*Article* **Species Diversity and Distribution Characteristics of** *Calonectria* **in Five Soil Layers in a** *Eucalyptus* **Plantation**

**LingLing Liu 1,2, WenXia Wu <sup>1</sup> and ShuaiFei Chen 1,\***


**Abstract:** The genus *Calonectria* includes pathogens of various agricultural, horticultural, and forestry crops. Species of *Calonectria* are commonly collected from soils, fruits, leaves, stems, and roots. Some species of *Calonectria* isolated from soils are considered as important plant pathogens. Understanding the species diversity and distribution characteristics of *Calonectria* species in different soil layers will help us to clarify their long-term potential harm to plants and their patterns of dissemination. To our knowledge, no systematic research has been conducted concerning the species diversity and distribution characteristics of *Calonectria* in different soil layers. In this study, 1000 soil samples were collected from five soil layers (0–20, 20–40, 40–60, 60–80, and 80–100 cm) at 100 sampling points in one 15-year-old *Eucalyptus urophylla* hybrid plantation in southern China. A total of 1037 isolates of *Calonectria* present in all five soil layers were obtained from 93 of 100 sampling points. The 1037 isolates were identified based on DNA sequence comparisons of the translation elongation factor 1-alpha (*tef1*), β-tubulin (*tub2*), calmodulin (*cmdA*), and histone H3 (*his3*) gene regions, as well as the combination of morphological characteristics. These isolates were identified as *C. hongkongensis* (665 isolates; 64.1%), *C. aconidialis* (250 isolates; 24.1%), *C. kyotensis* (58 isolates; 5.6%), *C. ilicicola* (47 isolates; 4.5%), *C. chinensis* (2 isolates; 0.2%), and *C. orientalis* (15 isolates; 1.5%). With the exception of *C. orientalis*, which resides in the *C. brassicae* species complex, the other five species belonged to the *C. kyotensis* species complex. The results showed that the number of sampling points that yielded *Calonectria* and the number (and percentage) of *Calonectria* isolates obtained decreased with increasing depth of the soil. More than 84% of the isolates were obtained from the 0–20 and 20–40 cm soil layers. The deeper soil layers had comparatively lower numbers but still harbored a considerable number of *Calonectria*. The diversity of five species in the *C. kyotensis* species complex decreased with increasing soil depth. The genotypes of isolates in each *Calonectria* species were determined by *tef1* and *tub2* gene sequences. For each species in the *C. kyotensis* species complex, in most cases, the number of genotypes decreased with increasing soil depth. The 0–20 cm soil layer contained all of the genotypes of each species. To our knowledge, this study presents the first report of *C. orientalis* isolated in China. This species was isolated from the 40–60 and 60–80 cm soil layers at only one sampling point, and only one genotype was present. This study has enhanced our understanding of the species diversity and distribution characteristics of *Calonectria* in different soil layers.

**Keywords:** fungal ecology; multi-gene phylogeny; plant pathogen; soil-borne fungi; tree disease

#### **1. Introduction**

Species in the genus *Calonectria* (*Hypocreales*, *Nectriaceae*) are phytopathogenic fungi that cause serious losses to plant crops in tropical and subtropical regions of the world [1–6]. Many species of *Calonectria* are important pathogens of agricultural, horticultural, and forestry crops and these species occur in approximately 335 plant species in nearly 100 plant families [1]. Species of *Calonectria* have been isolated from soils, fruits, leaves, stems, and roots [1,4,7–14]. The fungi are best known as foliar, shoot, and root pathogens [1,2,4,5], and

**Citation:** Liu, L.; Wu, W.; Chen, S. Species Diversity and Distribution Characteristics of *Calonectria* in Five Soil Layers in a *Eucalyptus* Plantation. *J. Fungi* **2021**, *7*, 857. https:// doi.org/10.3390/jof7100857

Academic Editors: Anush Kosakyan, Rodica Catana and Alona Biketova

Received: 31 August 2021 Accepted: 7 October 2021 Published: 13 October 2021

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they are commonly associated with disease symptoms, including seedling damping-off, seedling rot, cutting rot, leaf spots, leaf blight, shoot blight, crown cankers, stem lesions, collar and root rots, and tuber rot [1,14–23].

Some species of *Calonectria* isolated from soils are important plant pathogens. *Calonectria ilicicola* is a soil-borne fungal pathogen of worldwide importance that causes black rot disease in peanut and red crown rot in soybean [21,24–28]. Recently, we isolated five *Calonectria* species, namely *C. aconidialis*, *C. auriculiformis*, *C. hongkongensis*, *C. pseudoreteaudii*, and *C. reteaudii*, from soils in a plantation of *Eucalyptus* trees [14]. Inoculation results showed that all five species caused leaf spot, leaf blight, and seedling rot to the tested *Eucalyptus* genotypes within three days [14].

Previous research results indicated a high level of species diversity of *Calonectria* in southern China, especially in soils [9,11,13,14,23]. Currently, a total of 125 *Calonectria* species have been described using DNA sequence-based phylogenetic analyses and morphological comparisons [5,13,29–35]. A total of 25 species of *Calonectria* have been identified and described in China based on DNA sequence data [5,9,11,13,14,36]. Of these species, 17 have been isolated from soils, with 11 from soils under plantation *Eucalyptus* trees [5,9,11,14].

Some *Calonectria* species can survive in soil for long periods, and microsclerotia are the primary survival structures [37]. Microsclerotia of some *Calonectria* species can survive in the absence of hosts for 15 years or more [38,39]. *Calonectria* microsclerotia have been recorded at depths of up to 66 cm below the soil surface [40]. Long-term survival and deep soil presence of microsclerotia are serious threats to the management of diseases caused by *Calonectria* species.

Understanding the diversity and distribution characteristics of *Calonectria* species in different soil layers will help us to clarify their potential long-term harm to plants and potential dissemination patterns. Very little research has been conducted concerning the distribution characteristics of microsclerotia in soils, and the few published studies have focused only on the surface soil [38,41]. In the past several years, studies have been conducted to understand *Calonectria* species diversity in forest soils [9–11,13,14,36], but all of the soil samples obtained for *Calonectria* isolation were collected from the 0–20 cm soil layer. In this study, a relatively large number of soil samples were collected from five different soil layers up to 100 cm depth in one 15-year-old *Eucalyptus urophylla* hybrid plantation. Isolates of *Calonectria* from this plantation were obtained and identified. The aims of this study were as follows: (1) to understand the species diversity of *Calonectria* in different soil layers; and (2) to understand the distribution characteristics of each *Calonectria* species in different soil layers.

