Diversity and Distribution of Calonectria Species in Soils from Eucalyptus urophylla × E. grandis, Pinus massoniana, and Cunninghamia lanceolata Plantations in Four Provinces in Southern China

The species of Calonectria include many notorious plant pathogens and are widely distributed around the world. Leaf blight caused by Calonectria species is considered one of the most prominent diseases in Eucalyptus plantations in China. Some Calonectria species isolated from soils in Eucalyptus plantations are highly pathogenic to inoculated Eucalyptus genotypes. In southern China, the plantation trees Cunninghamia lanceolata, Eucalyptus spp., and Pinus massoniana are always adjacently planted, especially in FuJian, GuangDong, GuangXi, and YunNan Provinces. The aim of this study was to understand the diversity and distribution of Calonectria in soils from plantations of different tree species in different geographic regions. Soil samples were collected from 12 sampling sites in Eucalyptus urophylla × E. grandis, P. massoniana, and C. lanceolata plantations in FuJian, GuangDong, GuangXi, and YunNan Provinces. Approximately 250 soil samples were collected from each sampling site, and a total of 2991 soil samples were obtained. A total of 1270 Calonectria isolates were obtained from 1270 soil samples. The 1270 isolates were identified based on DNA sequence comparisons of the partial gene regions of act, cmdA, his3, rpb2, tef1, and tub2. These isolates were identified as 11 Calonectria species: Calonectria aconidialis (69.50%), C. kyotensis (13.10%), C. hongkongensis (10.80%), C. ilicicola (2.50%), C. asiatica (2.36%), C. curvispora (0.31%), C. chinensis (0.24%), C. pacifica (0.24%), C. yunnanensis (0.16%), and C. canadiana (0.08%) in the C. kyotensis species complex and C. eucalypti (0.71%) in the C. colhounii species complex. The three dominant species, C. aconidialis, C. kyotensis, and C. hongkongensis, were widely distributed. The richness of Calonectria (percentage of soil samples that yielded Calonectria) in soils in the eastern regions (relatively humid regions) was higher than that in the western regions. The Calonectria richness of E. urophylla × E. grandis, P. massoniana, and C. lanceolata plantations decreased gradually. For each of the three dominant species, its richness in the eastern regions was generally higher than that in the western regions; the species richness was highest in E. urophylla × E. grandis plantations for C. aconidialis, while for each of C. kyotensis and C. hongkongensis, its species richness was highest in P. massoniana plantations. The genetic variation in C. aconidialis, C. kyotensis, and C. hongkongensis was more greatly affected by geographic region than by plantation tree species. This study expanded our understanding of the richness, species diversity, and distribution characteristics of Calonectria in soils from the plantations of different tree species in different geographic regions in southern China. Results in this study enhanced our understanding of the influencing characteristics of geographic region and tree species on the species and genetic diversity of soilborne fungi.


DNA Extraction, PCR Amplifications, and Sequencing
All Calonectria morphological-like isolates obtained in this study were used for total genomic DNA extraction and sequence comparisons. Mycelia were scraped from 7-day-old cultures using a sterilized scalpel and transferred into 2 mL Eppendorf tubes. Total genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) protocol described by Van Burik and co-authors [36]. The extracted DNA was dissolved by adding 30 µL TE buffer (1 M Tris-HCl and 0.5 M EDTA, pH 8.0), and 2.5 µL RNase (10 mg/mL) was added to degrade the RNA. The mixture was incubated at 37 • C for 1 h. The DNA concentration was quantified using a NanoDrop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). All DNA samples were diluted to approximately 100 ng/uL with DNase/RNasefree ddH 2 O (Sangon Biotech Co., Ltd., Shanghai, China) and stored at -20 • C for further use.
To ensure the accuracy and integrity of all sequences, all PCR products were sequenced in both the forward and reverse directions using the same primers used for PCR amplification. Sequence reactions were performed by the Beijing Genomics Institute, Guangzhou, China. All obtained sequences were edited and assembled using MEGA v. 7.0 software [37] and deposited in GenBank (https://www.ncbi.nlm.nih.gov; accessed date: 24 January 2023).
For all the Calonectria morphological-like isolates, the tef1 gene regions were sequenced, and a standard nucleotide BLAST search was conducted using the tef1 sequences to preliminarily identify these fungi. For all isolates preliminarily identified as Calonectria, the tub2 gene regions were then sequenced. All obtained Calonectria isolates were genotyped by the tef1 and tub2 sequences. Based on the genotypes generated by tef1 and tub2 sequences, isolates for each tef1-tub2 genotype obtained from different regions and plantation tree species were selected for sequencing the act, cmdA, his3, and rpb2 gene regions.

