Two novel species of Calonectria isolated from soil in a natural forest in China

Abstract Species of Calonectria include important pathogens of numerous agronomic and forestry crops worldwide, and they are commonly distributed in soils of tropical and subtropical regions of the world. Previous research results indicated that species diversity of Calonectria in China is relatively high. Most Calonectria spp. reported and described from China were obtained from diseased Eucalyptus tissues or soils in Eucalyptus plantations established in tropical and subtropical areas in southern China. Recently, a number of Calonectria isolates were isolated from soils in a natural forest in the temperate region of central China. These isolates were identified by DNA sequence comparisons for the translation elongation factor 1-alpha (tef1), histone H3 (his3), calmodulin (cmdA) and β-tubulin (tub2) gene regions, combined with morphological characteristics. Two novel species of Calonectria were identified and described, and are named here as Calonectria lichi and Ca. montana, which reside in the Prolate Group and Sphaero-Naviculate Group, respectively. This study revealed that more species of Calonectria may occur in natural forests in central China than previously suspected.


Introduction
Calonectria species include many notorious plant pathogens and are widely distributed in tropical and subtropical areas of the world (Crous 2002, Lombard et al. 2010d, Aiello et al. 2013, Vitale et al. 2013, Alfenas et al. 2015).These species can cause serious plant epidemics on a wide range of plant hosts (Peerally 1991, Schoch et al. 2001, Crous 2002), and result in considerable economic losses to agriculture and forestry.Example include shoot blight on Pinus spp. in South African nurseries (Crous et al. 1991), root rot on Myrtus communis in Tunisia (Lombard et al. 2011), and leaf blight on Buxus sempervirens in Iran (Mirabolfathy et al. 2013).In addition, members of the genus Calonectria are responsible for red crown rot of Glycine max (soybean) in Japan (Yamamoto et al. 2017), fruit rot of Nephelium lappaceum (rambutan) in Puerto Rico (Serrato-Diaz et al. 2013) and root rot of Arbutus unedo (strawberry) in Italy (Vitale et al. 2009).As an important fast-growing tree species, Eucalyptus plays a significant role in the global pulpwood supply.Previous research showed that Calonectria leaf blight (CLB), associated with several species of Calonectria, is considered to be one of the most prominent Eucalyptus leaf diseases that has occurred in numerous countries such as Brazil (Alfenas et al. 2015, Lombard et al. 2016), China (Zhou et al. 2008, Chen et al. 2011), Colombia (Rodas et al. 2005), India (Sharma et al. 1984) and Vietnam (Old et al. 1999).Other fungal diseases of Eucalyptus spp.caused by Calonectria species include dampingoff, shoot blight, and root rot, which have been observed in Brazil (Ferreira 1989) and South Africa (Crous et al. 1991), and these diseases have received considerable attention.
Calonectria spp.are soil-borne fungi, they can form microsclerotia in soil and infected plant roots, stem and leaves as primary inoculum.After diseased tissues decompose or the plants are harvested, microsclerotia are released into the soil, which allows them to survive for extended periods even up to 15 years or more (Sobers andLittrell 1974, Crous 2002).Species of Calonectria are also rapidly dispersed via aerial dissemination and water movement, which leads to the transmission of Calonectria disease (Vitale et al. 2013).Based on previous studies, at least 145 Calonectria species have been identified using molecular data and have been described worldwide (Crous 2002, Crous et al. 2004, 2006, 2012, 2013, 2015, Lombard et al. 2010a, b, c, 2011, 2015, 2016, Chen et al. 2011, Xu et al. 2012, Alfenas et al. 2013a, b, 2015, Gehesquière et al. 2015).Sixty species were isolated from soil samples collected in subtropical or tropical regions (Crous 2002, Crous et al. 2004, Lombard et al. 2010a, b, c, 2015, 2016, Chen et al. 2011, Xu et al. 2012, Alfenas et al. 2015).
In China, Calonectria has a relatively high species diversity, and to date, 28 Calonectria species have been identified and described.Based on previous studies, Calonectria species have been reported in nine provinces and one Special Administrative Region (SAR), which with the exception of LiaoNing and ShanDong Provinces belong to temperate regions (Luan et al. 2006, Li et al. 2010).Most Calonectria have been isolated from agronomic crops or forestry plantations in subtropical and tropical regions, including FuJian, GuangDong, GuangXi, GuiZhou, HaiNan, JiangXi and YunNan Provinces, as well as Hong Kong SAR (Crous et al. 2004, Lombard et al. 2010a, 2015, Chen et al. 2011, Gai et al. 2012, Xu et al. 2012, Pei et al. 2015).
China has large areas of plantation and natural forests.To date 27 Calonectria species have been isolated from Eucalyptus tissues with CLB/leaf rot symptoms or from soils originating from Eucalyptus plantations in tropical or subtropical areas in Fu-Jian, GuangDong, GuangXi and HaiNan Provinces (Crous et al. 2004, Lombard et al. 2010a, 2015, Chen et al. 2011).However, little information is known about the species diversity of Calonectria in natural forests.In this study, a number of soil samples were collected from a natural forest in the temperate region of central China, and baited with alfalfa seeds for Calonectria.The aim of the current study was to identify these isolates using a combination of phylogenetic analyses and morphological characteristics and to gain a preliminary understanding of the species diversity of Calonectria in natural forests in China.

