Maximum parsimony (MP) was computed in PAUP* 4.0b10 with the default settings, using the heuristic search option with 1000 random sequence addition replicates and tree bisection-reconnection (TBR) as the branch swapping algorithm. Bayesian analysis (BI) was performed on the website of CIPRES Science Gateway v.3.3 platform (, last accessed on 13 October 2022 ) using MrBayes on XSEDE (3.2.7a) as a tool. The final ML search was conducted using the GTRGAMMA+I model. All free model parameters were estimated by RAxML with ML estimates of 25 per site rate categories. 3.3 (, last accessed on 13 October 2022 ) using RAxML v.8.2.8 as part of the “RAxML-HPC BlackBox” tool. Maximum likelihood (ML) analysis was performed at the CIPRES Science Gateway v. Finally, we chose “PhyML” in the third step, which is to select output format and convert. We chose “MAFFT” and “FASTA” in select format and then uploaded our alignment. The phylogeny website tool “ALTER” was used to transfer the alignment file from “.nex” to “.phy” file for RAxML analysis. Multiple sequence alignments were constructed and carried out using the MAFFT v.7.110 online program ( ) last accessed on 13 October 2022. All strains used in this study are listed in Table 1. The qualified sequences were submitted to GenBank and their accession numbers are shown in Table 1. Purification and sequencing of the PCR amplicons were undertaken by Sangon Biotech (Chengdu, China). The PCR thermal cycle program for ITS, tef1-ɑ, and act amplification was: initial denaturation at 95 ☌ for 5 min, followed by 40 cycles of denaturation at 95 ☌ for 30 s, annealing at 54 ☌ for 30 s, elongation at 72 ☌ for 1 min and the final extension at 72 ☌ for 10 min. The act region was amplified with primers ACT-512F and ACT-783R. Primers ITS4 and ITS5 were used to amplify the ITS, and EF-728F and EF-986R were used for tef1-ɑ. The tef1-ɑ, and act loci were also employed to support the species identification. For Cladosporium isolates, the ITS region was first sequenced, and a BLAST search of GenBank was used to reveal the closest matching taxa. PCR amplification was performed in a 25 μL reaction volume system. Petri dish, a sterile scalpel was used to transfer mycelium to a 1.5 mL centrifuge tube and the genomic DNA was extracted with PrepMan Ultra Reagent (Applied Biosystems, CA, USA) following the manufacturer’s protocol. Once the fungal colonies had grown to the edge of 90 mm diam. Dried holotype specimens were conserved in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP), while cultures were conserved in the Departmental Culture Collection (GUCC). All new taxa were registered in the Index Fungorum database ( (accessed on 13 October 2022). Morphological characteristics of the fungi were observed and photographed using a compound light microscope (Zeiss Scope 5) with an attached camera (AxioCam 208 color). After 12 h, the germination of conidium was observed, and they were then transferred to potato dextrose agar (PDA) and incubated at room temperature (28 ☌) for 10 days. Single conidia were picked off the leaves with a sterilized needle and placed on a drip board containing sterilized water. Abundant conidia were observed on the surface of the leaf spots examined using a dissecting microscope. To obtain pure cultures, leaf surfaces were disinfected according to Zhang et al. From 2021 to 2022, 94 samples were collected from orchards in six locations of Guizhou Province (Congjiang, Kaiyang, Longli, Luodian, Nayong and Wengan counties), including 10 host species (cherry, loquat, passionfruit, pitaya, plum, pomegranate, Rosa roxburghii, shaddock, tangerines and walnut).
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |