*Article* **Diversity and Distribution of** *Calonectria* **Species from Plantation and Forest Soils in Fujian Province, China**

**Qianli Liu 1,2, Michael J. Wingfield <sup>1</sup> , Tuan A. Duong <sup>1</sup> , Brenda D. Wingfield <sup>1</sup> and Shuaifei Chen 1,2,\***


**Abstract:** To meet the growing demand for wood and pulp products, *Eucalyptus* plantations have expanded rapidly during the past two decades, becoming an integral part of the southern China landscape. Leaf blight caused by various *Calonectria* spp., is a serious threat to these plantations. In order to explore the diversity and distribution of *Calonectria* spp. in Fujian Province soils, samples were collected in *Eucalyptus* plantations and adjacent plantings of *Cunninghamia lanceolata*, *Phyllostachys heterocycle* and *Pinus massoniana* as well as in natural forests. Three hundred and fiftythree *Calonectria* isolates were recovered from soil samples and they were identified based on a comparison of multilocus DNA sequence data for the *act* (actin), *cmdA* (calmodulin), *his3* (histone H3), *rpb2* (the second largest subunit of RNA polymerase), *tef1* (translation elongation factor 1-alpha) and *tub2* (β-tubulin) gene regions, as well as morphological characteristics. Six known taxa including *Calonectria aconidialis*, *Ca. hongkongensis*, *Ca. ilicicola*, *Ca. kyotensis*, *Ca. pacifica*, *Ca. pseudoreteaudii* and one novel species described here as *Ca*. *minensis* sp. nov. were identified. Of these, *Ca*. *aconidialis* and *Ca*. *kyotensis* were the most prevalent species, and found in eight and seven sites, and four and five forest types, respectively. *Calonectria* spp. were most abundant in soils from *Eucalyptus* stands, followed by *P. heterocycle* and natural forests. Relatively few species were found in the soils associated with *Cunninghamia lanceolata* and *Pinus massoniana*. The abundance of known *Calonectria* spp. suggests that these fungi have been relatively well sampled in Fujian. The results are also consistent with the fact that most Calonectria diseases are found on Angiosperm as opposed to Gymnosperm plants.

**Keywords:** Calonectria leaf blight; forest pathogens; fungal diversity; phylogeny; taxonomy

## **1. Introduction**

Species of *Eucalyptus* are the most important trees used to establish plantations in the tropics and Southern Hemisphere, where they provide substantial resources for the global fibre market [1]. These trees were first introduced into China as ornamentals in 1890 and plantations of *Eucalyptus* spp. had reached 5.46 million hm<sup>2</sup> by 2018 [1]. Plantations of these trees are mainly distributed in 11 provinces of China, and over 75% can be found in the Guangxi, Guangdong, Yunnan and Fujian Provinces of southern China [1]. The *Eucalyptus* plantations in China have been established with a relatively narrow genetic base and consequently many disease problems, caused by a variety of pathogens, have emerged as threats to their sustainability [2–6].

Among the diseases threatening *Eucalyptus* plantations, leaf blight caused by species of *Calonectria* De Not. has become a major constraint in southern China [4,7–10]. Symptoms of infection are characterised by water-soaked spots on leaves in the lower and middle parts of the tree crowns. These coalesce and gradually develop into extended necrotic areas, which result in blight and often severe defoliation [9]. In China, Calonectria Leaf Blight (CLB) has

**Citation:** Liu, Q.; Wingfield, M.J.; Duong, T.A.; Wingfield, B.D.; Chen, S. Diversity and Distribution of *Calonectria* Species from Plantation and Forest Soils in Fujian Province, China. *J. Fungi* **2022**, *8*, 811. https:// doi.org/10.3390/jof8080811

Academic Editors: Lei Cai and Cheng Gao

Received: 2 July 2022 Accepted: 25 July 2022 Published: 31 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

been observed in *Eucalyptus* plantations in Fujian, Guangdong, Guangxi, Hainan and Yunnan Provinces [4,7,9–11]. This is similar to the situation in Australia, Brazil, Indonesia, Thailand and Vietnam where *Eucalyptus* plantations have also suffered significant damage due to CLB [12–16].

The genus *Calonectria* includes many aggressive plant pathogens. These species are extensively distributed particularly in sub-tropical and tropical regions of the world, and they have a wide host range including more than 335 plant species [17]. *Calonectria* species are generally considered as soil-borne fungi and they can survive in the soil for extended periods due to their thick-walled microsclerotia [17].

A recent taxonomic revision of *Calonectria* by Liu and co-authors [18] accepted 120 species. Of these, 65 have been reported from soils samples; the remaining species are known from infections on plant tissues [10,18–22]. To date, 27 species of *Calonectria* have been recorded in China, 18 of which have been isolated from soil samples [4,7,10,11,18,21,23–26].

Plantations of *Eucalyptus* spp. are commonly established alongside those of *Cunninghamia lanceolata*, *Phyllostachys heterocycle* and *Pinus massoniana* and can also be in mixed plantings in the Fujian Province (Figure 1). In recent years, leaf blight has become a serious threat to *Eucalyptus* plantations in this province [7,8]. *Calonectria* species including *Ca. crousiana*, *Ca*. *eucalypti*, *Ca*. *fujianensis*, *Ca*. *pauciromosa* and *Ca*. *pseudoreteaudii* [7,8,18] have been isolated from diseased *Eucalyptus* tissues and are regarded as the important causal agents of CLB in Fujian. *Calonectria* infections initially arise from inoculum in the soil but very little is known regarding the species diversity and distribution of these fungi in Fujian soils. The aim of this study was thus to determine the identity and distribution of *Calonectria* spp. from a wide variety of soils in Fujian, with a particular focus on *Eucalyptus* spp. but also including other trees that are found in the area.