Multi-Gene Phylogenetic Analyses and Species Identification
All sequences of the six gene regions (act, cmdA, his3, rpb2, tef1, and tub2) generated in this study were compared with the sequences of type specimen strains of published Calonectria species. Sequences of all published species in the relevant species complexes were used for sequence comparisons and phylogenetic analyses. The datasets of Liu and co-authors [30] were used as templates, and the sequences of other recently described Calonectria species in the relevant species complexes were all used for sequence comparisons.
Sequences of each of the act, cmdA, his3, rpb2, tef1, and tub2 gene regions, as well as the combination of these six gene regions, were aligned using the online version of MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server; accessed date: 10 August 2022) with the alignment strategy FFT-NS-i (Slow; interactive refinement method). The alignments were manually edited using MEGA v. 7.0 software [37] when necessary. All alignments used for phylogenetic analyses were submitted to TreeBASE (http://treebase.org; accessed date: 15 August 2022).
The Maximum likelihood (ML) and Bayesian inference (BI) approaches were used for phylogenetic analyses of the sequence datasets of each of the six genes and the combined dataset of all six gene regions. ML analyses were conducted using RaxML v. 8.2.4 [38] on the CIPRES Science Gateway v. 3.3. BI analyses were conducted using MrBayes v. 3.2.6 [39] on the CIPRES Science Gateway v. 3.3. ML analyses were performed with a default GTR substitution matrix and 1000 bootstrap replicates. For BI analyses, four Markov chain Monte Carlo (MCMC) chains were run from a random starting tree for five million generations, and trees were sampled every 100th generation. The first 25% of the trees sampled were discarded as burn-in, and the remaining trees were used to determine the posterior probabilities. Two isolates of Curvicladiella cignea (CBS 109167 and CBS 109168) were used as outgroup taxa [30]. Phylogenetic trees generated by ML and BI analyses were viewed using MEGA v. 7.0. [37]

Calonectria Richness in Soils from Four Provinces and Plantations of Three Tree Species
The Calonectria isolates obtained in this study were identified. The numbers of Calonectria isolates obtained at each of the 12 sampling sites were counted. Furthermore, the percentage of soil samples that yielded Calonectria (Calonectria richness) at each sampling site was computed. The distribution characteristics of Calonectria in four regions (provinces) and plantations of three tree species were recorded, including the influencing characteristics of Calonectria richness by geographic region (provinces) and plantation tree species.

Calonectria Species Diversity in Four Provinces and Plantations of Three Tree Species
According to the species identification results of all isolates, the number of isolates of each Calonectria species obtained at each of the 12 sampling sites was counted. The percentage of soil samples that yielded each Calonectria species at each sampling site was also computed. The distribution characteristics of each Calonectria species in four provinces and plantations of three tree species were recorded, including the influencing characteristics of Calonectria species number and each species richness by geographic region (provinces) and plantation tree species.

Genotyping of Isolates within each Calonectria Species
The genotypes of the isolates within each identified Calonectria species were determined based on the tef1 and tub2 sequences. The number of genotypes of each species and the number of isolates belonging to each genotype were recorded. Furthermore, the number of genotypes of each Calonectria species in each of the 12 plantations (12 sampling sites) of three tree species in four provinces was counted.

Genotype Diversity of Calonectria Species in Four Provinces and Plantations of Three Tree Species
For each dominant Calonectria species, to preliminarily understand whether its genetic variation (based on shared genotype) was affected by geographical regions and plantation tree species, the numbers of shared genotypes among isolates at 12 sampling sites were counted. We further compared the number of shared genotypes for each dominant species to evaluate the influencing characteristics of geographical regions (provinces) and plantation tree species on the genetic variations of each dominant species.

Soil Sample Collection and Calonectria Isolation
A total of 2991 soil samples were collected, with 244-250 soil samples from each of the 12 sampling sites (Table 1). After the soil samples were incubated with alfalfa seeds, a singleconidium culture was obtained from each soil sample with white masses of conidiophores with typical morphological characteristics of Calonectria species. In total, 1308 Calonectria morphological-like isolates were obtained.