Fungal isolates
In April 2016, 17 soil samples were collected from a natural forestry area in central China.The collected soils were baited with surface-disinfested (30 s in 75% ethanol and washed several times with sterile water) Medicago sativa (alfalfa) seeds using the method described by Crous (2002).After one week, sporulating conidiophores were produced on infected alfalfa tissue.Using a dissection microscope AxioCam Stemi 2000C (Carl Zeiss, Germany), conidial masses were selected and 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 sterile needles.After incubation at 25 °C for one day, germinated spores were individually transferred onto fresh MEA under the dissection microscope and were incubated at 25 °C for one week.
Single conidial cultures were deposited in the Culture Collection of the China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), ZhanJiang, GuangDong Province, China.Representative isolates were stored in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China.The specimens (pure fungal cultures) were deposited in the Collection of Central South Forestry Fungi of China (CSFF), GuangDong Province, China.

DNA extraction, PCR and sequence reactions
Single conidial cultures grew on MEA for one week at 25 °C, after which actively growing mycelium was scraped using a sterilized scalpel and transferred into 2 mL Eppendorf tubes.Total genomic DNA was extracted following the protocols "Extraction method 5: grinding and CTAB" described by Van Burik et al. (1998).The extracted DNA was dissolved in 30 µL TE buffer (1 M Tris-HCl and 0.5 M EDTA, pH 8.0), and a Nano-Drop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the concentration.
Amplified fragments were sequenced in both directions using the same primer pairs used for amplification by the Beijing Genomics Institute, Guangzhou, China.Sequences were edited using MEGA v. 6.0.5 software (Tamura et al. 2013).All sequences of the isolates obtained in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov)(Table 1).

