a keystone forestry species.

Increased begomovirus disease incidence and severity in cucurbits associated with the emergence of CuLCrV in mixed infection with SLCuV in Baja California Sur, Mexico

Begomoviral diseases in cucurbits have been observed in Baja California Sur (BCS), Mexico recurrently since the 2017 detection of the Squash leaf curl virus (SLCuV) in squash. Two years later, samples of symptomatic plants were collected in three arid agricultural regions, where the most frequently observed symptoms were chlorosis, reduced leaf size, stunting and severe leaf deformation, observed mainly on squash and cantaloupe. The mean incidence of begomovirus-like symptoms differed among cucurbits, with 20% in cucumber, 28% in cantaloupe, 30% in watermelon and 50% in squash. Samples were tested by polymerase chain reaction for begomovirus infection with degenerate primers, and later the virus identities were confirmed by specific primer pairs. All symptomatic cucurbit samples tested positive for SLCuV, and in a second analysis of these previously tested samples, squash, and cantaloupe samples tested positive for Cucurbit leaf crumple virus (CuLCrV). Thus, these samples showed a mixed CuLCrV/SLCuV infection. Pairwise identity analysis of the DNA-A component of the viruses indicated the presence of CuLCrV at 99%, and SLCuV at 99.1%. Recombination analyses between these viruses were negative, indicating that they belong to well-defined phylogenetic groups. Additionally, the 5´-TGGTGTCC-3´ iteron was consistently observed in all SLCuV and CuLCrV variants, including in other close species of the SLCuV clade. However, an additional iteron was observed in CuLCrV-BCS that is not in the Arizona and California variants, indicating that more studies are needed to elucidate the role of the additional iteron and the importance of CuLCrV/SLCuVSLCV mixed infection in emerging viruses in Mexico and other parts of the world.

Two novel PCR-based assays for sexing of Silene latifolia and Silene dioica plants

Silene latifolia and S. dioica are model systems in studies of plant reproduction, chromosome evolution and sexual dimorphism, but sexing of plants based on morphology is only possible from flowering stage onwards. Both species show homogametic females (XX) and heterogametic males (XY).•Here we developed two assays (primer pairs ss816 and ss441) for molecular sexing of S. latifolia and S. dioica, targeting length polymorphisms between the X and Y-linked copies of the spermidine synthase gene.The two assays were successful in identifying known (flowering-stage) males and females from UK and Spanish populations, with an error rate of 3.1% (ss816; successful for both species) and 0% (ss441, only successful for S. latifolia). Our assays therefore represent novel tools for rapid, robust and simple determination of the genotypic sex of S. latifolia and S. dioica.

First Detection of Atractylodes Mild Mottle Virus in Atractylodes lancea (Thunb.) DC. in Hubei Province of China.

