Mild mosaic patterns appeared on the newly emerging leaves of inoculated plants after a 30-day incubation period. Three specimens from each of the two initial symptomatic plants and two specimens from each inoculated seedling reacted positively to Passiflora latent virus (PLV) testing using the Creative Diagnostics (USA) ELISA kit. In order to unequivocally determine the viral strain, RNA was extracted from leaves of one original symptomatic greenhouse plant and one inoculated seedling using the TaKaRa MiniBEST Viral RNA Extraction Kit (Takara, Japan). Two RNA samples underwent reverse transcription polymerase chain reaction (RT-PCR) analysis utilizing primers PLV-F (5'-ACACAAAACTGCGTGTTGGA-3') and PLV-R (5'-CAAGACCCACCTACCTCAGTGTG-3') as detailed by Cho et al. (2020). The 571-base pair RT-PCR products were obtained from the original greenhouse sample, as well as from the inoculated seedling. Using the pGEM-T Easy Vector, amplicons were cloned, followed by bidirectional Sanger sequencing of two clones per sample (performed by Sangon Biotech, China). The sequence of a clone from an initial symptomatic sample was submitted to NCBI (GenBank accession number OP3209221). The nucleotide sequence of this accession displayed an impressive 98% identity to a PLV isolate from Korea, specifically the one found in GenBank under accession number LC5562321. Two asymptomatic samples' RNA extracts, upon ELISA and RT-PCR testing, proved negative for PLV. In addition, the symptomatic sample originally collected was tested for common passion fruit viruses, including passion fruit woodiness virus (PWV), cucumber mosaic virus (CMV), East Asian passiflora virus (EAPV), telosma mosaic virus (TeMV), and papaya leaf curl Guangdong virus (PaLCuGdV), and the RT-PCR tests yielded negative results for all of these viruses. Given the leaf chlorosis and necrosis symptoms, we should keep open the possibility of a mixed infection with other viruses. PLV, a detrimental factor, influences fruit quality and potentially lessens its market worth. immunochemistry assay According to our current understanding, this Chinese report marks the initial documentation of PLV, offering a valuable reference for identifying, preventing, and controlling PLV. With the financial backing of the Inner Mongolia Normal University High-level Talents Scientific Research Startup Project (grant number ), this research was undertaken. Transform the sentence 2020YJRC010 into ten unique rewrites, each with a distinct structural arrangement, in a JSON array format. Figure 1 is presented in the supplementary material. Symptoms observed in PLV-infected passion fruit plants in China include: mottled leaves, distorted leaf shapes, puckered older leaves (A), mild puckering on young leaves (B), and ring-striped spots on the fruit (C).
A perennial shrub, Lonicera japonica, has held a long-standing role as a medicinal herb, used historically to counteract heat and toxins. To alleviate external wind heat or febrile conditions, the branches of L. japonica and unopened honeysuckle flower buds serve as traditional remedies (Shang et al., 2011). In July 2022, L. japonica plants grown at the experimental base of Nanjing Agricultural University (coordinates N 32°02', E 118°86') in Nanjing, Jiangsu Province, China, displayed a serious disease. Amongst the surveyed Lonicera plants, a count of over 200 exhibited an incidence of leaf rot exceeding eighty percent in the leaves. Chlorotic spots were the initial symptoms, subsequently followed by the gradual unfolding of visible white mycelial strands and powdery fungal spores on the foliage. Selleckchem Durvalumab On both the front and the back of the leaves, brown diseased spots appeared gradually over time. Thus, the accumulation of multiple disease areas induces leaf wilting and the separation of the leaves from the plant. The symptomatic leaves were harvested and converted into 5mm square fragments through precise cutting. To sterilize the tissues, 1% NaOCl was used for 90 seconds, followed by 75% ethanol for 15 seconds, and after that, three rinses with sterile water were carried out. On Potato Dextrose Agar (PDA) medium, at a temperature of 25 degrees Celsius, the treated leaves were grown. Mycelia that had encircled leaf pieces produced fungal plugs collected along the colony's outer edge, which were then transferred to fresh PDA plates utilizing a cork borer. Eight fungal strains were procured after three rounds of subculturing, displaying identical morphology. Within 24 hours, a white colony, demonstrating a substantial and rapid growth rate, colonized a culture dish having a 9-cm diameter. A gray-black discoloration became prominent in the colony during its later phases. Two days elapsed before minute black sporangia spots made their appearance on the hyphae. The sporangia, in their early stages, bore a yellow appearance which was replaced by a black one in their mature form. The size of oval spores, averaging 296 micrometers in diameter (224-369 micrometers), was determined from a sample of 50 spores. A BioTeke kit (Cat#DP2031) was utilized to extract the fungal genome from scraped