Phylogenetic analyses
The sequences generated from this study were added to other sequences of closely related Calonectria species downloaded from GenBank for phylogenetic analyses.All sequences used in this study were aligned using the online MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server) with the alignment strategy FFT-NS-i (Slow; interactive refinement method).The aligned sequences were manually edited using MEGA v. 6.0.5 and were deposited in TreeBASE (http://treebase.org).
Phylogenetic analyses were conducted on individual tef1, his3, cmdA and tub2 sequence datasets and on the combined datasets for the four gene regions, depending on the sequence availability.Two methods, maximum parsimony (MP) and maximum likelihood (ML) were used for phylogenetic analyses.
MP analyses were performed using PAUP v. 4.0 b10 (Swofford 2003), gaps were treated as a fifth character, and characters were unordered and of equal weight with 1000 random addition replicates.A partition homogeneity test (PHT) was conducted to determine whether data for the four genes could be combined.The most parsimonious trees were acquired using the heuristic search option with stepwise addition, tree bisection, and reconstruction branch swapping.MAXTREES was set to 5,000, and zero-length branches were collapsed.A bootstrap analysis (50% majority rule, 1,000 replicates) was carried out to determine statistical support for internal nodes in trees.The tree length (TL), consistency index (CI), retention index (RI) and homoplasy index (HI) were used to assess phylogenetic trees (Hillis and Huelsenbeck 1992).ML analyses were performed using PHYML v. 3.0 (Guindon and Gascuel 2003), and the best evolutionary model was obtained using JMODELTEST v. 2.1.5(Posada 2008).In PHYML, the maximum number of retained trees was set to 1,000, and nodal support was determined by non-parametric bootstrapping with 1,000 replicates.
Based on the morphological characteristics, datasets were separated into two groups: the Prolate Group and the Sphaero-Naviculate Group (Lombard et al. 2010b), and therefore phylogenetic analyses were performed with two separate sequence datasets.Calonectria hongkongensis (CBS 114711 and CBS 114828) and Ca.pauciramosa (CMW 5683 and CMW 30823) represented the outgroup taxa for the Prolate Group and Sphaero-Naviculate Group, respectively.The phylogenetic trees were viewed using MEGA v. 6.0.5 for both MP and ML analyses.

Sexual compatibility
Based on multi-gene phylogenetic analyses, isolates of each identified Calonectria species were crossed with each other in all possible combinations.Crosses were performed on minimal salt agar (MSA; Guerber and Correll 2001) on the surface of the medium using three sterile toothpicks.Isolates crossed with themselves were regarded as controls.These crosses were used to determine whether the identified species had a heterothallic or a homothallic mating system.The cultures were incubated at 25 °C for six weeks.When isolate combinations produced extruding viable ascospores, crosses were considered successful.

Morphology
To determine the morphological characteristics of the asexual morphs, representative isolates identified by DNA sequence comparisons were selected.Agar plugs from the periphery of actively growing single conidial cultures were transferred onto synthetic nutrient-poor agar (SNA; Nirenburg 1981) and incubated at 25 °C for one week (there were five replicates per isolate).Asexual structures that emerged on the surface of the SNA medium were mounted in one drop of 80% lactic acid on glass slides and examined under an Axio Imager A1 microscope (Carl Zeiss Ltd., Munchen, Germany) and an AxioCam ERc 5S digital camera with Zeiss Axio Vision Rel.4.8 software (Carl Zeiss Ltd., Munchen, Germany).Sexual morphs were studied by transferring perithecia obtained from the sexual compatibility tests into a tissue-freezing medium (Leica Biosystems, Nussloch, Germany) and were hand-sectioned using an HM550 Cryostat Microtome (Microm International GmbH, Thermo Fisher Scientific, Wall-dorf, Germany) at -20 °C.The 10-µm sections were mounted in 80% lactic acid and 3% KOH.
Fifty measurements were made for each morphological structure of the isolates selected as the holotype specimen, 30 measurements were made for the isolates selected as the paratype specimen.Minimum, maximum and average (mean) values were determined and presented as follows: (minimum-) (average -standard deviation) -(average + standard deviation) (-maximum).
The optimal growth temperature of the Calonectria species was determined by transferring the representative isolates to fresh 9 mm MEA Petri dishes, which were incubated under temperatures ranging from 5 to 35 °C at 5 °C intervals in the dark (there were five replicates per isolate).Colony colors were determined by inoculating the isolates on fresh MEA at 25 °C in the dark, after seven days incubation, a comparison was performed using the colour charts of Rayner (1970).

Fungal isolates
A total of 40 isolates with the typical morphological of Calonectria species were obtained from the infected alfalfa tissue cultivated in the soil samples.Based on preliminary phylogenetic analysis of the tef1 gene region (data not shown), 16 isolates from all soil samples were selected for further study (Table 1).