The cultivated aromatic medicinal herb Atractylodes lancea (Thunb.) DC. is widely used in the pharmaceuticals, nutraceuticals, and cosmetics industries (Na-Bangchang et al. 2014; Zhan et al. 2023). Huanggang in Hubei Province is a major production area for A. lancea (Huang et al. 2022; Wang et al. 2023). In April 2023, more than two-thirds of the surveyed plant leaves in this region exhibited virus-like symptoms, such as curling and mosaic patterns. To identify the underlying cause, 80 symptomatic plant leaf samples were collected from four fields (20 leaves per field) in this region and pooled for virome analysis. Total RNA, including ribosomal RNA, was extracted from the pooled samples using the Plant RNA Extraction Mini Kit (Onrew Biotech, Guangdong, China), for sequencing library construction. The Illumina NovaSeq 6000 platform was used to sequence the library and generate 150 bp paired-end reads. After processing the raw data with Trimmomatic software, a total of 44,354,650 high-quality clean reads were obtained. The clean reads were aligned against ribosomal RNA using BWA software (v0.7.17) to avoid interference and eliminate corresponding sequences. After removing potential contamination, contig assembly of the clean reads was performed using Megahit software (v1.2.9). The resulting contigs were compared with the virus NT database using the BLASTn program. Sequence pairwise comparison revealed 8 contigs (574 nt to 2243 nt) with identities ranging from 81.88% to 90.77% with Atractylodes mild mottle virus (AMMV, NC_027924.1, Lim et al., 2015). Additionally, contigs mapped to Carlavirus, Pelarspovirus, and other plant viruses in our virome dataset had low coverage and pairwise identity (less than 70%), which need to be further investigated. The presence of AMMV was confirmed by aligning the clean reads to the reference sequence (NC_027924.1) using BWA and SAMtools software, resulting in a consensus sequence (8024 nt) with gaps. DNA extraction from the pooled samples was performed using the Rapid Universal Genomic DNA Extraction Kit (Simgen, Zhejiang, China). Two pairs of specific primers, 3399F (5'-AAAGAAGAACCTCCTGATACGG-3')/5924R (5'-TGAACCTGATTCTCTTGGC-3') and 1830F (5'- CTCAGGAAATCCCAATGC -3')/3640R(5'-TTTCCCAATGTTCTTCGGG-3'), were designed to amplify the complete gene sequences of polymerase and coat protein (CP), based on the consensus sequence. The PCR products with the lengths of 2521 bp and 1814 bp were cloned into the pMD18-T vector (Takara Biotech, Dalian, China) for sequencing. The BLASTn analysis showed that the polymerase and CP gene sequences shared an identity of 94.51% (1929/2041 nt) and 88.41% (1419/1605 nt) with the AMMV isolate (NC_027924.1), respectively. The sequences have been deposited in GenBank under the accession numbers OR544810 and OR544811. We collected leaves from 32 A. lancea plants (16 symptomatic and 16 asymptomatic) in the fields. RT-PCR was conducted using CPF (5'-CTGCGAATATGAAAGTGC-3') and CPR (5'-GGTGAGCTTGTCTGTTAGG-3') primers, which were designed targeting a 527bp fragment of the CP gene (OR544811). Amplicons of the expected size (527bp) were detected in 24 plants (11 symptomatic and 13 asymptomatic), three of which were sequenced by Sanger sequencing, showing a 100% match to OR544811. These findings indicate that AMMV is prevalent in the major production area of A. lancea. Further research is needed to better characterize the potential risks of AMMV to A. lancea cultivation in China as well as other countries.

First Report of Sweet Potato Virus E(SPVE) Infecting Sweet Potato in China.