fungal hyphae, thereby identifying the pathogen. The fungal genome's internal transcribed spacer (ITS) region was amplified using ITS1/ITS4 primers, and the ITS sequence data was submitted to GenBank under accession number OP984201. With the aid of MEGA11 software, the phylogenetic tree was constructed by employing the neighbor-joining method. ITS-based phylogenetic analyses clustered the fungus with Rhizopus arrhizus (MT590591), characterized by high bootstrap support. Finally, the pathogen was correctly identified as *R. arrhizus*. To verify Koch's postulates, 12 healthy Lonicera plants were treated with a 60-milliliter spray of a spore suspension (1104 conidia/ml). A separate group of 12 plants received only sterile water as a control. Plants, all located in the greenhouse, experienced a constant temperature of 25 degrees Celsius and 60% relative humidity. After 14 days of infection, the infected plants exhibited symptoms that were strikingly similar to those in the original diseased plants. The diseased leaves of artificially inoculated plants yielded the strain, which was subsequently re-isolated and confirmed as the original strain via sequencing analysis. R. arrhizus was, from the analysis of the results, ascertained to be the pathogen that causes the rotting of Lonicera leaves. Research conducted previously has highlighted R. arrhizus as the source of garlic bulb rot (Zhang et al., 2022), and its role in the decay of Jerusalem artichoke tubers (Yang et al., 2020). To the best of our information, this is the first instance of R. arrhizus being implicated in the Lonicera leaf rot condition in China. Determining the identity of this fungus is crucial for effective leaf rot control strategies.
Evergreen, the Pinus yunnanensis tree, is a distinguished member of the Pinaceae family. From eastern Tibet to southwestern Sichuan, southwestern Yunnan, southwestern Guizhou, and northwestern Guangxi, the species can be found. This indigenous and pioneering tree species is crucial for establishing forests on barren mountains in southwest China. Anteromedial bundle Liu et al. (2022) underscored the substantial value of P. yunnanensis to the building and medical industries. Within the borders of Panzhihua City, Sichuan Province, China, in May 2022, P. yunnanensis plants displayed symptoms indicative of witches'-broom disease. Needle wither, coupled with plexus buds and yellow or red needles, was characteristic of the symptomatic plants. Twigs formed from the lateral buds of the afflicted pines. A collection of lateral buds developed, and a few needles were observed to have sprouted (Figure 1). The P. yunnanensis witches'-broom disease (PYWB) was located in selected areas within Miyi, Renhe, and Dongqu, respectively. In the three surveyed regions, the symptoms were seen in over 9% of the pine trees, with the disease demonstrating a rapid expansion. A total of 39 plant samples, sourced from three locations, included 25 specimens exhibiting symptoms and 14 that did not. Scanning electron microscopy (Hitachi S-3000N) was used to examine the lateral stem tissues of 18 samples. Spherical bodies were found within the phloem sieve cells of symptomatic pines, which are illustrated in Figure 1. The CTAB method (Porebski et al., 1997) was used for the extraction of total DNA from 18 plant samples, which were then analyzed through nested PCR. DNA from unaffected Dodonaea viscosa plants and double-distilled water were employed as negative controls; the DNA extracted from Dodonaea viscosa plants exhibiting witches'-broom disease acted as the positive control. Using nested PCR, the pathogen's 16S rRNA gene was amplified, generating a 12 kb segment. This amplified sequence has been submitted to GenBank (accessions OP646619; OP646620; OP646621). (Lee et al. 1993, Schneider et al., 1993). A PCR reaction targeting the ribosomal protein (rp) gene yielded a DNA fragment roughly 12 kb in size, as described by Lee et al. (2003), and stored in GenBank under accession numbers OP649589, OP649590, and OP649591. The positive control's fragment size was replicated in 15 samples, underscoring the correlation between phytoplasma and the disease. Comparative analysis of 16S rRNA sequences, using BLAST, showed the P. yunnanensis witches'-broom phytoplasma to have an identity of between 99.12% and 99.76% with the phytoplasma from Trema laevigata witches'-broom, corresponding to GenBank accession MG755412. A substantial degree of identity, falling between 9984% and 9992%, was observed in the rp sequence compared to that of the Cinnamomum camphora witches'-broom phytoplasma (GenBank accession OP649594). The analysis process integrated iPhyClassifier (Zhao et al.) for the investigation. According to a 2013 study, the virtual RFLP pattern originating from the 16S rDNA fragment (OP646621) of the PYWB phytoplasma exhibited a similarity coefficient of 100% when compared to the reference pattern of 16Sr group I, subgroup B, exemplified by OY-M (GenBank accession AP006628). It has been identified that the phytoplasma displays a relationship to 'Candidatus Phytoplasma asteris' and belongs to the 16SrI-B sub-group.