Phylogenetic analyses
Sequences for the 78 ex-type and other strains of 48 Calonectria species closely related to isolates obtained in this study were downloaded from GenBank (Table 1).For the 16 isolates collected in this study, nine resided in the Prolate Group, and seven were clustered in the Sphaero-Naviculate Group.Phylogenetic analyses of individual tef1, his3, cmdA and tub2 and the combined sequence datasets were conducted using both MP and ML method.For both the Prolate and Sphaero-Naviculate Groups, although the related position of some Calonectira species were slightly different between the MP and ML trees, the overall topologies were similar, and the ML trees were exhibited.
For the Prolate and Sphaero-Naviculate Groups, the PHT comparing the combined tef1, his3, cmdA and tub2 gene datasets generated P values of 0.141 and 0.333, respectively, which indicated that no significant difference existed between these datasets.These datasets were consequently combined and subjected to phylogenetic analyses.For each of the two groups, the sequence alignments of tef1, his3, cmdA, tub2 and the combination of the four genes were deposited in TreeBASE (TreeBASE No. 21357).The number of parsimony informative characters, the statistical values for the  phylogenetic trees of the MP analyses, and the parameters for the best-fit substitution models of ML analyses are shown in Table 2.
Phylogenetic analyses of each of the individual and combined sequence datasets indicated that in the Prolate Group, the nine isolates resided in the Ca.colhounii species complex and were closely related to Ca. colhounii, Ca. eucalypti, Ca. fujianensis, Ca. nymphaeae, Ca. paracolhounii and Ca.pseudocolhounii.In the his3 and cmdA phylogenetic trees, the nine isolates and Ca.fujianensis were clustered in the same clade (Suppl.materials 2, 3), while in the trees based on the tef1 and tub2 sequences, the nine isolates formed an independent clade (Supplementary Figures 1, 4).Based on the phylogenetic analyses of the combined sequences of the four genes, the nine isolates formed a new, strongly defined phylogenetic clade that was distinct from other Calonectria species and was supported by high bootstrap values (ML = 94%, MP = 93%) (Figure 1).Fixed unique single nucleotide polymorphisms (SNPs) were identified in the new phylogenetic clades of the nine isolates and their phylogenetically closed Calonectria species (Table 3).The total number of SNP differences between the new clade and the other closely related species varied between 10-34 for all four gene regions combined (Table 4).The results of these phylogenetic and SNP analyses indicate that the nine isolates in the Prolate Group represent a distinct, undescribed species.
Phylogenetic analyses of each of the individual and combined datasets indicated that in the Sphaero-Naviculate Group, the seven isolates were clustered in the Ca.kyotensis species complex and were closely related to Ca. canadiana.In the tef1 phylogenetic trees, the seven isolates were grouped in the same clade with Ca. canadiana (Suppl.material 5).In the phylogenetic trees based on the his3, cmdA and tub2 sequences, the seven isolates formed an independent clade distinct from Ca. canadiana and other species in the Ca.kyotensis species complex (Suppl.materials 6, 7 and 8).Based on the combined sequences of the four genes, the seven isolates formed a strongly defined phylogenetic clade that was distinct from Ca. canadiana and was supported by high bootstrap values (ML = 100%, MP = 100%) (Figure 2).The seven isolates obtained in this study were distinguished from Ca. canadiana using SNP analyses for each of the tef1, his3, cmdA and tub2 gene region sequences (Tables 5).The total number of SNP differences between the seven isolates and Ca.canadiana for all four genes was 51 (Table 6).The results indicate that the seven isolates in the Sphaero-Navivulate Group represent a novel species.

Sexual compatibility
After a six-week mating test on MSA, all 16 isolates and the crosses of isolates of each identified species failed to yield sexual structures, indicating that they were either selfsterile (heterothallic) or had retained the ability to recombine to produce fertile progeny.| Fixed polymorphisms for each group are shaded and in bold, those fixed but shared between two or more groups are only shaded.¶ "N/A" represents sequences that are not available.The order of the four genes: total (tef1, his3, cmdA and tub2).‡ "NA" represents sequences that are not available.