#### **2. Materials and Methods**

#### *2.1. Study Site, Soil Sampling, and Calonectria Isolation*

This study was performed in a *Eucalyptus urophylla* hybrid plantation (21◦15031.7400 N, 110◦0603500 E; altitude 90 m) located in the South China Experimental Nursery, China Eucalypt Research Centre (CERC), ZhanJiang, GuangDong Province, China. The *Eucalyptus* plantation is located on the northern edge of the tropics, with a maritime monsoon climate [42]. The average annual precipitation is 1777 mm, and the period from May to October accounts for 84.1% of the annual precipitation. The annual average temperature is 23.4 ◦C (http://en.weather.com.cn; accessed date: 10 August 2021). The soil type is Rhodi-Udic Ferralosols, according to the Chinese Soil Taxonomy Classification [42,43]. The area of the *Eucalyptus* plantation is about 6 ha (400 × 150 m), and the planting density of *Eucalyptus* trees is 3 × 2 m. The *Eucalyptus* trees were 15 years old.

One hundred points in the *Eucalyptus* plantation were selected for soil sampling. The 100 points were randomly distributed in the plantation, and the distance between adjacent sampling points was 10 m. Soil samples were collected from five layers at each sampling point: 0–20, 20–40, 40–60, 60–80, and 80–100 cm. Two soil samples were collected in each soil layer for each sampling point. In total, 1000 soil samples were collected from the 100 sampling points. Each of the soil samples was placed in a resealable plastic bag and transferred to the laboratory for *Calonectria* isolation. The soil samples were collected from July to August 2020.

For *Calonectria* isolation, the collected soil was transferred into a plastic cylinder sampling cup (diameter = 4.5 cm, height = 5 cm, and volume = 80 mL) (Chengdu Rich Science Industry Co., Ltd., Chengdu, China); the soil sample occupied two-thirds of the volume of the whole sampling cup volume. The soil sample was moistened by spraying with sterile water and stirred evenly with a sterilized bamboo stick. *Medicago sativa* (alfalfa) seeds were scattered onto the soil surface after it was surface-disinfested (30 s in 75% ethanol and washed several times with sterile water) in the sampling cup. The sampling cup with soil and alfalfa seeds was incubated at 25 ◦C under 12 h of daylight and 12 h of darkness. After one week, sporulating conidiophores with typical morphological characteristics of *Calonectria* species [1] were produced on infected alfalfa tissue. Using a dissection microscope (AxioCam Stemi 2000C, Carl Zeiss, Germany), the single conidial mass was scattered onto 2% malt extract agar (MEA) (20 g malt extract powder and 20 g agar powder per liter of water: malt extract powder was obtained from Beijing Shuangxuan microbial culture medium products factory, Beijing, China; the agar powder was obtained from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) using a sterile needle. After incubation at 25 ◦C for three to four hours, the germinated conidia were individually transferred onto fresh MEA under the dissection microscope and incubated at 25 ◦C for one week to obtain single-conidium cultures. For each soil sample, the soil was transferred into two plastic sampling cups for *Calonectria* isolation.

#### *2.2. DNA Extraction, PCR Amplification, and Sequencing*

All isolates obtained in this study were used for DNA extraction and sequence comparisons. DNA was extracted from 10-day-old cultures. Mycelia were collected using a sterilized scalpel and transferred to 2-mL Eppendorf tubes. The total genomic DNA was extracted using the CTAB protocol described by van Burik and co-authors [44]. The extracted DNA was dissolved in 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 at 37 ◦C for one hour to degrade RNA. Finally, the DNA concentration was measured using a NanoDrop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

According to previous research results, sequences of partial gene regions of translation elongation factor 1-alpha (*tef1*) and β-tubulin (*tub2*), as well as calmodulin (*cmdA*) and histone H3 (*his3*), were used to successfully identify *Calonectria* species [5,14]. These four partial gene regions were amplified using the primer pairs EF1-728F/EF2, T1/CYLTUB1R, CAL-228F/CAL-2Rd, and CYLH3F/CYLH3R, respectively. The PCR procedure was conducted as described by Liu and Chen [36] and Wang and Chen [23].

To obtain accurate sequences for each of the sequenced isolates, all of the PCR products were sequenced in both forward and reverse directions using the same primers used for PCR amplification by the Beijing Genomics Institute, Guangzhou, China. All of the sequences obtained in this study were edited using MEGA v. 7.0 software [45] and were deposited in GenBank (https://www.ncbi.nlm.nih.gov; accessed date: 18 September 2021). The *tef1* and *tub2* gene regions were sequenced for all *Calonectria* isolates. The isolates were genotyped by the *tef1* and *tub2* sequences. Based on the genotypes generated by *tef1* and *tub2* sequences, up to eight isolates for each *tef1*-*tub2* genotype were selected for sequencing the *cmdA* and *his3* gene regions.

#### *2.3. Multi-Gene Phylogenetic Analyses, Morphology, and Species Identification*

A standard nucleotide BLAST search was conducted using the *tef1*, *tub2*, *cmdA*, and *his3* sequences to preliminarily identify the species from which the isolates were obtained in this study. Sequences of *tef1*, *tub2*, *cmdA*, and *his3* gene regions obtained in this study were compared with sequences of type specimen strains of published *Calonectria* species. Sequences of all of the published species in the relevant species complexes were used for sequence comparisons and phylogenetic analyses. The datasets of Liu and co-authors [5]

were used as templates for analyses, while sequences of other recently described *Calonectria* species [13,32–35] were also used for sequence comparisons.

Sequences of each of the *tef1*, *tub2*, *cmdA*, and *his3* gene regions, as well as the combination of these four gene regions, were aligned using the online version of MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server; accessed date: 7 August 2021) with the alignment strategy FFT-NS-i (Slow; interactive refinement method). Sequence alignments were manually edited using MEGA v. 7.0 software [45] after initial alignments.