Sweet potato (Ipomoea batatas [L.] Lam.) is a versatile crop, cultivated in the subtropical and tropical areas, as food, fodder, and industrial raw material crop. In China, sweet potato has been used as a health-care food in recent years, as it contains a wide range of nutrients and xenobiotic phytochemicals. However, viral diseases are major constraint for the sweet potato yield and quality, especially the seed production and quality. Over 30 species of viruses infect sweet potato worldwide (Clark et al. 2012). More recently, a few new viruses infected sweet potato were identified, such as sweet potato virus E (SPVE), which was reported in Korea(Jo et al. 2020). In May 2022, a sweet potato sample (JSXZ1) with virus-like symptom, such as mosaic and vein clearing were collected from sweet potato germplasm Xuzhou resource nursery, Jiangsu Province, China (N34˚16', E117˚18') (Fig. S1A). To investigate the virus disease, the sample JSXZ1 showing the typical symptoms of disease was prepared for Small-RNA (sRNA) deep-sequencing. The sRNA library was constructed using TruSeq™ Small RNA Sample Prep Kits (Illumina, San Diego, USA) and sequenced using the Illumine Hiseq 2500 platform by LC-Bop Technologies (Hangzhou) CO., LTD. The sample was sequenced to obtain 26, 358, 439 raw reads and 22, 969, 139 clean reads after quality control trimming and analysis. The Velvet 1.0.5 software was used to de novo assemble the clean reads (18 to 28 nt) into larger contigs, which were then compared with the nucleotide sequences in the National Center for Biotechnology Information (NCBI) database using the BLASTn algorithm. Viruses found in the sample were sweet potato latent virus (SPLV), sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato badnavirus A (SPBV-A) and sweet potato badnavirus B (SPBV-B). Surprisingly, besides the viruses listed above, 28 contigs matched sequences of SPVE isolate GS (MH388502). To verify the result, total RNA was extracted from the sample JSXZ1 and from other leave samples (JSXZ2-JSXZ5) that contained SPFMV, SPVC, SPLV, SPVG respectively stored in lab using FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co., LTD, Nanjing, China). cDNA was synthesized using random primer (hexadeoxyribonucleotide mixture; pd(N)6). The cDNA serves as template in PCR using a newly designed primer pairs based on SPVE p1 gene (SPVE-F: 5'- TCACCAAAAAGAATGCTACAAC-3'/SPVE-R: 5'-GAAATCCTCCCACTCTCCATA-3'). An expected ~500-bp PCR fragment was obtained in JSXZ1, while none of the fragment was obtained from JSXZ2-JSXZ5 (Fig. S1B). The PCR fragment was cloned into pMD18-T vector (Takara Bio Inc., Beijing, China) and plasmid DNA from transformed Escherichia coli DH5α cell (n=3) were commercially sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequences of the three fragment clones we obtained were 100% identical when compared. A BLASTN analysis of the sequences revealed that they are specific to SPVE and shared 98.62% nucleotide identity to SPVE GS isolate (MH388502) and one sequence was submitted to GenBank (Accession number OQ948331). To determine the occurrence of SPVE in infected sweet potato plants, a total of 37 leaves samples with viral symptom collected from Shandong Province (n=6) and Jiangsu Province (n=31) were indexed by RT-PCR as described before. Only 9 (24.3%) out of 37 from Shandong (n=1) and Jiangsu (n=8) were positive to SPVE respectively. In addition, five additional viruses (SPFMV, SPVC, SPVG, SPLV, SPCSV) were detected among these 37 samples and always in a mixed infection of two or more viruses. To our knowledge, this is the first report of SPVE infecting sweet potato in China. Sweet potato is an important crop in China and other countries (Zhang et al. 2023). China is the largest sweet potato producer all over the world. In addition, as sweet potato is produced through the vegetative propagation mode, thus, more attention should be paid to detection and monitoring of occurrence of SPVE in China.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Acalypha indica in China.

Acalypha indica L. is an annual erect herb of the Euphorbiaceae family. This plant is found widely in the tropics and parts of Africa and Asia (Chakraborty et al. 2023). In China, A. indica is a vegetable and also used as a folk medicine due to its antipyretic and hemostatic, antibacterial and anti-inflammatory properties. In February 2022 and 2023, powdery mildew symptoms were observed on 70% of A. indica plants 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, 66 to 150 × 10 to 15 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 31 to 59 µm long. Conidia (n =100) were ellipsoid-ovoid to doliiform, 20 to 33 ×12 to 20 µm (length/width ratio = 1.3 to 2.4), 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 HMAI-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. OR775733). A BLASTn search in GenBank of this sequence showed 99% similarity with the ITS sequences of P. xanthii on plants of Fabaceae, Malvaceae and Cucurbitaceae family from China (MH143485, MT242593, MK439611 and MH143483), Thailand (LC270779 and LC270778), Korea (MG754404), Vietnam (KM260704), and Puerto Rico (OP882310). Additionally, the 28S rDNA region was amplified using the primer pairs NL1 and NL4 (O´Donnell 1993; accession no. OR784547). This region shared 99% similarity with P. xanthii isolates (LC371333, LC270780, AB936277, and OP765401) as well. To confirm pathogenicity, five healthy potted plants of A. indica were inoculated by gently pressing a powdery mildew-infected leaf onto 15 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. In different countries and regions, P. xanthii has been previously reported on A. indica from Sudan and India (Amano 1986). To our knowledge, this is the first record of P. xanthii infecting A. indica in China. We are concerned that the pathogen could become a threat to the widespread planting of A. indica in the future.

First report of Pseudomonas cichorii causing bacterial leaf blight of pocketbook plant (Calceolaria hybrida) in Taiwan.