Taxonomy
Based on DNA sequence comparisons, the 16 isolates collected in this study presented two strongly defined phylogenetic clades in both the Prolate Group and the Sphaero-Naviculate Group.Morphological differences were observed between each phylogenetic clade and its phylogenetically closed species, especially with respect to the size of the macroconidia (Table 7).Based on the phylogenetic analyses, as well as morphological characteristics, the fungi isolated from the soil in this study represent two novel species of Calonectria, they are described as follows: Calonectria lichi Q.L. Liu & S.F.Chen, sp.nov.MycoBank MB821348 Figure 3 Etymology.lichi, which is Calonectria in Chinese.
Calonectria montana Q.L. Liu & S.F.Chen, sp.nov.MycoBank MB821349 Figure 4 Etymology.montis, meaning mountain in Latin, referring to the location where this fungus was collected.
Diagnosis.Calonectria montana can be distinguished from the phylogenetically closely related species Ca. canadiana by the size of macroconidia.

Culture characteristics.
Colonies forming abundant buff and wooly aerial mycelium on MEA at 25 °C after seven days, with feathery, irregular margins at the edges, sporulation moderate and more concentrated in the colony centre.Surface with buff to sienna (8) outer margins, reverse sienna (8) to umber (9), and chesnut (9'm) inner region, abundant chlamydospores throughout the medium, forming microsclerotia.Optimal growth temperature at 30 °C, no growth at 5 °C and 35 °C, after seven days, colonies at 10 °C, 15 °C, 20 °C, 25 °C and 30 °C reached 22.9 mm, 31.5 mm, 51.1 mm, 61.9 mm and 77.2 mm, respectively, this is a high-temperature species.

Discussion
This study identified two novel species of Calonectria from soil in a natural forest in the temperate region of central China.The identification of the fungi was supported by DNA sequence comparisons and morphological features.The two species were named Calonectria lichi and Ca.montana.
Calonectria lichi is a new addition to the Ca.colhounii complex that belongs to the Prolate Group.Based on phylogenetic analyses of four gene sequences, Ca. lichi formed a distinct and well-supported phylogenetic clade closely related to Ca. fujianensis, Ca. nymphaeae and Ca.paracolhounii, but it can be distinguished from these species by its larger macroconidia.To date, 10 species in the Ca.colhounii complex have been identified and described.Other than Ca.lichi described in this study, the other species include Ca. colhounii, Ca. eucalypti, Ca. fujianensis, Ca. macroconidialis, Ca. monticola, Ca. nymphaeae, Ca. paracolhounii, Ca. parva and Ca. pseudocolhounii (Crous 2002, Lombard et al. 2010b, 2016, Chen et al. 2011, Xu et al. 2012, Crous et al. 2015).Of these species, Ca. colhounii, Ca. eucalypti, Ca. fujianensis, Ca. nymphaeae and Ca.pseudocolhounii have been shown to be homothallic and always produce bright yellow perithecia (Crous 2002, Lombard et al. 2010b, Chen et al. 2011, Xu et al. 2012).In China, four species in the Ca.colhounii complex have been reported: except for Ca.lichi, which was isolated from a natural forest in the temperate zone in central China, the other species, including Ca. fujianensis, Ca. pseudocolhounii and Ca.nym-phaeae, were previously isolated from tropical or subtropical regions in southern China (Chen et al. 2011, Xu et al. 2012).
Calonectria montana adds a new species to the Ca.kyotensis complex that belongs to the Sphaero-Naviculate Group.Phylogenetic analyses showed that Ca. montana, which formed an independent clade with a high bootstrap value, is closely related to Ca. canadiana.Morphological differences were observed between Ca. montana and Ca.canadiana, especially with respect to the size of the macroconidia and the shape of the vesicles (Kang et al. 2001, Crous 2002).Species in the Ca.kyotensis complex are characterized by having sphaeropedunculate vesicles with lateral stipe extensions on a conidiogenous apparatus (Crous et al. 2004, Lombard et al. 2010b, 2015, 2016).No lateral stipe extensions were produced by Ca. montana, indicating that this species is different from other species in the Ca.kyotensis complex.In this study, Ca. montana was isolated from soil in central China, 14 species residing in the Ca.kyotensis complex were previously reported in China, and all of them were isolated from soil in southern China (Crous et al. 2004, Lombard et al. 2015).The results from this study suggest that more species in Ca. kyotensis complex have yet to be discovered from China.
Species of Calonectria are important plant pathogens that can cause devastating diseases on various plant hosts worldwide, especially on horticultural, agronomic and forestry crops (Polizzi et al. 2001, 2009, Crous 2002, Saracchi et al. 2008, Chen et al. 2011, Pan et al. 2012).In China, Calonectria species have been reported as pathogens of various important agronomic and forestry crops.In agriculture, the Fabaceae and Arecaceae plant families are susceptible to infection by Calonectria species, including Ca. ilicicola, which causes black rot (CBR) of Arachis hypogaea (peanut) and Medicago sativa (Gai et al. 2012, Pan et al. 2012, Pei et al. 2015), Ca. ilicicola causes red crown rot of Glycine max (soybean) (Guan et al. 2010), and Ca.colhounii and Ca.pteridis cause leaf spot on Phoenix canariensis and Serenoa repens, respectively (Luo et al. 2009, Yang et al. 2014).In forestry, leaf blight caused by Calonectria species is considered as one of the most serious threats to Eucalyptus plantations and nurseries in southern China (Zhou et al. 2008, Lombard et al. 2010a, Chen et al. 2011).The leaf inoculations showed that all tested Calonectria species were pathogenic to the tested Eucalyptus clones, including the clones that are widely planted in southern China (Chen et al. 2011, Li et al. 2014a, b).These research results suggest that species of Calonectria need to be monitored carefully, both in agronomic crops and forests.
Accurate diagnosis of plant diseases and identification of their casual agents provide the foundation for developing effective disease management strategies (Booth et al. 2000, Crous 2002, Old et al. 2003, Vitale et al. 2013, Wingfield et al. 2015).
Based on previous research results, the majority of Calonectria species identified and described in China were isolated from diseased plant tissues or soil under forestry plantations in subtropical and tropical regions (Crous et al. 2004, Lombard et al. 2010a, 2015, Chen et al. 2011).In this study, two novel Calonectria species were described, and they were isolated from soil in a natural forest in the temperate zone.The results from this study suggest that more extensive surveys need to be conducted to collect Calonectria in more geographic regions with different climate zones, which will help to clarify the species diversity of Calonectria in China.