For *Calonectria* species, maximum parsimony (MP) and maximum likelihood (ML) are frequently used for phylogenetic analyses [5,9,12,14]. Both MP and ML were used for phylogenetic analyses of sequence datasets of each of the four genes and the combination of the four gene regions in order to test whether the analysis results between the two methods were consistent. The MP and ML analyses were conducted by the methods described by Liu and Chen [36]. Phylogenetic trees were viewed by MEGA v. 7.0 [45]. Sequence data of two isolates of *Curvicladiella cignea* (CBS 109167 and CBS 109168) were used as outgroups [5].

The isolates selected for sequencing *tef1*, *tub2*, *cmdA*, and *his3* gene regions were used for morphological studies. Size of macroconidia and width of vesicles are the most typical asexual characteristics used for morphological comparisons for species of *Calonectria* [5,9,11,13,14,29,36]. In order to induce asexual structures, isolates were cultured on 2% MEA in Petri dishes at 25 ◦C for 10 days. Sterile water was then added to the Petri dishes, and a sterilized, soft-bristled paintbrush was used to dislodge the mycelium from the agar surface. The water was then removed, and the dishes were placed upside down and incubated at 25 ◦C for 2–3 days. This resulted in asexual structures being produced on the surface of the cultures for some *Calonectria* isolates, a pattern that has been noted for *Calonectria pteridis* by Graça and co-authors [46] and for *Calonectria pentaseptata* (synonymized as a synonym of *C. pseudoreteaudii* in Liu and co-authors [5]) by Wang and Chen [23]. Fifty measurements of macroconidia and vesicles were measured for the selected isolates that produced abundant macroconidia and vesicles.

#### *2.4. Calonectria Species Diversity in Different Soil Layers*

After all of the *Calonectria* isolates were identified, the number of isolates present in each identified species was counted. The species diversity associated with soil layers was computed. The distribution characteristics of each *Calonectria* species in each soil layer were recorded, including the number of sampling points from which each *Calonectria* species was obtained and the number of isolates of each *Calonectria* species in each of the five soil layers.

#### *2.5. Genotyping of Isolates within Each Calonectria Species*

After all of the *Calonectria* isolates were identified, we examined the genotype diversity of each identified *Calonectria* species in the five different soil layers. The genotypes of isolates within each species were determined based on *tef1* and *tub2* sequences, and the number of isolates belonging to each genotype was recorded.

#### *2.6. Genotype Diversity of Calonectria Species in Different Soil Layers*

Based on the results of genotype analysis of each isolate determined by the sequences of *tef1* and *tub2* gene regions, the numbers of genotypes of each *Calonectria* species in different soil layers were counted. To investigate possible evolutionary relationships among the observed *tef1*–*tub2* genotypes for the *Calonectria* species identified in this study with the most dominant species, minimum spanning networks (MSN) were constructed using Bruvo's distance with the R packages poppr and ape [47,48].

#### **3. Results**

#### *3.1. Soil Sampling and Calonectria Isolation*

One thousand soil samples from 100 sample points were collected from the *E. urophylla* hybrid plantation, with 200 soil samples from each of the five soil layers. For each soil sample, two plastic sampling cups with soil and alfalfa seeds were used for the incubation of *Calonectria*. After the conidia were transferred onto fresh MEA and incubated at 25 ◦C, more than 90% of the conidia germinated within four hours. For each sampling cup, one to four single conidia were transferred onto fresh MEA to obtain one to four singleconidium cultures. In total, *Calonectria* fungi were isolated from 93 sampling points in the plantation; the totals were 92, 40, 20, 7, and 5 from the 0–20, 20–40, 40–60, 60–80, and 80–100 cm soil layers, respectively (Supplementary Table S1, Supplementary Figure S1). One thousand and thirty-seven isolates of *Calonectria* were obtained, with 564 (54.4%), 310 (29.9%), 107 (10.3%), 28 (2.7%), and 28 isolates (2.7%) from the 0–20, 20–40, 40–60, 60–80, and 80–100 cm soil layers, respectively, and 84.3% of the isolates were distributed in the 0–20 and 20–40 cm soil layers (Table 1, Supplementary Table S2, Figure 1). From the results, it was clear that the number of sampling points that yielded *Calonectria* and the number (and percentage) of *Calonectria* isolates obtained decreased with increasing soil depth (Supplementary Figure S1, Figure 1).

**Table 1.** Number of isolates obtained for each *Calonectria* species from each soil layer.


**Figure 1.** Numbers and percentages of *Calonectria* isolates obtained in each of the five soil layers.

#### *3.2. Sequencing*

The *tef1* and *tub2* genes were amplified for all the 1037 isolates obtained in this study (Supplementary Table S2). Twenty-two genotypes were generated based on *tef1* and *tub2* gene sequences (Table 2). Depending on the isolate number of each *tef1*-*tub2* genotype, one to eight isolates of each genotype were selected; finally, 85 isolates in total were selected to sequence the *cmdA* and *his3* gene regions (Table 3). The sequence fragments were approximately 500, 565, 685, and 440 bp for the *tef1*, *tub2*, *cmdA*, and *his3* gene regions, respectively.


**Table 2.** Isolate numbers of each genotype from each *Calonectria* species.

#### *3.3. Multi-Gene Phylogenetic Analyses, Morphology, and Species Identification*

The standard nucleotide BLAST search results conducted using the *tef1*, *tub2*, *cmdA*, and *his3* sequences showed that the isolates obtained in the current study belonged to two species complexes of *Calonectria*, namely, the *C. kyotensis* species complex and the *C. brassicae* species complex. The 85 *Calonectria* isolates with four gene regions sequenced were used for phylogenetic analyses (Table 3). Based on the recently published results in Liu and co-authors [5] and Crous and co-authors [34], sequences of *tef1*, *tub2*, *cmdA*, and *his3* of published species in the *C. kyotensis* species complex and *C. brassicae* species complex, respectively, were used for sequence comparisons and phylogenetic analyses (Table 4).