Sequencing
For all 1308 Calonectria morphological-like isolates obtained from soil samples, the tef1 gene sequences were amplified and used to conduct a standard nucleotide BLAST search to preliminarily identify the species. Ultimately, 1270 isolates were identified as Calonectria species (Appendix A Table A1). The majority of the remaining 38 isolates were grouped into the genus Cylindrocladiella. The tub2 gene region was also amplified and sequenced for the 1270 Calonectria isolates (Appendix A Table A1). Ninety-seven tef1-tub2 genotypes were generated based on the tef1 and tub2 gene sequences (Table 2). Subsequently, 207 isolates were selected to amplify the act, cmdA, his3, and rpb2 gene regions. These 207 isolates presented all three tree species in all four sampling regions (provinces), and presented all 97 genotypes based on tef1 and tub2 gene sequences (Table 3). One to ten isolates of each genotype revealed by the tef1 and tub2 sequences were selected (Tables 2 and 3). Amplicons generated for the act, cmdA, his3, rpb2, tef1, and tub2 gene regions were approximately 235, 680, 430, 1030, 500, and 620 bp, respectively.       a Genotype within each Calonectria species, determined by sequences of the tef1, tub2, cmdA, his3, rpb2 and act regions; "-" means not available. b Code of 12 sampling sites connecting to "Site and Tree species code" in Table 1. c CSF: Culture Collection located at Research Institute of Fasting-growing Trees (RIFT), Chinese Academy of Forestry, ZhanJiang, GuangDong Province, China. d Information associated with sample point and isolate, for example, "20210525-1-(4)" indicates sample number "20210525-1-(4)" and isolate from this sample. e tef1 = translation elongation factor 1-alpha; tub2 = β-tubulin; cmdA = calmodulin; his3 = histone H3; rpb2 = the DNA-directed RNA polymerase II second largest subunit; act = actin. f "-" represents the relative locus that was not successfully amplified in this study.

Multi-Gene Phylogenetic Analyses and Species Identification
The standard nucleotide BLAST search results conducted using the act, cmdA, his3, rpb2, tef1, and tub2 gene sequences showed that the isolates obtained in the current study belonged to two species complexes of Calonectria, the C. kyotensis species complex and the C. colhounii species complex. The 207 Calonectria isolates with six sequenced gene regions were used for phylogenetic analyses (Table 3). Based on the published results in Liu and co-authors [30] and several recent publications [29,32,33,[40][41][42], sequences of act, cmdA, his3, rpb2, tef1, and tub2 of 44 published species in the C. kyotensis species complex and C. colhounii species complex were downloaded from GenBank and used for sequence comparisons and phylogenetic analyses (Table 4).    Phylogenetic analyses based on the six individual gene regions and the combination dataset for those six gene regions were conducted using both ML and BI methods. The overall topologies generated from the BI analyses were essentially similar to those from the ML analyses for each dataset. Consequently, only the ML tree with bootstrap support values of ML and posterior probabilities of BI was presented. The ML tree generated based on a combination of six gene sequences is presented in Figure 3, and the ML trees generated based on each of the six gene sequences were presented in Appendix F Figures A1-A6. Phylogenetic analyses showed that the 207 Calonectria isolates were clustered in 11 groups (Groups A-K) based on combined tef1/tub2/cmdA/his3/rpb2/act gene sequence analyses ( Figure 3). The analyses showed that isolates in Groups A-J belong to the C. kyotensis species complex and that isolates in Group K belong to the C. colhounii species complex (    , and absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30]. Curvicladiella cignea (CBS 109167 and CBS 109168) was used as the outgroup taxon.

Isolates in the Calonectria kyotensis Species Complex
Isolates in Groups A and B were clustered with C. kyotensis and C. hongkongensis, respectively, based on the tef1, tub2, cmdA, his3, rpb2, act, and combined tef1/tub2/cmdA/his3/rpb2/act trees ( Figure 3, Appendix F Figures A1-A6). Therefore, isolates in Groups A and B were identified as C. kyotensis and C. hongkongensis, respectively. C. aciculata, and C. minesis in the act tree (Appendix F Figure A6). The isolates were clustered with or close to C. eucalypti, C. shaoguanensis, and C. honghensis in the combined tef1/tub2/cmdA/his3/rpb2/act tree ( Figure 3). The isolates in Group K were consistently clustered with or close to C. eucalypti in all analyses ( Figure 3, Appendix F Figures A1-A6). Isolates in Group F were identified as C. eucalypti.