Figure 1 .
Figure 1.Phylogenetic tree of Calonectria species in the Prolate group based on maximum likelihood (ML) analysis of combined DNA dataset of tef1, his3, cmdA and tub2 gene sequences.ML and MP (maximum parsimony) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -.Isolates representing ex-type material are marked with "T", isolates highlighted in bold were sequenced in this study and novel species were covered in blue.The tree was rooted to Ca. hongkongensis(CBS 114711 and CBS 114828).
only in all of the isolates are shown, not alleles that partially occur in individuals per phylogenetic group.‡ Numerical positions of the nucleotides in the DNA sequence alignments are indicated.§ Ex-type isolates are indicated in bold.

Figure 2 .
Figure 2. Phylogenetic tree of Calonectria species in the Sphaero-Naviculate group based on maximum likelihood (ML) analysis of combined DNA dataset of tef1, his3, cmdA and tub2 gene sequences.ML and MP (maximum parsimony) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -.Isolates representing ex-type material are marked with "T", isolates highlighted in bold were sequenced in this study and novel species were covered in orange.The tree was rooted to Ca. pauciramosa (CMW 5683 and CMW 30823).

Table 1 .
The species of Calonectria used in this study.

Table 2 .
Statistics resulting from phylogenetic analyses.

Table 3 .
Single nucleotide polymorphism comparisons in four gene regions between Calonectria lichi and the phylogenetically closest related species.

Table 4 .
Number of unique alleles found in Calonectria lichi and the phylogenetically closest related species in total and in the four gene regions.

Table 6 .
Number of unique alleles found in Calonectria montana and Ca.canadiana in total and in the four gene regions.

Table 7 .
Morphological comparisons of Calonectria lichi, Ca. montana and their phylogenetically closely related species.