The partition homogeneity test (PHT) comparing the *tef1*, *tub2*, *cmdA*, and *his3* gene combination datasets generated a *p*-value of 0.001, indicating that the accuracy of the combined datasets did not suffer relative to the individual partitions [60]. Thus, sequences of the four loci were combined for analyses. Between the MP and ML trees, the overall topologies were similar for the phylogenetic trees based on *tef1*, *tub2*, *cmdA*, and *his3* individually and the combination datasets, but the relative positions of some *Calonectria* species slightly differed. The five ML trees are presented in Figure 2 and Supplementary Figures S2–S5. The numbers of taxa and parsimony-informative characters, statistical values of the MP analyses, and parameters of the best-fit substitution models of ML analyses are provided in Table 5.

**Identity Genotype 1 Isolate No. 2 Sampling Point No. 3 Soil Layer Sample and Isolate Information 4 Collectors GenBank Accession No. 5** *tef1 tub2 cmdA his3 C. aconidialis* AAAA CSF20325 6 0–20 cm 20200711-1-(3)\_0–20 cm\_A\_R2\_SC2 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167700 OK168737 OK169148 OK169232 *C. aconidialis* AAAA CSF21348 98 0–20 cm 20200816-1-(6)\_0–20 cm\_A\_R2\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167855 OK168892 OK169151 OK169235 *C. aconidialis* AACA CSF20378 9 0–20 cm 20200711-1-(6)\_0–20 cm\_A\_R2\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167701 OK168738 OK169149 OK169233 *C. aconidialis* AACA CSF20447 11 0–20 cm 20200715-1-(1)\_0–20 cm\_B\_R2\_SC2 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167704 OK168741 OK169150 OK169234 *C. aconidialis* ABBA CSF20985 6 68 20–40 cm 20200811-1-(4)\_0–40 cm\_B\_R1\_SC3 L.L. Liu, J.L. Han, and L.S. Sun OK167856 OK168893 OK169152 OK169236 *C. aconidialis* ABBA CSF21262 93 20–40 cm 20200816-1-(1)\_0–40 cm\_B\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167857 OK168894 OK169153 OK169237 *C. aconidialis* ABBA CSF21266 93 20–40 cm 20200816-1-(1)\_0–40 cm\_B\_R2\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167861 OK168898 OK169154 OK169238 *C. aconidialis* ABBA CSF21349 98 0–20 cm 20200816-1-(6)\_0–20 cm\_A\_R2\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167864 OK168901 OK169155 OK169239 *C. aconidialis* ACAA CSF20257 1 0–20 cm 20200709-1-(1)\_0–20 cm\_A\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167865 OK168902 OK169156 OK169240 *C. aconidialis* ACAA CSF20323 6 6 0–20 cm 20200711-1-(3)\_0–20 cm\_A\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167866 OK168903 OK169157 OK169241 *C. aconidialis* ACAA CSF20376 6 9 0–20 cm 20200711-1-(6)\_0–20 cm\_A\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167868 OK168905 OK169158 OK169242 *C. aconidialis* ACAA CSF21346 98 0–20 cm 20200816-1-(6)\_0–20 cm\_A\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167946 OK168983 OK169159 OK169243 *C. chinensis* AAAA CSF20756 6 52 0–20 cm 20200809-1-(2)\_0–20 cm\_A\_R2\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK168055 OK169092 OK169184 OK169268 *C. chinensis* AAAA CSF20759 6 52 0–20 cm 20200809-1-(2)\_0–20 cm\_A\_R2\_SC4 L.L. Liu, J.L. Han, and L.S. Sun OK168056 OK169093 OK169185 OK169269 *C. hongkongensis* AAAA CSF20258 1 0–20 cm 20200709-1-(1)\_0–20 cm\_A\_R1\_SC2 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167035 OK168072 OK169109 OK169194 *C. hongkongensis* AAAA CSF20271 2 0–20 cm 20200709-1-(2)\_0–20 cm\_A\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167044 OK168081 OK169110 OK169195 *C. hongkongensis* AAAA CSF20291 3 0–20 cm 20200709-1-(3)\_0–20 cm\_A\_R2\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167056 OK168093 OK169111 OK169196 *C. hongkongensis* AAAA CSF21370 100 0–20 cm 20200816-1-(8)\_0–20 cm\_A\_R2\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167588 OK168625 OK169112 OK169197 *C. hongkongensis* ABA- CSF20758 52 0–20 cm 20200809-1-(2)\_0–20 cm\_A\_R2\_SC3 L.L. Liu, J.L. Han, and L.S. Sun OK167596 OK168633 OK169113 – 7 *C. hongkongensis* ACAA CSF20524 17 0–20 cm 20200715-1-(7)\_0–20 cm\_B\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167597 OK168634 OK169114 OK169198 *C. hongkongensis* ACAA CSF20525 17 0–20 cm 20200715-1-(7)\_0–20 cm\_B\_R1\_SC2 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167598 OK168635 OK169115 OK169199 *C. hongkongensis* ACAB CSF21368 100 0–20 cm 20200816-1-(8)\_0–20 cm\_A\_R1\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167599 OK168636 OK169116 OK169200 *C. hongkongensis* ACAB CSF21372 100 0–20 cm 20200816-1-(8)\_0–20 cm\_B\_R1\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167600 OK168637 OK169117 OK169201 *C. hongkongensis* ADAA CSF20412 10 0–20 cm 20200711-1-(7)\_0–20 cm\_B\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167601 OK168638 OK169118 OK169202 *C. hongkongensis* ADAA CSF20454 11 20–40 cm 20200715-1-(1)\_0–40 cm\_A\_R2\_SC3 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167602 OK168639 OK169119 OK169203 *C. hongkongensis* ADAA CSF20834 60 0–20 cm 20200810-1-(4)\_0–20 cm\_B\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167604 OK168641 OK169120 OK169204 *C. hongkongensis* ADAA CSF21304 96 0–20 cm 20200816-1-(4)\_0–20 cm\_A\_R2\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167607 OK168644 OK169121 OK169205 *C. hongkongensis* AEAA CSF20923 65 0–20 cm 20200811-1-(1)\_0–20 cm\_A\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167608 OK168645 OK169122 OK169206 *C. hongkongensis* AEAA CSF20924 6 65 0–20 cm 20200811-1-(1)\_0–20 cm\_A\_R1\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167609 OK168646 OK169123 OK169207 *C. hongkongensis* AFAA CSF20259 1 0–20 cm 20200709-1-(1)\_0–20 cm\_A\_R2\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167610 OK168647 OK169124 OK169208 *C. hongkongensis* AFAA CSF20309 4 0–20 cm 20200711-1-(1)\_0–20 cm\_A\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167611 OK168648 OK169125 OK169209 *C. hongkongensis* AFAA CSF20470 12 0–20 cm 20200715-1-(2)\_0–20 cm\_A\_R2\_SC1 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167615 OK168652 OK169126 OK169210 *C. hongkongensis* AFAA CSF21233 90 0–20 cm 20200815-1-(3)\_0–20 cm\_B\_R2\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167629 OK168666 OK169127 OK169211 *C. hongkongensis* AGAA CSF20380 9 0–20 cm 20200711-1-(6)\_0–20 cm\_B\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167630 OK168667 OK169128 OK169212 *C. hongkongensis* AGAA CSF20441 11 0–20 cm 20200715-1-(1)\_0–20 cm\_A\_R1\_SC2 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167631 OK168668 OK169129 OK169213 *C. hongkongensis* AGAA CSF20528 17 40–60 cm 20200715-1-(7)\_0–60 cm\_A\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167632 OK168669 OK169130 OK169214 *C. hongkongensis* AGAA CSF21018 71 0–20 cm 20200811-1-(7)\_0–20 cm\_B\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167644 OK168681 OK169131 OK169215 *C. hongkongensis* AHAA CSF20760 52 0–20 cm 20200809-1-(2)\_0–20 cm\_B\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167645 OK168682 OK169132 OK169216 *C. hongkongensis* AHAA CSF20761 6 52 0–20 cm 20200809-1-(2)\_0–20 cm\_B\_R1\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167646 OK168683 OK169133 OK169217 *C. hongkongensis* AHAA CSF21155 82 0–20 cm 20200813-1-(2)\_0–20 cm\_B\_R2\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167647 OK168684 OK169134 OK169218 *C. hongkongensis* AHAA CSF21156 82 0–20 cm 20200813-1-(2)\_0–20 cm\_B\_R2\_SC2 L.L. Liu, J.L. Han, and L.S. Sun OK167648 OK168685 OK169135 OK169219 *C. hongkongensis* BAAA CSF20472 12 0–20 cm 20200715-1-(2)\_0–20 cm\_B\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167649 OK168686 OK169136 OK169220 *C. hongkongensis* BAAA CSF20734 51 0–20 cm 20200809-1-(1)\_0–20 cm\_A\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167652 OK168689 OK169137 OK169221 *C. hongkongensis* BAAA CSF21183 86 0–20 cm 20200814-1-(2)\_0–20 cm\_B\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167657 OK168694 OK169138 OK169222 *C. hongkongensis* BAAA CSF21359 99 0–20 cm 20200816-1-(7)\_0–20 cm\_A\_R2\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167660 OK168697 OK169139 OK169223 *C. hongkongensis* CAAA CSF20353 6 7 0–20 cm 20200711-1-(4)\_0–20 cm\_A\_R2\_SC2 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167664 OK168701 OK169140 OK169224 *C. hongkongensis* CAAA CSF20358 7 20–40 cm 20200711-1-(4)\_0–40 cm\_B\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167665 OK168702 OK169141 OK169225 *C. hongkongensis* CAAA CSF20359 7 20–40 cm 20200711-1-(4)\_0–40 cm\_B\_R1\_SC2 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167666 OK168703 OK169142 OK169226 *C. hongkongensis* CAAA CSF20360 6 7 20–40 cm 20200711-1-(4)\_0–40 cm\_B\_R1\_SC3 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167667 OK168704 OK169143 OK169227 *C. hongkongensis* DAAA CSF20334 6 20–40 cm 20200711-1-(3)\_0–40 cm\_B\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167669 OK168706 OK169144 OK169228 *C. hongkongensis* DAAA CSF20383 6 9 0–20 cm 20200711-1-(6)\_0–20 cm\_B\_R2\_SC2 S.F. Chen, L.L. Liu, J.L. Han, Y Liu, and X.Y. Liang OK167673 OK168710 OK169145 OK169229 *C. hongkongensis* DAAA CSF20444 11 0–20 cm 20200715-1-(1)\_0–20 cm\_B\_R1\_SC1 S.F. Chen, L.L. Liu, J.L. Han, L.S. Sun, and W.W. Li OK167678 OK168715 OK169146 OK169230 *C. hongkongensis* DAAA CSF21367 100 0–20 cm 20200816-1-(8)\_0–20 cm\_A\_R1\_SC1 L.L. Liu, J.L. Han, and L.S. Sun OK167699 OK168736 OK169147 OK169231