**Figure 1.** Different forest plantations and natural forests in southern China. (**a**). mixed species plantations in Zhangzhou Region, Fujian Province, 1: *Eucalyptus* sp., 2: *Pinus massoniana*, 3: *Cunninghamia lanceolata*; (**b**). mixed species plantations in Jiangxi Province, 1: *Eucalyptus* sp., 3: *Cunninghamia lanceolata*; 4: *Phyllostachys heterocycle*; (**c**). *Eucalyptus* sp. in Yongan Region, Fujian Province; (**d**). *Cunninghamia lanceolata* in JiangXi Province; (**e**). *Phyllostachys heterocycle* in Nanping Region, Fujian Province; (**f**). natural forests in Nanping Region, Fujian Province. Soil samples in this study were collected from Fujian Province.

## **2. Materials and Methods**

## *2.1. Sample Collection and Fungal Isolation*

Soil samples were collected from *Eucalyptus* plantations and adjacent plantings, including those of *Cunninghamia lanceolata*, *Phyllostachys heterocycle* and *Pinus massoniana* as well as in natural forests (Figure 1). These plantations and forests were distributed in nine counties or districts in five regions of Fujian Province (one site in Nanping Region, two sites in Fuzhou Region, two sites in Sanming Region, three sites in Longyan Region, one site in Zhangzhou Region) of southern China (Figure 2). These forests typically have thick layers of leaf litter, which was removed before collecting soil samples from the upper 0–20 cm of the humid soil profile. Between three and 37 soil samples (Table 1) were collected randomly at each site. The soil samples were placed in re-sealable plastic bags to maintain moisture and transported to the laboratory for further study.

Soil samples were placed in plastic cups and moistened using distilled water. *Medicago sativa* (alfalfa) seeds were surface-disinfested in 75% ethanol for 30 s and scattered onto the surface of the moistened soil to bait for *Calonectria* spp. as described by Crous [17]. After eight to ten days at 25 ◦C, conidiophores typical of *Calonectria* spp. were observed with a Zeiss Stemi 2000C dissection microscope on the germinating alfalfa plants. Conidial masses were transferred to 2% MEA (Malt Extract Agar) using a sterile needle. After 12 h of incubation at 25 ◦C, single hyphal tips were transferred to fresh MEA plates using a sterile needle and these cultures were incubated at 25 ◦C for seven days. Cultures were sorted based on their morphological characteristics and one to five isolates were retained for each of the soil samples.

Cultures were deposited in the Culture Collection (CSF) at the Research Institute of Fastgrowing Trees (RIFT) (previous institution: China Eucalypt Research Centre, CERC), Chinese Academy of Forestry (CAF), ZhanJiang, Guangdong Province, China. Representative isolates have also been maintained in the China General Microbiological Culture Collection Centre (CGMCC), Beijing, China. Dried specimens were deposited in the Mycological Fungarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS), Beijing, China.

**Figure 2.** *Calonectria* species collected from nine counties (districts) in Fujian Province. (**a**–**i**). the percentage of each species in nine different counties (districts). Different species are indicated by numbers with different colours.



<sup>a</sup> N/A refers to samples that did not yield *Calonectria* isolates.

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

Mycelium was collected from axenic cultures grown on MEA for 5–7 days using a sterilised scalpel. Genomic DNA was extracted from the cultures using the CTAB method "5" described by Van Burik et al. [27]. Partial gene sequences were determined for the actin (*act*), calmodulin (*cmdA*), histone H3 (*his3*), the second largest subunit of RNA polymerase (*rpb2*), translation elongation factor 1-alpha (*tef1*) and β-tubulin (*tub2*) regions. Primer pairs ACT-512F/ACT-783R, CAL-228F/CAL-2Rd, CYLH3F/CYLH3R, fRpb2-5F/fRpb2-7cR, EF1- 728F/EF2 and T1/CYLTUB1R [18] were used to amplify the six gene regions, respectively.

The PCR reaction mixtures contained 17.5 µL TopTaqTM Master Mix, 1 µL of each primer (10 mM), 2 µL DNA sample and RNase-Free H2O to a final volume of 35 µL. The amplifications were conducted under conditions described by Liu and co-authors [18]. All PCR products were sequenced in both directions using the same primers used for amplification. Raw sequences were inspected and manually corrected in Geneious v. 9.1.4 (Biomatters, Auckland, New Zealand) [28]. All sequences generated in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov; accessed on 24 July 2022) (Table 2, Appendix A Table A1).

## *2.3. Phylogenetic Analyses*

To obtain the preliminary identification of the isolates, a standard nucleotide BLAST search was conducted using sequences of the six (*act*, *cmdA*, *his3*, *rpb2*, *tef1* and *tub2*) gene regions. Furthermore, sequences obtained in this study (Table 2) and sequences of other phylogenetically closely related *Calonectria* species downloaded from NCBI (http: //www.ncbi.nlm.nih.gov; accessed on 24 July 2022) (Table 3) were used in the analyses. Sequence alignments were conducted online with MAFFT v. 7 (Suita, Janpan) [29] and were manually adjusted in MEGA v. 6.0.5 software (Auckland, New Zealand) [30] when necessary. The final alignments used in phylogenetic analyses were submitted to TreeBASE (http://treebase.org; accessed on 3 October 2021).