Taxonomy
Based on the results of multi-gene phylogenetic analyses and consideration of the morphological characteristics, C. shaoguanensis recently described in Zhang and co-authors [33] is reduced to synonymy with existing taxon as follows: Calonectria eucalypti L. Lombard Notes: Calonectria shaoguanensis was identified as a new species based on DNA sequence comparisons of the tef1, tub2, and cmdA gene regions and the morphological characteristics in Zhang and co-authors [33]. Calonectria shaoguanensis was treated as a synonym with C. eucalypti in this study. In comparison of DNA sequences for the tef1, tub2, and cmdA gene regions, there was only one base difference between the extype isolate of C. shaoguanensis (ZHKUCC 21-0036) and the ex-type isolate of C. eucalypti (CMW 18444 = CBS 125275) in the tub2 sequences. Both of the species produce clavate vesicles with overlapping dimensions (C. shaoguanensis: 2-7 µm [33]; C. eucalypti: 4-6 µm [44]). The macroconidia of C. shaoguanensis (av. 65 × 6.5 µm) are shorter than those of C. eucalypti (av. 72 × 6 µm) [33,44], which were considered to represent intraspecific variation justifying this synonymy.

Calonectria Richness in Soils from Four Provinces and Plantations of Three Tree Species
A total of 1270 isolates of Calonectria were obtained from 2991 soil samples collected from 12 sampling sites of three plantations in four provinces (Table 5, Figure 4). Calonectria isolates were obtained from 42.5% of the soil samples (Table 5, Figure 4). When considering the 12 sampling sites, 0.4 to 87.2% of the soil samples yielded Calonectria ( Figure 4); the highest percentage of soil samples that yielded Calonectria was P. massoniana in GuangDong (87.2%), followed by E. urophylla × E. grandis from FuJian (86.8%) and E. urophylla × E. grandis from GuangDong (85.2%); the lowest percentages of soil samples that yielded Calonectria were from P. massoniana (0.4%) and C. lanceolata (0.4%) in YunNan (Table 5, Figure 4).

Number of Soil Sample
Yielded Calonectria

Number of Soil Sample
Yielded Calonectria

Calonectria Species Diversity in Four Provinces and Plantations of Three Tree Species
Based on the sequence comparisons of act, cmdA, his3, rpb2, tef1, and tub2 sequences, the 1270 Calonectria isolates were identified as 11 species. These species were C. aconidialis (883 isolates; 69.50%), C. kyotensis (166 isolates; 13.10%), C. hongkongensis (137 isolates; 10.80%), C. ilicicola (32 isolates; 2.50%), C. asiatica (30 isolates; 2.36%), C. eucalypti (9 isolates; 0.71%), C. curvispora (4 isolates; 0.31%), C. chinensis (3 isolates; 0.24%), C. pacifica (3 isolates; 0.24%), C. yunnanensis (2 isolates; 0.16%), and C. canadiana (1 isolate; 0.08%) (Table 6, Figure 5). Calonectria aconidialis was most dominant, followed by C. kyotensis and C. hongkongensis. Three species accounted for 93.4% of all Calonectria isolates obtained in this study ( Figure 5). These three species were regarded as the dominant species (Table 6, Figure 5). A relatively small number of isolates were obtained for C. ilicicola and C. asiatica. Less than 10 isolates were obtained for each of the remaining six species (Table 6, Figure 5).   When considering the 12 sampling sites, each of C. aconidialis, C. kyotensis, C hongkongensis and C. ilicicola was isolated from more than half of all the 12 sampling sites Calonectria aconidialis and C. ilicicola were distributed at all sampling sites in four prov inces, with the exception of P. massoniana and C. lanceolata plantations in YunNan Calonectria kyotensis was distributed at all sampling sites in FuJian, GuangDong, and GuangXi Provinces, with the exception of C. lanceolata plantations in FuJian and GuangXi Calonectria hongkongensis was distributed at all sampling sites in FuJian, GuangDong, and GuangXi Provinces, with the exception of C. lanceolata plantations in GuangDong and GuangXi. The remaining seven species were isolated only from the soils of one or two tree species plantations in a single province ( Table 6, Figure 2b-m).
When considering the four sampled geographic regions, five, six, four, and six Calonectria species were isolated from soil samples in FuJian, GuangDong, GuangXi, and YunNan, respectively (Table 6). Calonectria aconidialis and C. ilicicola were found in all fou provinces. Calonectria kyotensis and C. hongkongensis were found in three provinces, ex When considering the 12 sampling sites, each of C. aconidialis, C. kyotensis, C. hongkongensis and C. ilicicola was isolated from more than half of all the 12 sampling sites. Calonectria aconidialis and C. ilicicola were distributed at all sampling sites in four provinces, with the exception of P. massoniana and C. lanceolata plantations in YunNan. Calonectria kyotensis was distributed at all sampling sites in FuJian, GuangDong, and GuangXi Provinces, with the exception of C. lanceolata plantations in FuJian and GuangXi. Calonectria hongkongensis was distributed at all sampling sites in FuJian, GuangDong, and GuangXi Provinces, with the exception of C. lanceolata plantations in GuangDong and GuangXi. The remaining seven species were isolated only from the soils of one or two tree species plantations in a single province ( Table 6, Figure 2b-m).
When considering the four sampled geographic regions, five, six, four, and six Calonectria species were isolated from soil samples in FuJian, GuangDong, GuangXi, and Yun-Nan, respectively (Table 6). Calonectria aconidialis and C. ilicicola were found in all four provinces. Calonectria kyotensis and C. hongkongensis were found in three provinces, excluding YunNan. Each of the remaining seven species was found in only one province ( Table 6, Figures 2 and 6). For C. aconidialis, the percentage of soil samples that yielded Calonectria decreased from the eastern to the western provinces, with the exception of GuangDong Province (Table 6, Figure 6). For each species of C. kyotensis and C. hongkongensis, the percentage of soil samples that yielded Calonectria decreased from regions in the eastern to the western provinces (Table 6, Figure 6). The percentages of soil samples containing C. ilicicola in regions in the eastern and western provinces were similar (Table 6, Figure 6).