**Table 3.** Isolates sequenced and used for phylogenetic analyses and morphological studies in this study.



β-tubulin; *cmdA* = calmodulin; *his3* = histone H3. 6

study.

Isolates used for measuring macroconidia and vesicles in the current study. 7 "–" represents the relative locus was not successfully amplified in the current


**Table 4.** Isolates from other studies used in the phylogenetic analyses in this study.






 Codes B1 to B120 of the 120 accepted *Calonectria* species resulting from Liu and co-authors [5], "B124" indicated *C. singaporensis* described in Crous and co-authors [34]. 2 T: ex-type isolates of the species. 3 AR: Amy Y. Rossman working collection; ATCC: American Type Culture Collection, Virginia, USA; CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CERC: China Eucalypt Research Centre, ZhanJiang, GuangDong Province, China; CMW: Culture collection of the Forestry and Agricultural Biotechnology Institute FABI, University of Pretoria, Pretoria, South Africa; CPC: Pedro Crous working collection housed at Westerdijk Fungal Biodiversity Institute; IMI: International Mycological Institute, MUCL: Mycotheque, Laboratoire de Mycologie Systematique st Appliqee, I'Universite, Louvian-la-Neuve, Belgium; STE-U: Department of Plant Pathology, University of Stellenbosch, South Africa; "–" represents no other collection number. 4 *tef1*: translation elongation factor 1-alpha; *tub2*: β-tubulin; *cmdA*: calmodulin; *his3*: histone H3; for GenBank Accession No. in bold, the sequences were submitted in this study. 5 N/A represents data that is not available.