Genotypes of all the isolates were determined based on the sequences for the six gene regions. Representative isolates for all the genotypes were selected for the phylogenetic analyses. All the isolates of the novel species were used in the analyses. Maximum Parsimony (MP) and Maximum Likelihood (ML) approaches were used for phylogenetic analyses. The sequence datasets for the six individual gene regions and a concatenated dataset for those regions were used to determine the phylogenetic relatedness of all the isolates. PAUP v. 4.0 b10 [31] was used to perform the MP analyses, and PhyML v. 3.0 [32] was applied to conduct the ML analyses. A partition homogeneity test (PHT) [33] was performed to assess whether the datasets for the six gene regions could be combined.

For MP analyses, all characters were unordered and equally weighted. Gaps were regarded as fifth character and phylogenetic trees were obtained using a heuristic tree search criterion including 1000 random stepwise additions and tree-bisection-reconstruction (TBR) branch swapping. Branches of zero-length were collapsed. Supports for tree-branching points were determined using bootstrap analyses with 1000 replicates [34]. Tree length (TL), retention index (RI), consistency index (CI), rescaled consistency indexes (RC) and homoplasy index (HI) (Table 4) were calculated for parsimony trees. For ML analyses, the best substitution model for each dataset was determined using JModeltest 2.1.7 [35]. Sequence data for two isolates of *Curvicladiella cignea* (CBS 109167 and CBS 109168) were used as outgroup taxa (Table 3).

**Table 2.** Isolates sequenced in this study and used for phylogenetic analyses and morphological studies.




CSF9794 ABAAAA

CSF9799 ABAAAA

Soil (*Eucalyptus* plantation)

Hua'an, Zhangzhou, Fujian, China

24◦53049.36900 N, 117◦32045.07000 E

S.F. Chen, Q.L. Liu and F.F. Liu

Soil (*Eucalyptus* plantation)

Hua'an, Zhangzhou, Fujian, China

24◦53049.36900 N, 117◦32045.07000 E

S.F. Chen, Q.L. Liu and F.F. Liu

OK253083 OK253194 OK253338 OK253440 OK253671 OK253902

OK253084 OK253195 OK253339 OK253441 OK253672 OK253903










forest area)

Soil (natural forest area)

CSF9978 AAAAAA

CSF9933 g,h;

Soil (*Eucalyptus* plantation)

Xinluo, Longyan, Fujian, China

25◦07008.59700 N, 116◦44042.25700 E

S.F. Chen, Q.L. Liu and F.F. Liu

CGMCC3.18875 ABBABB

CSF9934 ABBABB

Soil (*Eucalyptus* plantation)

Xinluo, Longyan, Fujian, China

25◦07008.59700 N, 116◦44042.25700 E

S.F. Chen, Q.L. Liu and F.F. Liu

Fujian, China

Liancheng, Longyan, Fujian, China

25◦26014.34800 N, 116◦38042.40000 E

and F.F. Liu

S.F. Chen, Q.L. Liu and F.F. Liu

OK253126 OK253264 OK253408 OK253482 OK253819 OK253972

OK253127 OK253265 OK253409 OK253483 OK253820 OK253973

OK253128 OK253266 OK253410 OK253484 OK253821 OK253974


**Table 2.** *Cont.*


*rpb2*, *tef1* and *tub2* regions; '-' means not available. e

i

factor 1-alpha; *tub2 =* β-tubulin. f *N/A* represents sequences that are not available. g

Isolates that represent ex-type cultures are indicated in bold.

*act* = actin; *cmdA* = calmodulin; *his3* = histone H3; *rpb2* = the second largest subunit of RNA polymerase; *tef1* = translation elongation

Isolates used in morphological and culture growth studies. h

Isolates used for mating studies.


**Table 3.** Isolates from other studies and used in the phylogenetic analyses.






<sup>a</sup> Codes (B1 to B120) of the 120 accepted *Calonectria* species resulting from Liu and co-authors [18]. <sup>b</sup> *ATCC* = American Type Culture Collection, Virginia, USA; *CBS* = Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; *CERC* = China Eucalypt Research Centre, ZhanJiang, Guangdong Province, China; *CGMCC* = China General Microbiological Culture Collection Center, Beijing, 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; *CSF* = Culture Collection from Southern Forests (CSF), ZhanJiang, Guangdong Province, China; *IMI* = International Mycological Institute, CABI Bioscience, Egham, Bakeham Lane, UK; *MUCL* = Mycotheque, Laboratoire de Mycologie Systematique st Appliqee, I'Universite, Louvian-la-Neuve, Belgium; *PPRI* = Plant Protection Research Institute, Pretoria, South Africa; *STE-U* = Department of Plant Pathology, University of Stellenbosch, South Africa; '–' represent no other collection number. <sup>c</sup> *T* = ex-type isolates of the species. <sup>d</sup> *act* = actin; *cmdA* = calmodulin; *his3* = histone H3; *rpb2* = the second largest subunit of RNA polymerase; *tef1* = translation elongation factor 1-alpha; *tub2* = β-tubulin. <sup>e</sup> *N/A* represents information not available.

**Table 4.** Statistics resulting from phylogenetic analyses in this study.



a *bp* = Base pairs. <sup>b</sup> *PIC* = Number of parsimony informative characters. <sup>c</sup> *CI* = Consistency index. <sup>d</sup> *RI* = Retention

index. <sup>e</sup> *RC* = Rescaled consistency index. <sup>f</sup> *HI* = Homoplasy index. <sup>g</sup> *Subst. model* = best fit substitution model. <sup>h</sup> *NST* = Number of substitution rate categories.