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Calonectria decreased from the eastern to the western provinces, with the exception of GuangDong Province (Table 6, Figure 6). For each species of C. kyotensis and C. hongkongensis, the percentage of soil samples that yielded Calonectria decreased from regions in the eastern to the western provinces (Table 6, Figure 6). The percentages of soil samples containing C. ilicicola in regions in the eastern and western provinces were similar (Table 6, Figure 6). When considering the plantation tree species, eight, seven, and seven species were identified in E. urophylla × E. grandis, P. massoniana, and C. lanceolata plantations, respectively (Table 6). Calonectria aconidialis, C. kyotensis, C. hongkongensis, and C. ilicicola were isolated from soils in all three tree species. Each of the remaining seven species was isolated only from soils with one or two tree species (Table 6, Figures 2 and 7). For C. aconidialis, the highest percentage of soil samples that yielded Calonectria was in E. urophylla × E. grandis plantations, followed by P. massoniana plantations and C. lanceolata plantations (Table 6, Figure 7). For each species of C. kyotensis and C. hongkongensis, the percentage of soil samples that yielded Calonectria was highest in P. massoniana plantations, followed by E. urophylla × E. grandis plantations and C. lanceolata plantations (Table 6, Figure 7). For C. ilicicola, the percentage of soil samples that yielded Calonectria was similar among the plantations of three tree species (Table 6, Figure 7). When considering the plantation tree species, eight, seven, and seven species were identified in E. urophylla × E. grandis, P. massoniana, and C. lanceolata plantations, respectively (Table 6). Calonectria aconidialis, C. kyotensis, C. hongkongensis, and C. ilicicola were isolated from soils in all three tree species. Each of the remaining seven species was isolated only from soils with one or two tree species (Table 6, Figures 2 and 7). For C. aconidialis, the highest percentage of soil samples that yielded Calonectria was in E. urophylla × E. grandis plantations, followed by P. massoniana plantations and C. lanceolata plantations (Table 6, Figure 7). For each species of C. kyotensis and C. hongkongensis, the percentage of soil samples that yielded Calonectria was highest in P. massoniana plantations, followed by E. urophylla × E. grandis plantations and C. lanceolata plantations (Table 6, Figure 7). For C. ilicicola, the percentage of soil samples that yielded Calonectria was similar among the plantations of three tree species (Table 6, Figure 7).
The tef1-tub2 genotypes of each Calonectria species in each of the 12 sampling sites are listed in Appendix B Table A2 and Table 7. For the three dominant species, C. aconidialis, C. kyotensis, and C. hongkongensis, the overall data showed that the number of genotypes of each Calonectria species at each sampling site positively correlated with the number of isolates (Tables 6 and 7, Appendix B Table A2). For each species of C. aconidialis and C. hongkongensis, the dominant genotype (genotype AA) existed in most of the sampling sites (Appendix B Table A2). For example, the dominant genotype AA accounted for 61.7 to 100% of the C. aconidialis isolates obtained from sampling sites 1-9 (Appendix B Table A2). There was no dominant genotype for C. kyotensis from the seven sampling sites that had Calonectria (Appendix B Table A2).
The tef1-tub2 genotypes of each Calonectria species in each of the 12 sampling sites are listed in Appendix B Tables 7 and A2. For the three dominant species, C. aconidialis, C. kyotensis, and C. hongkongensis, the overall data showed that the number of genotypes of each Calonectria species at each sampling site positively correlated with the number of isolates ( Table 6, Table 7 and Appendix B Table A2). For each species of C. aconidialis and C. hongkongensis, the dominant genotype (genotype AA) existed in most of the sampling sites (Appendix B Table A2). For example, the dominant genotype AA accounted for 61.7 to 100% of the C. aconidialis isolates obtained from sampling sites 1-9 (Appendix B Table A2). There was no dominant genotype for C. kyotensis from the seven sampling sites that had Calonectria (Appendix B Table A2).