Pocketbook plants (Calceolaria spp.) are flowering ornamentals often grown as potted plants (Poesch 1937). In December 2022, leaf blight symptoms were observed on 2-mo-old plants of C. hybrida F1 'Dainty'. The disease was found in a nursery in Ren'ai Township, Nantou, and about 20% of the plants exhibited symptoms. Symptomatic plants had brown or gray necrotic lesions of different sizes and shapes, mostly around leaf margins. Lower leaf wilting was also observed (Fig. S1, A and B). Three plants were sampled. Leaf lesions were surface-disinfected with 75% ethanol and cut into smaller pieces in 10 mM MgCl2. After observing bacterial streaming under a microscope, the bacteria were streaked onto nutrient agar (NA). Following 2 days at 28°C, a type of round, creamy white colony predominated on all the plates. Three strains (Calc-A, Calc-B, and Calc-C) were obtained, one from each plant. The strains produced fluorescent pigments on King's B medium and were tested Gram-negative. The strains were characterized with the LOPAT scheme (Schaad et al. 2001). They did not exhibit activities of pectic enzymes, arginine dihydrolase and levan sucrase, but produced oxidase and induced the hypersensitive response in tobacco. DNA was extracted from the strains for PCR amplification of the 16S rDNA with primer pair 27f/1492r as described by Lane (1991). The 16S rDNA sequences were compared with entries in the GenBank database. The sequences obtained (GenBank accession no. OR824302) matched that of Pseudomonas cichorii MAFF 301158 (accession no. AB724288; 1,403/1,403 bp) and were 99% identical to that of DSM 50259T (accession no. CP074349; 1,391/1,405 bp). The strains were also tested with the species-specific primers hrp1a and hrp2a (Cottyn et al. 2011). The amplicons were sequenced and a BLASTn search showed that the sequences (accession no. OR827305) shared the highest identity (99.3%) with that of P. cichorii strain 83-1 (accession no. DQ168848; 848/854 bp) and were 97.3% identical to the sequence of DSM 50259T (accession no. CP074349; 831/854 bp). Calc-A was selected as a representative strain and deposited in the Bioresource Collection and Research Center, Taiwan (reference no. BCRC 81432). Koch's postulates were fulfilled by spray-inoculating a suspension of Calc-A on three 2-mo-old C. hybrida F1 'Dainty' plants. The inoculum was prepared by suspending NA-grown cells in 10 mM MgCl2 including 0.02% Silwet L-77 (OD600 = 0.3; 1.5 x 108 CFU/ml). For the controls, three plants were sprayed with bacteria-free solution. The plants were bagged throughout the experiment and kept in a growth chamber (14/10 h light/dark; 26/24°C day/night). Leaf blight and wilting symptoms developed on all leaves of the inoculated plants after 30 h, but not the controls (Fig. S1, C and D). The pathogen was reisolated from the treatment group, and colony PCR with hrp1a/hrp2a showed that the reisolated strain shared the same sequence with Calc-A to Calc-C. Repeating the inoculation assay produced consistent results. This is the first report of P. cichorii affecting Calceolaria in Taiwan. The bacterium has been reported infecting diverse crops in Taiwan, such as tomato and lettuce (Tsai et al. 2014). Expanding the understanding of the pathogen's potential hosts could help prevent its spread across important crops.

First report of leaf blight caused by Stemphylium lycopersici on Salvia splendens in China.