## *2.4. Sexual Compatibility*

The mating system as either homothallic or heterothallic was determined for the novel species identified in this study. Representative isolates of this species were crossed with each other in all possible combinations. These crosses were made on minimum salt agar (MSA) [54] with autoclaved toothpicks randomly placed on the agar surface. Petri dishes were then incubated at 25 ◦C for 2–8 wk, and they were observed regularly for the appearance of perithecia. When perithecia extruding ascospores emerged, germination tests were conducted to determine if the spores were viable. Production of viable ascospores was accepted as an indication of successful mating.

## *2.5. Morphology*

Representative isolates of the novel species identified in this study were selected for morphological characterisation. Synthetic nutrient-poor agar (SNA) [55] was used to induce the asexual morphs. Agar plugs from axenic cultures were transferred to SNA and incubated at 25 ◦C for seven days. Fungal structures were lifted from the plates using a sterile needle and transferred to a drop of 85% lactic acid on microscope slides. Microscopic structures were examined under a Zeiss Axio Imager A1 microscope (Carl Zeiss Ltd., Jena, Germany).

In the case of sexual structures, the perithecia were transferred to Jung tissue freezing medium (Leica Biosystems, Wetzlar, Germany), which was frozen at −20 ◦C for ten minutes. Vertical sections (10 µm thick) were cut through the perithecia on a HM550 cryostat microtome (Microme International GmbH, Termo Fisher Scientifc, Walldorf, Germany) at −20 ◦C and examined under an Axio Imager A1 microscope.

For cultures selected as the ex-type isolates, 50 replicate measurements were made for each taxonomically characteristic structure. For other isolates, 30 replicate measurements were made. Minimum, maximum and average (mean) measurements were recorded as (minimum–) (average–standard deviation)–(average + standard deviation) (–maximum).

Optimal growth temperatures for the novel species were determined on MEA. Agar plugs were removed from the actively growing edges of 7-day-old cultures with a 5 mm diam. cork borer and transferred to the centres of 90 mm Petri dishes containing MEA. Cultures were grown at seven different temperatures ranging from 5 ◦C to 35 ◦C, at 5 ◦C intervals with five replicates per isolate. Colony diameters were measured after seven days. Colony colours were described using the colour charts of Rayner [56] using seven-dayold cultures on MEA incubated at 25 ◦C. All descriptions were deposited in MycoBank (www.mycobank.org, accessed on 3 October 2021).

## **3. Results**

## *3.1. Sample Collection and Fungal Isolation*

A total of 209 soil samples were collected and 353 isolates having a morphology typical of *Calonectria* were isolated from 79 of these samples (Table 1, Appendix A Table A1). Of these, 121 soil samples were from seven *Eucalyptus* plantations, of which 57 samples yielded 253 *Calonectria* isolates. Forty-three soil samples were collected from four natural forests, of which 14 samples yielded 61 *Calonectria* isolates; 21 soil samples were collected from two *C. lanceolata* plantations, two of which yielded nine *Calonectria* isolates; and 14 soil samples collected from a single *P. heterocycle* plantation, of which five samples yielded 25 *Calonectria* isolates. In addition, ten soil samples were collected from the *Pi. massoniana* plantation, only one of which yielded five *Calonectria* isolates (Table 1).

## *3.2. Phylogenetic Analyses*

The *tef1* fragment was amplified for all of the 353 isolates (Appendix A Table A1), and based on sequence differences for this region and the sampling sites, 144 isolates were selected to amplify the *cmdA*, *his3* and *tub2* gene regions. Subsequently, based on the 37 genotypes revealed by these four gene regions, 71 representative isolates were chosen to amplify the *act* and *rpb2* gene regions (Appendix A Table A1). All of the 71 isolates, representing the 40 genotypes determined from the sequence data for the six gene regions, were used for phylogenetic inference (Table 2). Amplicons generated for the *act*, *cmdA*, *his3*, *rpb2*, *tef1*, and *tub2* gene regions were approximately 300, 700, 500, 860, 550, and 600 bp, respectively.

Sequence data for 46 *Calonectria* species closely related to those collected in this study were downloaded from GenBank and a total of 78 sequences (for ex-type and other strains) from previous studies were included in the phylogenetic analyses (Table 3). Phylogenetic analyses based on the six individual gene regions and the concatenated dataset for those regions were conducted using both MP and ML methods. The results showed that the overall topologies generated from the MP analyses were essentially similar to those from the ML analyses, and consequently, only the ML trees are presented (Figure 3, Appendix B Figures A1–A6).

**Figure 3.** *Cont*.

**Figure 3.** Phylogenetic tree of *Calonectria* species based on maximum likelihood (ML) analyses of combined DNA dataset of *act*, *cmdA*, *his3*, *rpb2*, *tef1*, and *tub2* gene sequences. Bootstrap value ≥70% for ML and MP analyses are presented above the branches. Bootstrap values lower than 70% are marked with "\*", and absent analyses values are marked with "-". Ex-type isolates are marked with "T". Isolates sequenced in this study are highlighted in blue and bold type. The "B" species codes are consistent with the recently published results in Liu and co-authors [18]. The tree was rooted to *Curvicladiella cignea* (CBS 109167 and CBS 109168).

The partition homogeneity test carried out on the datasets, for the combined six gene regions, generated *p* values of 0.001. This showed that the accuracy of the combined data did not suffer relative to the individual partitions [57]. Sequence data for the six gene regions were thus combined for analyses. The sequence alignments based on the individual six gene regions and the combination of these were deposited in TreeBASE (No. S28845). Statistics and important parameters emerging from the phylogenetic analyses are presented in Table 4.