Genotype Diversity of Calonectria Species in Four Provinces and Plantations of Three Tree Species
Calonectria aconidialis, C. kyotensis, and C. hongkongensis were the dominant species in this study. The statistical results of the number of shared genotypes of C. aconidialis isolates indicated that the ratio of shared genotypes among the sites of "the same region but different plantation tree species" (30 shared genotypes/12 pairs of comparison sampling sites = 2.5) was much bigger than that of the sites of "different geographical region but the same plantation tree species" (12 shared genotypes/18 pairs of comparison sampling sites = 0.67) and also bigger than that of the sites of "different geographical region and different plantation tree species" (24 shared genotypes/36 pairs of comparison sampling sites = 0.67) (Appendix C Table A3). The statistical results of the number of shared genotypes of C. kyotensis isolates indicated that the ratio of shared genotypes among the sites of "the same region but different plantation tree species" (19 shared genotypes/12 pairs of comparison sampling sites = 1.58) was much bigger than that of the sites of "different geographical region but the same plantation tree species" (12 shared genotypes/18 pairs of comparison sampling sites = 0.67) and also bigger than that of the sites of "different geographical region and different plantation tree species" (15 shared genotypes/36 pairs of comparison sampling sites = 0.42) (Appendix D Table A4). The statistical results of the number of shared genotypes of C. hongkongensis isolates indicated that the ratio of shared genotypes among the sites of "the same region but different plantation tree species" (13 shared genotypes/12 pairs of comparison sampling sites = 1.08) was much bigger than that of the sites of "different geographical region but the same plantation tree species" (11 shared genotypes/18 pairs of comparison sampling sites = 0.61) and also bigger than that of the sites of "different geographical region and different plantation tree species" (17 shared genotypes/36 pairs of comparison sampling sites = 0.47) (Appendix E Table A5). These results suggest that the genetic variations of each species of C. aconidialis, C. kyotensis, and C. hongkongensis are likely to be more affected by geographical region than plantation tree species.