**Table 5.** Statistical values of datasets for maximum parsimony and maximum likelihood analyses in this study.

<sup>1</sup> bp = base pairs. <sup>2</sup> PIC = number of parsimony informative characters. <sup>3</sup> CI = consistency index. <sup>4</sup> RI = retention index. <sup>5</sup> RC = rescaled consistency index. <sup>6</sup> HI = homoplasy index. <sup>7</sup> Subst. model = best fit substitution model. <sup>8</sup> NST = number of substitution rate categories.

> The phylogenetic analyses showed that the 85 *Calonectria* isolates were clustered in six groups (Group A, Group B, Group C, Group D, Group E, and Group F) based on *tef1*, *tub2*, *cmdA*, *his3,* and combined *tef1*/*tub2*/*cmdA*/*his3* analyses (Figure 2, Supplementary Figures S2–S5). The analyses showed that isolates in Groups A, B, C, D, and E belonged to the *C. kyotensis* species complex. Isolates in Groups A, B, C, and E were clustered with or were closest to *C. hongkongensis*, *C. kyotensis*, *C. chinensis,* and *C. ilicicola*, respectively, based on the *tef1*, *tub2*, *cmdA*, *his3,* and combined *tef1*/*tub2*/*cmdA*/*his3* trees (Figure 2, Supplementary Figures S2–S5). Therefore, isolates in Groups A, B, C, and E were identified as *C. hongkongensis*, *C. kyotensis*, *C. chinensis,* and *C. ilicicola*, respectively. Isolates in Group D were clustered in two sub-groups, sub-group D1 and sub-group D2, in the *tub2* tree. Isolates in sub-group D1 were clustered with or were closest to *C. aconidialis*; isolates in sub-group D2 were clustered with *C. asiatica* (Supplementary Figure S3); isolates in Group D were clustered with or were closest to *C. aconidialis* based on the *tef1*, *cmdA*, *his3,* and combined *tef1*/*tub2*/*cmdA*/*his3* trees (Figure 2, Supplementary Figures S2, S4, and S5). Isolates in Group D were identified as *C. aconidialis*. Isolates in Group F belonged to the *C. brassicae* species complex. These isolates were consistently only clustered with *C. orientalis* based on the *tef1*, *tub2*, *his3,* and combined *tef1*/*tub2*/*cmdA*/*his3* trees and were clustered with both *C. orientalis* and *C. brassicae* in the *cmdA* tree (Figure 2, Supplementary Figures S2–S5). Isolates in Group F were identified as *C. orientalis*.

**Figure 2.** Phylogenetic tree of *Calonectria* species based on maximum likelihood (ML) analyses of the dataset of combined *tef1*, *tub2*, *cmdA*, and *his3* gene sequences in this study. Bootstrap support values ≥ 70% are presented above the branches as follows: ML/MP. Bootstrap values < 70% or absent are marked with "\*". Isolates highlighted in six different colors, and bold were obtained in this study. Ex-type isolates are marked with "T". The "B" species codes are consistent with the recently published results in Liu and co-authors [5]. *Curvicladiella cignea* (CBS 109167 and CBS 109168) was used as the outgroup taxon.

#### 131

Based on the results of phylogenetic analyses and induction of asexual structures, 17 isolates representing six *Calonectria* species were selected for macroconidia and vesicle morphological comparisons (Tables 3 and 6). These representative isolates could be classified into two groups based on the shape of the vesicles. Isolates of *C. aconidialis*, *C. chinensis*, *C. hongkongensis*, *C. ilicicola*, and *C. kyotensis* produce sphaeropedunculate vesicles, while the vesicles of *C. orientalis* are typically clavate. With the exception of *C. ilicicola* isolates, which produce 1(–3) septate macroconidia, isolates of the other five species all produced one septate macroconidium (Table 6). The shape of the vesicle and the number of macroconidia septations for each of the six *Calonectria* species found in this study were consistent with the described strains of relevant species in previous studies [1,9,29,49] (Table 6).

The morphological comparisons showed that significant variation existed in the size of macroconidia or width of vesicles among some isolates of each species of *C. aconidialis*, *C. hongkongensis*, and *C. kyotensis* identified in this study. For example, the macroconidia of *C. aconidialis* isolate CSF20985 were much longer than those of the other two tested *C. aconidialis* isolates CSF20323 and CSF20376. In *C. hongkongensis*, the macroconidia of isolate CSF20383 were longer than those of the other four isolates; the vesicles of isolate CSF20924 were wider than those of other isolates. In *C. kyotensis*, the macroconidia of isolate CSF20276 were much longer than those of isolate CSF21191 (Table 6).

The measurement results further showed that macroconidia size and vesicle width of isolates of some species obtained in this study were not always similar to the originally described strains of the same *Calonectria* species. For example, the macroconidia lengths of isolates of *C. chinensis* and *C. orientalis* obtained in this study were much shorter than the originally described strains of the relevant species [29,49] (Table 6).

#### *3.4. Calonectria Species Diversity in Different Soil Layers*

Based on the sequence comparisons of *tef1*, *tub2*, *cmdA,* and *his3* sequences, the 1037 *Calonectria* isolates were identified as *C. hongkongensis* (665 isolates; 64.1%), *C. aconidialis* (250 isolates; 24.1%), *C. kyotensis* (58 isolates; 5.6%), *C. ilicicola* (47 isolates; 4.5%), *C. chinensis* (2 isolates; 0.2%), and *C. orientalis* (15 isolates; 1.5%) (Table 1). *Calonectria hongkongensis* was dominant, followed by *C. aconidialis.* Each of the two dominant species was isolated from more than or close to 50% of all of the sampling points, and the two species accounted for 88.2% of all of the *Calonectria* isolates obtained in this study (Table 1, Supplementary Table S1, Figure 3). Both *C. chinensis* and *C. orientalis* were only isolated from one sampling point; *C. chinensis* was only isolated from the 0–20 cm soil layer, and only two isolates were obtained; *C. orientalis* was isolated from the soil layers of 40–60 and 60–80 cm, and 11 and 4 isolates in the two soil layers were obtained, respectively (Table 1, Supplementary Table S1, Figure 3).