Based on the six-gene combined phylogenetic tree (Figure 3), for the 71 isolates used in the phylogenetic analyses, eight isolates resided in the *Ca*. *colhounii* species complex, two isolates in the *Ca*. *reteaudii* species complex and 61 isolates in the *Ca*. *kyotensis* species complex.

## *3.3. Species in the Calonectria colhounii Species Complex*

Six isolates (CSF9941, CSF9974, CSF9975, CSF9976, CSF9977 and CSF9978), representing one genotype, formed a distinct lineage in the *cmdA* and *tub2* analyses as well as in the six-gene combined phylogenetic tree (Figure 3, Appendix B Figures A2 and A6). The total number of SNP differences between the six isolates and other phylogenetically closely related species [*Ca. aciculata* (ex-type isolate CERC 5342), *Ca. colhounii* (ex-type isolate CBS 293.79), *Ca. eucalypti* (ex-type isolate CMW 18444) and *Ca. honghensis* (ex-type isolate CERC 5572)] for six gene regions combined, varied between 13 and 31. Thus, this fungus can be regarded as a novel species. Two isolates (CSF9933 and CSF9934) formed an independent clade and were phylogenetically most closely related to the six isolates in the six-gene phylogenetic tree (Figure 3). These two isolates were consequently considered as the same species as the six isolates CSF9941, CSF9974, CSF9975, CSF9976, CSF9977 and CSF9978 and were identified as the novel species.

## *3.4. Species in the Calonectria reteaudii Species Complex*

Two isolates (CSF10059 and CSF10060) were phylogenetically closely related to *Ca. pseudoreteaudii* and various other species based on *act* and *cmdA* trees (Appendix B Figures A1 and A2), and clustered with *Ca. pseudoreteaudii* based on *his3*, *rpb2*, *tef1*, *tub2* and the six-gene combined trees (Figure 3, Appendix B Figures A3–A6). In comparisons of DNA sequences for these six gene regions, all the sequences for the two isolates (CSF10059 and CSF10060) were 100% identical to the ex-type isolate (CMW 25310) of *Ca. pseudoreteaudii*. Consequently, they were identified as *Ca. pseudoreteaudii* (Figure 3).

## *3.5. Species in the Calonectria kyotensis Species Complex*

Thirty-four isolates representing 20 genotypes were phylogenetically closest to *Ca. kyotensis* in each of the *cmdA*, *his3*, *rpb2* (sequence data for the *rpb2* were not available for isolate CSF9834), *tef1*, *tub2* and the six-gene combined trees (Figure 3, Appendix B Figures A2–A6), and clustered with *Ca. kyotensis* based on the *act* tree (Appendix B Figure A1). Some isolates formed distinct clades based on the six-gene combined trees (Figure 3), while the total number of SNP differences between the 34 isolates and the extype isolate of *Ca. kyotensis* (CBS 114525) for six gene regions combined varied between 2 and 8. Based on the phylogenetic analyses, these 34 isolates were identified as *Ca. kyotensis*.

Four isolates (CSF7124, CSF9784, CSF9794 and CSF9799), representing two genotypes, were phylogenetically closest to *Ca. hongkongensis* in each of the *cmdA*, *tub2* and six-gene combined tree (Figure 3, Appendix B Figures A2 and A6), and clustered with *Ca. hongkongensis* based on *act*, *his3*, *rpb2* and *tef1* trees (Appendix B Figures A1 and A3–A5). There were only three or four SNP differences between these four isolates and the ex-type isolate of *Ca. hongkongensis* (CBS 114828) when sequences for six gene regions were combined. Thus, these four isolates were identified as *Ca. hongkongensis*.

Two isolates (CSF9862 and CSF9863), representing one genotype clustered with *Ca. ilicicola* in the *his3* tree (Appendix B Figure A3), formed independent clades but closely related to *Ca. ilicicola* in the *act*, *cmdA*, *rpb2*, *tef1* and six-gene combined trees (Figure 3, Appendix B Figures A1, A2 and A4–A6). There were only six SNP differences between the

two isolates and the ex-type isolate of *Ca. ilicicola* (CMW 30998) for five gene regions (*tub2* sequence data were not available for *Ca. ilicicola*) combined. Consequently, these isolates were regarded as *Ca. ilicicola*.

Four isolates (CSF10024, CSF10070, CSF10077 and CSF10129), representing three genotypes, were phylogenetically related to *Ca. pacifica* and various other closely related species based on *act* and *tef1* trees (Appendix B Figures A1 and A5). They were, however, phylogenetically closest to *Ca. pacifica* based on *his3* and six-gene combined trees (Appendix B Figure A3), and clustered with *Ca. pacifica* based on *cmdA* and *rpb2* trees (Appendix B Figures A2 and A4). There were only one or three SNP difference(s) between the four isolates and the ex-type isolate of *Ca. pacifica* (CMW 16726) for five gene regions (*tub2* sequence data were not available for *Ca. pacifica*) combined. These four isolates were thus identified as *Ca. pacifica*.

Seventeen isolates representing 11 genotypes were phylogenetically closest to *Ca. aconidialis* based on *cmdA*, *his3*, *tef1* and six-gene combined trees (Figure 3, Appendix B Figures A2, A3 and A5), and clustered with *Ca. aconidialis* based on *act* and *rpb2* (*rpb2* sequence data were not available for CSF9779 and CSF9875) trees (Appendix B Figures A1 and A4). Some isolates formed distinct clades based on the six-gene combined trees (Figure 3), while the total number of SNP differences between the 17 isolates and the ex-type isolate of *Ca. aconidialis* (CMW 35174) for five gene regions (sequence data for the *tub2* region were not available for *Ca. aconidialis*) combined varied between 0 and 4. Therefore, the 17 isolates were identified as *Ca. aconidialis*.

