接第二篇：
Diversity analysis
All samples shall be homogenized according to the minimum sequence number. OTU with 97% similarity shall be selected, and Mothur method shall be used (http://www.mothur.org/wiki/). Calculate the Ace index, Shannon index, Simpson index, Chao index, and Coverage index of each group. In this study, the sclerotium and mycoderm samples in the stage of sclerotia, sterile cultivation, and mulching cultivation in the stage of mycoderm formation and maturity were divided into nine groups of samples, with three replicates in each group. The average diversity index of each group was calculated by SPSS software, and the significant differences between the groups were compared by one-way analysis of variance (ANOVA). Using the unweighted group average method to cluster and calculate the beta diversity index of different samples, principal coordinate analysis (PCoA) was conducted to clarify the distance matrix between different groups.
Analysis of bacterial community structure
Use the R language tool to make a percentage stacking column chart for the bacterial groups whose relative abundance is greater than 1.0% in each group of samples. If the relative abundance is less than 1.0%, it should be attributed to others (the relative abundance is the average of three repeats of each group of samples). The common OTU and unique OTU of C. chanhua samples in different stages under the same environment and in different cultivation environments at the same stage were analyzed by Venn diagram, and the similarity and overlap of species composition of each sample were analyzed. The petal map was drawn for all C. chanhua samples, and the common and endemic genera in each sample were analyzed; based on the bacterial community abundance data in the samples, the significant differences between groups of the top 15 genera of bacterial community abundance in different samples were analyzed using one-way ANOVA, and significant species of differences between sample groups were obtained.
Bacterial function prediction
Via PICRUSt (http://picrust.github.io/picrust/), the 16S series of bacteria were annotated with Clusters of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functions to obtain the annotation information of OTU at each functional level of COG and KEGG and its abundance in different samples.
Data analysis
Other test data were collated by Excel and statistically analyzed by SPSS software.
ACKNOWLEDGMENTS
This work was supported by grants from the National Natural Science Foundation of China (no. 31860037), Department of Science and Technology of Guizhou Province (no. [2020]1Z009), and Guizhou Qianxinan Tobacco Company (no. 2022-04).
AUTHOR AFFILIATIONS
1Institute of Fungal Resources, College of Life Sciences, Guizhou University, Guiyang, Guizhou, China
2Department of Humanities, Business College of Guizhou University of Finance and Economics, Qiannan, Guizhou, China
3Tea College, Guizhou University, Guiyang, Guizhou, China
AUTHOR ORCIDs
Gongping Hu http://orcid.org/0009-0000-9356-2683
Xiao Zou http://orcid.org/0000-0002-3666-5536
AUTHOR CONTRIBUTIONS
Gongping Hu, Writing – original draft.
DATA AVAILABILITY
The raw sequence data have been deposited in the SRA database under accession number PRJNA944443.
ADDITIONAL FILES
The following material is available online.
Supplemental Material
Supplemental material (Spectrum01179-23-s0001.docx). Fig. S1 to Fig. S9; Tables S1 to S3.
Open Peer Review
PEER REVIEW HISTORY (review-history.pdf). An accounting of the reviewer comments and feedback.
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