With the exception of *C. orientalis* in the *C. brassicae* species complex, the diversity of species in the *C. kyotensis* species complex decreased with increasing soil depth. Five, four, four, four, and two species were identified in the soil layers of 0–20, 20–40, 40–60, 60–80, and 80–100 cm, respectively (Table 1, Supplementary Table S1).

For each of the five species in the *C. kyotensis* species complex, the number of sampling points containing *Calonectria* decreased with increasing depth of the soil, with the exception of *C. hongkongensis* in soil layers of 60–80 cm (2 sampling points) and 80–100 cm (4 sampling points) (Supplementary Table S1, Figure 4A); the number of isolates obtained decreased with increasing soil depth, with the exception of *C. hongkongensis* in the 60–80 cm soil layer (8 isolates) and 80–100 cm (20 isolates) as well as *C. ilicicola* in the 0–20 cm (16 isolates) and 20–40 cm (19 isolates) soil layers (Table 1, Figure 4B). Most isolates were obtained from the soil layers 0–20 and 20–40 cm, accounting for 86.6%, 85.6%, 81%, 74.5%, and 100% of all of the obtained isolates within each species of *C. hongkongensis*, *C. aconidialis*, *C. kyotensis*, *C. ilicicola*, and *C. chinensis*, respectively (Figure 5).


**Table 6.** Morphological comparisons of *Calonectria* isolates and species obtained in the current study.

represents data that are not available.

**Figure 3.** Numbers and percentages of isolates obtained for each *Calonectria* species from all soil samples collected.

**Figure 4.** Number of sampling points yielded different *Calonectria* species in each of the five soil layers (**A**), and numbers of isolates obtained for different *Calonectria* species in each of the five soil layers (**B**).

**Figure 5.** Relative abundances of each *Calonectria* species in each of the five soil layers. Relative abundance was based on the proportional frequencies of isolates of each *Calonectria* species in each soil layer.

#### *3.5. Genotyping of Isolates within Each Calonectria Species*

For the 1037 *Calonectria* isolates obtained and identified in this study, the genotype results based on *tef1* and *tub2* sequences indicated that 11, 3, 3, 3, 1, and 1 genotype(s) existed in *C. hongkongensis*, *C. aconidialis*, *C. kyotensis*, *C. ilicicola*, *C. chinensis*, and *C. orientalis*, respectively (Table 2). The isolates presenting the dominant genotype (genotype AA) accounted for 84.4%, 62.4%, 56.9%, 55.3%, 100%, and 100% of all of the isolates obtained from *C. hongkongensis*, *C. aconidialis*, *C. kyotensis*, *C. ilicicola*, *C. chinensis*, and *C. orientalis*, respectively (Table 2).

#### *3.6. Genotype Diversity of Calonectria Species in Different Soil Layers*

The *tef1*-*tub2* genotypes of each *Calonectria* species in each soil layer are listed in Table 7 and are shown in Figure 6. For each species in the *C. kyotensis* species complex, the results showed that the number of genotypes decreased with increasing soil depth, with the exception of *C. hongkongensis* and *C. aconidialis* in the 60–80 cm (one genotype) and 80–100 cm (two genotypes) soil layers (Table 7, Figure 6A,B); the 0–20 cm soil layer contained all of the genotypes of each species in the *C. kyotensis* complex (Table 7, Figure 6A–E). For the genotype with the most isolates of each species in the *C. kyotensis* complex, the majority of isolates were obtained from 0–20 cm soil layer, with the exception of *C. ilicicolla* (Table 7, Figure 6A–E). Only one genotype of *C. orientalis* was present in the 40–60 and 60–80 cm soil layers (Table 7, Figure 6F).


#### **Table 7.** Isolate numbers of each genotype in each soil layer for each *Calonectria* species.

**Figure 6.** The isolate numbers of each genotype of each *Calonectria* species in five soils layers. The genotypes were determined by DNA sequences of *tef1* and *tub2* gene regions. (**A**): *C. hongkongensis*; (**B**): *C. aconidialis*; (**C**): *C. kyotensis*; (**D**) *C. ilicicola*; (**E**): *C. chinensis*; (**F**): *C. orientalis*.

The minimum spanning network (MSN) analysis was conducted for *C. hongkongensis*, which was considered as the dominant species identified in this study. The analysis revealed that most isolates of *C. hongkongensis* were genotype AA (561 isolates), followed by genotypes DA (31 isolates) and AF (20 isolates); genotype AA was present in the isolates from all five soil layers; genotypes AB, AC, AE, AH, and BA were present only in the isolates from the 0–20 cm soil layer, and the other genotypes were present in isolates from two to four soil layers. Isolates from the 0–20 cm soil layer contained all of the genotypes (Figure 7).

**Figure 7.** Minimum spanning network constructed using Bruvo's distances showing that the *C. hongkongensis* isolates were grouped into 11 genotypes based on *tef1* and *tub2* sequences. The size of a node is proportional to the number of represented *tef1*-*tub2* genotypes.

#### **4. Discussion**

In this study, more than 1000 *Calonectria* isolates were obtained from five soil layers at 100 sampling points in one *Eucalyptus* plantation. All of the isolates were identified based on DNA sequence comparisons of multiple gene regions. Six *Calonectria* species were identified, namely, *C. aconidialis*, *C. chinensis*, *C. hongkongensis*, *C. ilicicola,* and *C. kyotensis* in the *C. kyotensis* species complex, and *C. orientalis* in the *C. brassicae* species complex. *Calonectria hongkongensis* (64.1% of all of the isolates) was the dominant species, followed by *C. aconidialis* (24.1% of all of the isolates). To our knowledge, this is the first report of *C. orientalis* in China. The species diversity and distribution characteristics of the six species

in different soil layers were clarified. The results showed that the number of sampling points from which *Calonectria* was obtained, and the number of *Calonectria* isolates obtained decreased with increasing depth of the soil. The majority of isolates (84.3% of all the isolates) were obtained from soil layers of 0–20 and 20–40 cm. The diversity of the five species in the *C. kyotensis* species complex decreased with increasing soil depth. For each species in the *C. kyotensis* species complex, in most cases, the number of genotypes decreased with increasing soil depth, and the 0–20 cm soil layer contained all of the genotypes of each species.