Seventy-one of the 353 isolates collected in this study were identified based on the DNA sequence of the six gene regions. According to the species identification results, we further identified the remaining 282 isolates based on the DNA sequences for two or four gene regions (Appendix A Table A1). Consequently, for the entire collection of 353 isolates, these were identified as *Ca. aconidialis* (178), *Ca. kyotensis* (103), *Ca. hongkongensis* (37), *Ca. pacifica* (17), *Ca. ilicicola* (five), *Ca. pseudoreteaudii* (five) and a novel species (eight), respectively.

## *3.6. Sexual Compatibility*

Three isolates (CSF9933, CSF9941 and CSF9975) of the novel species were used in the mating tests (Table 2). All of these isolates formed protoperithecia readily within two weeks, and perithecia with viable ascospores were produced within four weeks. This was irrespective of whether they were crossed with each other or with themselves. The species was thus shown to be homothallic.

## *3.7. Morphology and Taxonomy*

Based on multi-gene phylogenetic analyses (Figure 3, Appendix B Figures A1–A6) and morphological characteristics, seven *Calonectria* species were identified in this study, including six described species, i.e., *Ca. aconidialis*, *Ca. kyotensis*, *Ca. hongkongensis*, *Ca. pacifica*, *Ca. ilicicola*, *Ca. pseudoreteaudii* and one novel species. To facilitate future studies, complete morphological descriptions and illustrations have been made for the known species and these are presented in Appendix C (Figures A7–A12). The novel species can be distinguished from the phylogenetically most closely related species (*Ca. aciculata*, *Ca. colhounii*, *Ca. eucalypti* and *Ca. honghensis*) by the dimensions of its macroconidia and ascospores (Table 5). This species is described as follows:


**Table 5.** Morphological comparisons of *Calonectria* species obtained in this study and other phylogenetically closely related species.

<sup>a</sup> All measurements are in <sup>µ</sup>m. <sup>b</sup> <sup>L</sup> <sup>×</sup> W = length <sup>×</sup> width. <sup>c</sup> Measurements are presented in the format [(minimum–) (average–standard deviation)–(average + standard deviation) (–maximum)]. <sup>d</sup> N/A represents data that is not available.

## Taxonomy

## *Calonectria minensis* Q.L. Liu and S.F. Chen, sp. nov.

MycoBank MB841412. (Figure 4).

Etymology: Name refers to the short name of Fujian Province in Chinese "Min", where this fungus was isolated.

Diagnosis: *Calonectria minensis* can be distinguished from the phylogenetically closely related species *Ca. aciculata*, *Ca. colhounii*, *Ca. eucalypti* and *Ca. honghensis* by its distinct ascospore and macroconidia dimensions.

Type: China: Fujian Province, Longyan Region, Xinluo District (25◦07008.59700 N, 116◦44042.25700 E), from soil collected in a *Eucalyptus* plantation, 6 November 2016, *S.F. Chen*, *Q.L. Liu* and *F.F. Liu* (HMAS249935—–holotype, CSF9941 = CGMCC3.18877—ex-type culture).

Description: *Ascomata* perithecial, solitary or in groups of four, bright yellow, becoming orange with age; in section, apex and body yellow, base red-brown, sub-globose to ovoid, 258–395 µm high, 227–330 µm diam, body turning dark yellow, and base dark red-brown in 3% KOH+; ascomatal wall rough, consisting of two thick-walled layers; outer layer of *textura globulosa*, 22–66 µm thick, cells becoming more compressed towards the inner layer of *textura angularis*, 9–21 µm thick, cells becoming thin-walled and hyaline towards the centre; outermost cells 16–31 × 8–16 µm, cells of inner layer 8–33 × 2–8 µm; ascomatal base up to 196 µm wide, consisting of dark red, angular cells, merging with an erumpent stroma; cells of the outer wall layer continuous with the pseudoparenchymatous cells of the erumpent stroma. *Asci* 4-spored, clavate, 80–163 × 11–27 µm, tapering into a long thin stalk. *Ascospores* aggregated in the upper third of the ascus, hyaline, guttulate, fusoid with rounded ends, straight to slightly curved, (1–)3-septate, constricted at the septum, (38.5–)46.5–64.5(–80.5) × (6–)6.5–8(–8.5) µm (av. = 55.5 × 7 µm). *Macroconidiophores* consisting of a stipe, a suite of penicillately arranged fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, 33–144 × 4–9 µm, stipe extension septate, straight to flexuous 63–240 µm long, 2–3 µm wide at the apical septum, terminating in a clavate vesicle, 3–5 µm diam; lateral stipe extensions (90◦ to main axis) absent. *Conidiogenous apparatus* 28–97 µm wide, and 35–83 µm long; primary branches aseptate, 13–40 × 3–7 µm; secondary branches aseptate, 9–31 × 3–6 µm; tertiary branches aseptate, 8–14 × 3–5 µm, quaternary branches aseptate, 7−12 × 3–5 µm, each terminal branch producing 2–4 phialides; phialides allantoid to elongate doliiform to reniform, hyaline, aseptate, 4–14 × 2–7 µm, apex with minute periclinal thickening and inconspicuous collarette. *Macroconidia* cylindrical, rounded at both ends, straight, (51–)55–66(–79) × (4.5–)5–6(–7.5) µm (av. = 60.5 × 5.5 µm), (1–)3-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

**Figure 4.** *Calonectria minensis*. (**a**) Perithecium; (**b**) vertical section through a perithecium; (**c**) cells around ostiolar region of perithecium; (**d**) section through lateral perithecial wall; (**e**,**f**) asci; (**g**,**h**) ascospores; (**i**,**j**) macroconidiophore; (**k**,**m**) clavate vesicles; (**n**,**o**) conidiogenous apparatus with conidiophore branches and elongate doliiform to reniform phialides; (**p**,**q**) macroconidia.—Scale bars: a = 200 µm; b = 100 µm; c, d and f = 20 µm; e and i, j = 50 µm; g, h and n–q = 10 µm; k, m = 5 µm.