Salvia splendens is a popular ornamental plant in China with extensive potentials, including value in traditional Chinese medicine and in environmental restoration function (Li et al. 2008). In September 2019, leaf blight disease was observed on road side plants of S. splendens in Bayi park, Nanchang city, Jiangxi province, China. The typical symptoms appeared as irregular necrotic spots or leaf blight, accompanied by extensive scorch necrosis or ultimately defoliation. Small segments cut from diseased leaves were surface sterilized in a 2% sodium hypochlorite solution for 2 min and rinsed three times with sterile distilled water. Then, the samples were placed on potato dextrose agar (PDA) plates incubated at 25°C in darkness. Pure cultures were obtained by the hyphal tip method. Morphologically, all 11 colonies were identical to each other on PDA. Two strains, YZU 191468 and YZU 191481, were selected for further study and deposited in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. The 7-day-old colonies were circular, 53 to 56 mm in diameter, and consisted of white mycelium with a buff margin, and were cinnamon colored in the center of the reverse side. To examine conidial morphology, the mycelium was transferred onto potato carrot agar (PCA) and incubated at 23°C with a period of 8 h light/16 h dark for 7 days. Conidia were normally solitary or two in a chain, ellipsoid or long ellipsoid, beakless, 10 to 23×30 to 60 µm in size (n=50). Based on morphology, the isolates were consistent with Stemphylium lycopersici (Yamamoto 1960). To confirm the identification, genomic DNA was extracted from both isolates and used to amplify the internal transcribed spacer rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and calmodulin (CAL) genes with primer pairs ITS5/ITS4, gpd1/gpd2, and CALDF1/CALDR2, respectively (Woudenberg et al. 2017). Sequences were deposited in GenBank with accession numbers OP564983 and OP564984 (ITS), OP892529 and OP892530 (GAPDH), OP584970 and OP584971 (CAL). A neighbor-joining tree was constructed with Mega 7.0 based on the combined dataset with 1,000 bootstrap replicates. The resulting phylogenetic tree showed that the strains from S. splendens clustered with S. lycopersici (CBS 122639 and CBS 124980) supported with 100% bootstrap values. The molecular analyses confirmed that the species causing leaf blight symptoms was S. lycopersici. To test pathogenicity, healthy leaves of S. splendens were surface sterilized and inoculated by mycelium blocks (6 mm in diameter) and spore suspension (1×106 spore/mL) of representative strains YZU 191468 and YZU 191481, respectively. Controls were inoculated with blocks of PDA and sterile water. Each strain was inoculated on three leaves of a plant. One clean plant was used as control. The test was replicated three times. After inoculation, the plants were covered with plastic bags and incubated in a greenhouse (25℃, 80 % relative humidity, 8 h light/16 h dark). After 5 days, the inoculated leaves exhibited dark brown spots with white mycelium, followed by withering of necrotic tissues. There were no symptoms observed on the controls. The fungal isolates inoculated leaves had the same morphological characteristics as the strains used for inoculation. S. lycopersici has been found on eggplant and Zinnia elegans in China (He et al. 2019; Yang et al. 2017). To the best of our knowledge, this is the first report of S. lycopersici causing leaf blight on S. splendens in China. This finding offers a new reference for the management and control of S. splendens leaf diseases in China.

Abstract LB111: CORSA study - Telomere length measurement of 5371 Austrian colorectal cancer and adenoma patients and healthy controls: Relationships to tumor stage, course of disease and to clinical, life style and health parameters

Abstract CORSA study - Telomere length measurement of 5371 Austrian colorectal cancer and adenoma patients and healthy controls: relationships to tumor stage, course of disease and to clinical, life style and health parametersPetr Hanak1, Michal Kroupa1,3,4, Uttarilli Anusha1, Ludmila Vodickova1,3,4 Andrea Gsur2, Stefanie Brezina2, Pavel Vodicka1,3,4 1 Department of Molecular Biology of Cancer, Institute of Experimental Medicine of the Czech Academy ofSciences, Videnska 1083, 142 20 Prague, Czech Republic.2 Center for Cancer Research, Medical University of Vienna, Vienna, Austria;3 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic 4Faculty of Medicine and Biomedical center in Pilsen, Pilsen, Czech Republic Presenting author: Petr Hanák (e-mail: petr.hanak@iem.cas.cz)Telomeres are important parts of human chromosomes, playing a pivotal role in maintenance of genomic integrity. If their function is perturbed, it may trigger many pathologies, including colorectal cancer (CRC). In the present study, comparison of assessed relative telomere length (RTL) and tumor stage, progression of the disease and various health status and lifestyle parameters is investigated. Relative telomere length was measured in samples of DNA from peripheral blood lymphocytes of CRC patients by monochromatic multiplex qPCR. Repetitive telomere motif TTAGG was amplified from specific primer pair simultaneously with the reference albumin gene, the second one amplified from another set of primers specific to this gene. The qPCR reaction is based on intercalating dye Syto 9, which does not distinguish specific target. A separation of amplification of one target from the other one is based on division in time of both amplifications and on different levels of annealing temperatures of primer pairs. Relative telomere length was assessed in all patient samples. Obtained data correspond to our previously observed values. Here, we observe significant decreasing in RTL in the order healthy controls, adenoma and CRC patients (controls vs adenoma patients P&amp;lt;0.0001, controls vs CRC patients P=0.0001). Detailed statistical analyses and assignment to all relevant groups including health and lifestyle parameters is currently in progress. Acknowledgement &amp; Funding: The study was funded by the Grant Agency of Czech Republic (21-27902S), National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union – Next Generation EU and Ministry of Health of the Czech Republic (NU22J-03-00028) Citation Format: Petr Hanák. CORSA study - Telomere length measurement of 5371 Austrian colorectal cancer and adenoma patients and healthy controls: Relationships to tumor stage, course of disease and to clinical, life style and health parameters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB111.

Development of PCR-based assays for the detection of the evident and latent infection with Stilbocrea banihashemiana, the causal agent of fruit tree cankers

The fig Bionectria canker, caused by Stilbocrea banihashemiana, is rapidly spreading in fruit trees, posing a significant threat to the horticultural industry. Early and specific detection of this pathogen is crucial for effective management. We developed a PCR-based assay using species-specific primers designed by exploiting sequence differences in the translation elongation factor 1-alpha (tef1) gene for early, rapid, and reliable diagnostics of this pathogen. The primers successfully amplified the target pathogen and did not react with other non-target fig canker-causing agents in direct and nested PCR. The designated primer pair, TEF-Sb3, with a 577 bp amplicon, demonstrated its efficacy by successfully detecting the pathogen with the lowest detectable concentration of 1 × 10−15 and 1 × 10−17 g.L−1 in direct and nested PCR, respectively. Furthermore, the selected primer pair had high performance in detecting the target pathogen in naturally infected samples with evident canker symptoms in nested PCR. Moreover, our molecular tool has effectively identified latent infection of S. banihashemiana in asymptomatic samples from fig and walnut trees from three different regions of Fars Province. This is a notable advancement, as traditional culture-based methods have failed to detect these infections. Our results suggested that the designed species-specific PCR assays were sufficiently sensitive, specific, and reliable for identifying S. banihashemiana from pure cultures and detecting the pathogen in symptomatic and asymptomatic trees. This assay can be a rapid and cost-effective tool for monitoring and managing Bionectria canker caused by S. banihashemiana in natural and potential host trees.

The development and utilization of two SCAR markers linked to the resistance of banana (Musa spp. AAA) to Fusarium oxysporum f. sp. cubense race 4

Banana (Musa spp.) production worldwide is seriously threatened by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). The best way to control this disease is to grow resistant cultivars. However, it requires large-scale field evaluations and labor- and time-consuming to obtain disease-resistant germplasm. Development of early, reliable, and reproducible selection strategies are considered as the efficient approach which could speed up the selection procedure. In this study, two pairs of sequence-related amplified polymorphism (SRAP) primers related to banana Foc resistance/susceptibility were screened from 100 pairs of random primers. Correspondingly, two pairs of sequence characterized amplified region (SCAR) markers (namely SC4-F/SC4-R and SC14-F/SC14-R, respectively) were successfully generated from these two SRAP markers using 30 cultivars either resistant or susceptible to Foc. Both SCAR markers were located in mitochondrion genome and showed discriminatory power of 96.67% and 100%, respectively. Mitochondrial proteins possibly play a very important role in banana resistance to Foc. In additional, these two SCAR markers were employed simultaneously to screen potential resistant germplasm from 53 accessions with unknown resistance to Foc, and the results revealed a consistency of 83.0% with each other, further indicating their high reliability and reproducibility. These results suggest that both SCAR markers could be used in molecular marker-assisted selection for banana germplasm resistant to Fusarium.