Five species, namely, *C. aconidialis*, *C. chinensis*, *C. hongkongensis*, *C. ilicicola*, and *C. kyotensis,* in the *C. kyotensis* species complex were isolated from the soil of the *Eucalyptus* plantation in this study. These five species have been frequently isolated from soils in several other regions in southern China, especially from soils in *Eucalyptus* plantations [9,11,14,49]. *Calonectria ilicicola* is considered as a soil-borne fungal pathogen that has been isolated from a number of diseased plant species in China [21,61]. This study presents the first record of *C. ilicicola* isolated from soil in a *Eucalyptus* plantation. Results of this and previous studies suggest that all five of the species in the *C. kyotensis* species complex are potentially widely distributed in *Eucalyptus* plantation soils in other regions of southern China [9,11,14].

This study is the first report of *C. orientalis* in China, and this species is the first *Calonectria* species in the *C. brassicae* species complex found in China. *Calonectria orientalis* has been isolated from soil in Indonesia [29]. Some other species in the *C. brassicae* species complex have also been frequently isolated from soils. With the exception of *C. orientalis*, the other species in the *C. brassicae* species complex isolated from soils were all from Ecuador and Brazil in South America [5,10,29–31,56]. Most of the *Calonectria* species in the *C. brassicae* species complex have only been isolated from South America but not from Asia [5] and *C. orientalis*, in this study, was only isolated from one of the 100 sampling points. These results suggest that *C. orientalis* is not widely distributed in China.

For the five species in the *C. kyotensis* species complex, the results of this study indicate that the diversity of the five species decreased with increasing soil depth, and the number of sampling points containing *Calonectria* and the number of *Calonectria* isolates obtained also decreased with soil depth. Most isolates were obtained from the 0–20 and 20–40 cm soil layers. In most cases, the number of genotypes decreased with increasing soil depth for each species, and the 0–20 cm soil layer contained all of the genotypes of each species. These results suggest that 0–20 cm is the best soil depth for *Calonectria* isolation and for examining the species and genotype diversity of *Calonectria* in soils in *Eucalyptus* plantations in southern China. In several previous studies specialized in the research on *Calonectria* species diversity, soil samples were also exclusively collected from the surface layer, all from the 0–20 cm layer [9–11,13,14,36]. These studies have characterized the diversity of *Calonectria* species well. Results of a number of other studies indicated that microbial diversity and richness are typically affected by the soil depth [62–67], and shallower layers usually have a higher level of microbial diversity [62,63,66–68]. This pattern is consistent with the results of the present study. A possible reason for the vertical distribution of soil microbes is the harsher environment in deeper soil layers, where the soil density is higher, oxygen concentrations are lower, and carbon and nutrients are less available [69]. For *Calonectria*, which includes some important pathogens of various agricultural, horticultural, and forestry crops worldwide, as well as for other genera of fungi in forests, no systematic research has been conducted to examine the species diversity and distribution characteristics in different soil layers. This study showed that the deeper soil layers had comparatively fewer but still contained many *Calonectria*. It remains unknown whether the *Calonectria* were originally distributed in deeper soil layers or whether the fungi in deeper soil layers migrated from surface layers, perhaps through the infiltration of rainwater. Studies on the population diversity differences among different soil layers should be conducted to address this question. Furthermore, the *Calonectria* distributed in deeper soil layers increase the challenge of controlling the diseases caused by these fungi.

This study examined the species diversity and distribution characteristics of *Calonectria* in five soil layers in a *Eucalyptus* plantation in southern China. Six species were isolated from soils in a relatively small *Eucalyptus* plantation, indicating that the diversity of *Calonectria* species in these soils in southern China is relatively high. This study also revealed that the species diversity and number of genotypes of each *Calonectria* species decreased with increasing soil depth, a pattern that helps us to understand the distribution characteristics of *Calonectria* species in different layers of soil. For some *Calonectria* species, there were relatively large numbers of isolates obtained from different soil layers, especially for *C. hongkongensis* and *C. aconidialis* in the 0–20, 20–40, and 40–60 cm soil layers. The genetic structures and population biology of these species in the different soil layers are unknown, but additional studies may increase our understanding of the distribution characteristics and dissemination patterns of *Calonectria* species.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jof7100857/s1, Table S1: Number of sampling points containing each *Calonectria* species in each soil layer, Table S2: All 1037 isolates obtained and sequenced in this study, Figure S1: Number of sampling points that yielded *Calonectria* in each of the five soil layers, Figure S2: Phylogenetic tree of *Calonectria* species based on maximum likelihood (ML) analyses of the *tef1* gene sequences, Figure S3: Phylogenetic tree of *Calonectria* species based on ML analyses of the *tub2* gene sequences, Figure S4: Phylogenetic tree of *Calonectria* species based on ML analyses of the *cmdA* gene sequences, Figure S5: Phylogenetic tree of *Calonectria* species based on ML analyses of the *his3* gene sequences.

**Author Contributions:** S.C. conceived and designed the experiments. L.L. and S.C. collected the samples. L.L. performed the laboratory work. L.L., W.W. and S.C. analyzed the data. S.C. and L.L. wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was initiated through the bilateral agreement between the Governments of South Africa and China and supported by The National Key R&D Program of China (China-South Africa Forestry Joint Research Centre Project; project No. 2018YFE0120900), the National Ten-thousand Talents Program (Project No. W03070115) and the GuangDong Top Young Talents Program (Project No. 20171172).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and Supplementary Materials.

**Acknowledgments:** We thank JiaLong Han, LanSen Sun, Ying Liu, and XueYing Liang for their assistance in collecting samples. We thank QianLi Liu for sequencing the *tub2* gene region of some isolates in Table 4. We thank FeiFei Liu for analyzing the genotype data. We thank GuoQing Li, WenWen Li, and QuanChao Wang who provided assistance in laboratory work and checking the data. We thank LetPub (www.letpub.com; accessed date: 27 August 2021 and 6 October 2021) for providing linguistic assistance during the preparation of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

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