Discussion
In this study, a relatively large number of soil samples were collected from 12 plantations of E. urophylla × E. grandis, P. massoniana, and C. lanceolate in FuJian, GuangDong, GuangXi, and YunNan Provinces in southern China. A total of 1270 Calonectria isolates were obtained. Based on multi-gene sequence phylogenetic analyses, these isolates were identified as 11 Calonectria species. Except for C. eucalypti, which resides in the C. colhounii species complex, the remaining 10 species belong to the C. kyotensis species complex. The most dominant species was C. aconidialis, followed by C. kyotensis and C. hongkongensis.
The richness of Calonectria in soils (percentage of soil samples that yielded Calonectria) among the four geographical regions, as well as among the three tree species, differed. Calonectria richness in the eastern regions was higher than that in the western regions. A possible reason for this phenomenon is that the annual rainfall in the eastern regions was greater than in the western regions, where the soil in plantations in the eastern regions was under continuous high humidity [59,60]. Previous research results have shown that Calonectria species are more likely to exist in soils with consistently high levels of moisture [61]. The richness of Calonectria in soils of E. urophylla × E. grandis, P. massoniana, and C. lanceolata plantations decreased gradually. The richness of Calonectria in soils is probably affected by the litter of different tree species [62][63][64].
This study indicated that Calonectria species are widely distributed in soils of E. urophylla × E. grandis, P. massoniana, and C. lanceolata plantations. Previous research results have shown that Calonectria species, especially those in the C. kyotensis species complex, are widely distributed in the soils of Eucalyptus plantations in southern China [19,31]. Recent research results have indicated that Calonectria is also frequently isolated from soils in plantations of multiple tree species [32]. We suppose that Calonectria species are widely distributed in forest soils in southern China.
The distribution characteristics of the 11 Calonectria species at 12 sampling sites from 12 plantations of three tree species in four provinces differed. The three dominant species, C. aconidialis, C. kyotensis, and C. hongkongensis, as well as C. ilicicola, were distributed much more widely than the remaining seven species. This is consistent with recent research results [31,32]. Both C. aconidialis and C. ilicicola were isolated from 10 of the 12 sampling sites, while the richness of C. aconidialis at these sites was much higher than that of C. ilicicola. These results highlight the distribution differences in Calonectria species in soils. This study resulted in the first report of C. curvispora in China, and it was isolated only from soils in P. massoniana plantations in GuangDong Province. Our results suggest that C. curvispora may not be widely distributed in plantation soil in southern China.
The distribution of the three dominant species, C. aconidialis, C. kyotensis, and C. hongkongensis, was affected by geographic regions and plantation tree species, although their distribution patterns were not the same. The richness of these three species was generally higher in eastern regions than in western regions. However, the influencing characteristics of species richness, affected by plantation tree species, were not the same. Species richness was highest in E. urophylla × E. grandis plantations for C. aconidialis, while richness was highest in P. massoniana plantations for both C. kyotensis and C. hongkongensis. Species richness was lowest for these three species in the C. lanceolate plantations. Our research results suggest that the distribution patterns differ among Calonectria species associated with soils in angiosperm and gymnosperm plants [32].
Calonectria aconidialis is the most dominant species obtained from forest soils in this study. Since this species was first isolated and described from soils in Eucalyptus plantation in HaiNan Province in southern China [18], it has been frequently isolated from soils in Eucalyptus plantations in GuangXi, GuangDong and FuJian Provinces [15,19,31,32]. Besides Eucalyptus, C. aconidialis was also obtained from soils in C. lanceolata, Phyllostachys heterocycle and natural forests [32]. We speculate that C. aconidialis is widely distributed in soils in forests of multiple tree species in southern China and neighboring countries.
The distribution characteristics of Calonectria in YunNan differed from those in FuJian, GuangDong, and GuangXi. The percentages of soil samples that yielded Calonectria in plantations of E. urophylla × E. grandis, P. massoniana, and C. lanceolata in YunNan were significantly lower than those in the other three provinces. A possible reason is that the climate in YunNan is relatively drier than that of the other three provinces [65]. Among the 11 species identified in this study, C. asiatica, C. yunnanensis, C. eucalypti, and C. canadiana were isolated only from YunNan Province. Based on several previous studies conducted on Calonectria in China, C. asiatica and C. yunnanensis have been collected only from soils in Eucalyptus plantations in YunNan [10,15,19,[29][30][31][32]. Calonectria eucalypti has been isolated only from the leaves of Eucalyptus plantations in the FuJian and YunNan Provinces [15,16]. This study reported the first record of C. eucalypti isolated from soils. In China, C. canadiana has only been isolated from soil in northern regions, including HeNan, HeiLongJiang, and HeBei Provinces [28,66,67]. Calonectria canadiana is considered a temperate climatedistributed species. In this study, it was isolated from YunNan Province in southern China. For the region in YunNan Province where C. canadiana was obtained, the climate was similar to these regions in northern China, since the region in YunNan in this study is located in the Yunnan-Guizhou Plateau, and the average annual temperature in this region is relatively low. A possible reason for the differences in Calonectria richness and species diversity between YunNan and the other provinces is the special climate in YunNan compared with the other three provinces [68].