Culture characteristics: Colonies forming abundant woolly white to sienna (8) aerial mycelium at 25 ◦C on MEA, profuse sporulation; surface rust-coloured (39); reverse sienna (8) to rust-coloured (39) after 7 d. Chlamydospores extensive throughout the medium forming microsclerotia. Optimal growth temperature 25 ◦C, no growth at 5 ◦C and 35 ◦C, after 7 d, colonies at 10 ◦C, 15 ◦C, 20 ◦C, 25 ◦C and 30 ◦C reached 18.1 mm, 27.0 mm, 58.2 mm, 69.5 mm and 42.4 mm, respectively.

Additional specimens examined: China: Fujian Province, Longyan Region, Xinluo District (25◦07008.59700 N, 116◦44042.25700 E), from soil collected in a *Eucalyptus* plantation, 6 November 2016, S.F. Chen, Q.L. Liu and F.F. Liu (HMAS249936, culture CSF9933 = CGMCC3.18875); Fujian Province, Longyan Region, Liancheng County (25◦26014.34800 N, 116◦38042.40000 E), from soil under a natural forest, 6 November 2016, S.F. Chen, Q.L. Liu and F.F. Liu (HMAS249937, culture CSF9975 = CGMCC3.18881).

Notes: *Calonectria minensis* is a new species in the *Ca. colhounii* species complex. It is closely related to *Ca. aciculata*, *Ca. colhounii*, *Ca. eucalypti*, and *Ca. honghensis*, and can be distinguished from those species by the dimensions of its ascospores and macroconidia. The ascospores of *Ca. minensis* (av. = 55.5 × 7 µm) are larger than those of *Ca. eucalypti* (av. = 33 × 6 µm) [37] and *Ca. honghensis* (av. = 49 × 6 µm) [4]. The macroconidia of *Ca. minensis* (av. = 60.5 × 5.5 µm) are shorter than those of *Ca. aciculata* (av. = 69 × 5.5 µm) [4], *Ca. colhounii* (av. = 65 × 5 µm) [17] and *Ca. eucalypti* (av. = 72 × 6 µm) [37], but longer than those of *Ca. honghensis* (av. = 54 × 5.5 µm) [4]. The total number of SNP differences between the ex-type isolate of *Ca. minensis* (CSF9941), and the ex-type isolates of *Ca. aciculata* (CERC 5342), *Ca. colhounii* (CBS 293.79), *Ca. eucalypti* (CMW 18444) and *Ca. honghensis* (CERC 5572) for six gene regions combined, varied between 13 and 31.

## *3.8. Distribution of Calonectria Species in Fujian Province*

Of the seven *Calonectria* species identified, *Ca. aconidialis* accounted for 50.4% of all the isolates. This was followed in order of occurrence by *Ca. kyotensis* (29.2%), *Ca. hongkongensis* (10.5%), *Ca. pacifica* (4.8%), *Ca. minensis* (2.3%), *Ca. ilicicola* (1.4%) and *Ca. pseudoreteaudii* (1.4%) (Figure 5). *Calonectria aconidialis* and *Ca. kyotensis* can be regarded as the most prevalent species (Figure 5).

**Figure 5.** *Calonectria* species collected from soils of five different types of forests in Fujian Province. (**a**). the percentage of each *Calonectria* species accounted for all of the species isolated in this study. Different species are indicated by numbers with different colours; (**b**–**f**). the percentage of each *Calonectria* species obtained from five different types of forests.

Between two and four *Calonectria* species were isolated from soils sampled at each of the nine Counties or Districts (Figure 2). *Calonectria aconidialis* was found at all sites other than Cangshan District, *Ca. kyotensis* was found at all sites other than Yanping District and Zhangping County, and the remaining five species were found at between one and three sampling sites (Figure 2).

All seven species were isolated from soils collected in *Eucalyptus* plantations. Five of the species were isolated from soils in natural forests, the exception being *Ca. ilicicola* and *Ca. pesudoreteaudii*. Only *Ca. aconidialis* and *Ca. kyotensis* were isolated from soils in *P. heterocycle* and *C. lanceolata* plantations, and only *Ca. kyotensis* was collected from soils in the *Pi. massoniana* plantation (Figure 5). Based on the percentage of soil samples that

obtained *Calonectria* from each of the five forest types, the results showed that *Calonectria* was widely distributed in *Eucalyptus* plantation soils (47.1%, 57 of 121 sampled soils), followed by *P. heterocycle* (35.7%, 5 of 14 sampled soils) and natural forests (32.6%, 14 of 43 sampled soils), only 10% of soil samples obtained *Calonectria* from *C. lanceolata* (2 of 21 sampled soils) or *Pi. massoniana* (1 of 10 sampled soils).

*Calonectria kyotensis* was detected in soils in all of the soil types sampled, while *Ca. aconidialis* was isolated from soils in all forest types other than *Pi. massoniana*. *Calonectria hongkongensis*, *Ca. pacifica* and *Ca. minensis* were found both in *Eucalyptus* plantations and natural forests and the remaining two species were found only in *Eucalyptus* plantations (Figure 5).