The distribution patterns of Calonectria fungi in forest soils in different continents and countries are not consistent. In South America, the majority of Calonectria fungi isolated from forest soils resided in the C. brassicae and C. candelabrum species complexes [30,41]. In Asia, Calonectria species in the C. kyotensis, C. reteaudii, C. colhounii, C. cylindrospora and C. brassicae species complexes were isolated from forest soils in China and southeastern Asian countries; most of these obtained species resided in the C. kyotensis species complex [9,15,19,31,32,42]. In this study, ten of eleven obtained Calonectria species resided in C. kyotensis species complex.
All the three, eight of eleven, and six of eight Calonectria species isolated from soils resided in C. kyotensis species complex in Indonesia, Vietnam and Malaysia, respectively [9,42]. The dominant species in the C. kyotensis species complex among different Asian countries were not the same. For example, C. aconidialis is the most dominant species isolated from forest soils in China, while this species has never been isolated from other Asian countries [9,15,19,[30][31][32]42].
This study explored the richness, species diversity, and distribution characteristics of Calonectria from soils in the plantations of three tree species in four provinces in southern China. Our research results indicate that Calonectria richness is affected by geographic regions and plantation tree species. For the dominant species, their distribution patterns affected by geographic regions and plantation tree species are not the same, and their genetic variations may be more greatly affected by geographic region than by plantation tree species. For the dominant species, additional studies need to be conducted to clarify the genetic diversity and population differences among isolates from soils in different geographic regions and plantations of different tree species, which will help us to understand the influencing characteristics of geographic regions and plantation tree species on their genetic variations.
C. kyotensis C. aconidialis AA--  C. kyotensis  C. kyotensis C. aconidialis C  C. kyotensis C. aconidialis C   C. kyotensis C. aconidialis C                   Figure A1. Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analysis of the DNA dataset of the tef1 gene sequences. Bootstrap support values ≥ 70% from ML analysis and posterior probabilities values ≥ 0.95 obtained from Bayesian inference (BI) are indicated at the nodes as ML/BI. Bootstrap values < 70% or posterior probabilities values < 0.95 are marked with "*", and absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates Figure A1. Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analysis of the DNA dataset of the tef1 gene sequences. Bootstrap support values ≥ 70% from ML analysis and posterior probabilities values ≥ 0.95 obtained from Bayesian inference (BI) are indicated at the nodes as ML/BI. Bootstrap values < 70% or posterior probabilities values < 0.95 are marked with "*", and absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30]. Curvicladiella cignea (CBS 109167 and CBS 109168) was used as the outgroup taxon. obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30].    Bootstrap support values ≥ 70% from ML analysis and posterior probabilities values ≥ 0.95 obtained from Bayesian inference (BI) are indicated at the nodes as ML/BI. Bootstrap values < 70% or posterior probabilities values < 0.95 are marked with "*", and absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30]. Curvicladiella cignea (CBS 109167 and CBS 109168) was used as the outgroup taxon.
absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30].   Bootstrap support values ≥ 70% from ML analysis and posterior probabilities values ≥ 0.95 obtained from Bayesian inference (BI) are indicated at the nodes as ML/BI. Bootstrap values < 70% or posterior probabilities values < 0.95 are marked with "*", and absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30]. Curvicladiella cignea (CBS 109167 and CBS 109168) was used as the outgroup taxon. and absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T".
The "B" species codes are consistent with the recently published results of Liu and co-authors [30].    Bootstrap support values ≥ 70% from ML analysis and posterior probabilities values ≥ 0.95 obtained from Bayesian inference (BI) are indicated at the nodes as ML/BI. Bootstrap values < 70% or posterior probabilities values < 0.95 are marked with "*", and absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30]. Curvicladiella cignea (CBS 109167 and CBS 109168) was used as the outgroup taxon.
absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30].
Curvicladiella cignea (CBS 109167 and CBS 109168) was used as the outgroup taxon.   Bootstrap support values ≥ 70% from ML analysis and posterior probabilities values ≥ 0.95 obtained from Bayesian inference (BI) are indicated at the nodes as ML/BI. Bootstrap values < 70% or posterior probabilities values < 0.95 are marked with "*", and absent analysis values are marked with "-". "*/*", "*/-", "-/*", and "-/-" are not displayed. Isolates obtained in this study are highlighted in blue and bold. Ex-type isolates are indicated with "T". The "B" species codes are consistent with the recently published results of Liu and co-authors [30]. Curvicladiella cignea (CBS 109167 and CBS 109168) was used as the outgroup taxon.