## **4. Discussion**

A total of 353 *Calonectria* isolates were collected from soils in *Eucalyptus* plantations and adjacent plantations of other species or natural forests in Fujian Province. Multilocus phylogenetic inference and morphological characteristics revealed seven *Calonectria* species including *Ca. aconidialis*, *Ca. hongkongensis*, *Ca. ilicicola*, *Ca. kyotensis*, *Ca. pacifica* and *Ca. pseudoreteaudii*, and a novel species described here as *Ca. minensis*.

Results in this study showed that *Ca. aconidialis* and *Ca. kyotensis* were the most prevalent species in the soils sampled. *Calonectria aconidialis* accounted for 50.4% of all the isolates, which was found in eight of the nine sampled sites and soils of all forest types other than those of *Pi. massoniana*. The next most common species was *Ca. kyotensis*, accounting for 29.2% of the isolates, which was isolated from seven sites and soils of all five different forest types. The remaining five species were less common, and isolated only from one to three sites, either from *Eucalyptus* plantations or natural forests, or from both of these forest types.

Among the identified species, *Ca. aconidialis* is newly reported in Fujian Province and *Ca. pacifica* represents a first record for China. Eight *Calonectria* species were previously known in Fujian Province. These include *Ca. crousiana*, *Ca. eucalypti*, *Ca. fujianensis*, *Ca. pauciramosa* and *Ca. pseudoreteaudii* collected from diseased *Eucalyptus* leaves [7,8], *Ca. hongkongensis* and *Ca. kyotensis* isolated from soils in unknown forest types [4,18] and *Ca. ilicicola* collected from diseased peanuts (*Arachis hypogaea*) in Longyan Region [58].

The *Calonectria* species diversity in soils was clearly dependent on the forest types sampled. Of the seven species detected, all were obtained from *Eucalyptus* plantations, five were obtained from natural forests and only one or two species were from other forest types. While these observations are convincing in terms of broad patterns, they must be tempered by the fact that the greatest number of soil samples were from *Eucalyptus* plantations and natural forests, which could have influenced the results.

The newly described *Ca. minensis* isolated from soils both in *Eucalyptus* plantations and natural forest, adds a new species to the *Ca. colhounii* species complex. As a consequence, 13 species are now accommodated in this complex [4,7,17,18,21,25,37,39,46,49]. With the exception of *Ca. macroconidialis* [46], *Ca. madagascariensis* [17] and *Ca. paracolhounii* [39], all of the other 10 species have been recorded in southeastern Asia [4,7,17,21,25]. Species in this complex include some important causal agents of CLB on *Eucalyptus* spp. including *Ca. aciculata*, *Ca. eucalypti* and *Ca. fujianensis*, which have all been reported from diseased *Eucalyptus* trees in China plantations [4,7].

Five species residing in the *Ca. kyotensis* species complex were identified in the present study. Of these, *Ca. aconidialis* accounted for more than half of all the isolates collected, and has previously been shown to be widely distributed in soils of *Eucalyptus* plantation in many regions of southern China, including Guangdong [11,18], Guangxi [4,10,11] and Hainan Provinces [11]. In the present study, *Ca. aconidialis* was collected from soils of four types of forests and in eight of the nine sampling sites in Fujian Province (Figure 2), providing new geographic records for this pathogen in China. This species has previously been shown to infect inoculated *Eucalyptus* seedlings [10] and could pose a threat to *Eucalyptus* plantation forestry. *Calonectria pacifica* was isolated from soils both in the *Eucalyptus* plantations (Minhou and Yongan Counties) and natural forests (Yanping District) in this study. This species was originally described on *Araucaria heterophylla* from Hawaii, USA [40], and this is the first report of the fungus in China.

This study elucidated the diversity and distribution characteristics of *Calonectria* species in soils collected from plantations and natural forests in Fujian Province. Broad patterns of occurrence were clear with *Eucalyptus* soils yielding the largest number of species. The conifer forests had the lowest number of species, which is consistent with the fact that most *Calonectria* spp. are known from Angiosperm hosts or from soils associated with these plants. The results of the present study bring the number of *Calonectria* species recorded in Fujian to 11. Most of these species have also been shown to be pathogenic to *Eucalyptus* in previous studies [7,9,10]. The surprisingly high species diversity in this region suggests that *Calonectria* species will pose long-term challenges for the development of *Eucalyptus* forestry in southern China.

**Author Contributions:** Conceptualization, Q.L. and S.C.; methodology, Q.L. and S.C.; software, Q.L.; validation, Q.L., M.J.W., T.A.D., B.D.W. and S.C.; formal analysis, Q.L.; investigation, Q.L. and S.C.; resources, Q.L. and S.C.; data curation, Q.L. and S.C; writing—original draft preparation, Q.L.; writing—review and editing, Q.L., M.J.W., T.A.D., B.D.W. and S.C.; visualization, Q.L.; supervision, M.J.W., T.A.D., B.D.W. and S.C.; project administration, S.C.; funding acquisition, S.C. 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 special fund for basic scientific research of State Key Laboratory of Tree Genetics and Breeding (SKLTGB) of China (project No. TGB2017001), the National Ten-thousand Talents Program (Project No. W03070115) and the Guangdong Top Young Talents Program in China (Project No. 20171172).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The sequences from the current study were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 24 July 2022) and the accession numbers were listed in Table 2.

**Acknowledgments:** We thank FeiFei Liu for her assistance in collecting soil samples.

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