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Research Article
Two novel species of Calonectria isolated from soil in a natural forest in China
expand article infoQianLi Liu, ShuaiFei Chen
‡ China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), ZhanJiang, China
Open Access

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.

Key words

C ylindrocladium, pathogen, phylogeny, taxonomy

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 damping-off, 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 and Littrell 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 FuJian, 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.

Materials and methods

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.

Based on previous research (Lombard et al. 2010d, Alfenas et al. 2015), partial gene regions including translation elongation factor 1-alpha (tef1), histone H3 (his3), calmodulin (cmdA) and β-tubulin (tub2), were used as successful DNA barcodes at species, being able to clearly distinguish between intra- and inter-specific divergence. The primer pairs EF1-728F/EF2, CYLH3F/CYLH3R, CAL-228F/CAL-2Rd and T1/CYLTUB1R were used to amplify the fragments of the respective tef1, his3, cmdA and tub2 genes (Lombard et al. 2010d).

The PCR reaction mixture used to amplify the different loci consisted of TopTaqTM Master Mix 12.5 µL (Qiagen Inc., Hilden, Germany), forward primer 1 µL, 10 µM (Invitrogen, Shanghai, China), reverse primer 1 µL, 10 µM (Invitrogen, Shanghai, China), and RNase-Free H2O 8.5 µL (Qiagen Inc., Hilden, Germany), and 2 µL (100 ng/μL) of the DNA samples was added as the template to each PCR reaction. The amplifications were performed in 25 µL reaction volumes on an MJ Mini Cycler (BIO-RAD, Hercules, CA, USA) under the conditions described by Groenewald et al. (2013). The amplification products were separated by 1.5% agarose gel electrophoresis and visualized with SYBR Safe DNA gel stain (Thermo Fisher Scientific Inc., USA).

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).

The species of Calonectria used in this study.

Species Isolate No.†,‡ Substrate Sampling site Collector GenBank accession No.§,| Reference
tef1 his3 cmdA tub2
Calonectria acicola CBS 114813 Pinus radiata New Zealand H. Pearson GQ267292 DQ190693 GQ267360 DQ190591 Gadgil and Dick 2004
CBS 114812 P. radiata New Zealand H. Pearson GQ267291 DQ190692 GQ267359 DQ190590 Gadgil and Dick 2004
Ca. aconidialis CBS 136086 Soil in Eucalyptus plantation HaiNan, China X. Mou & S.F. Chen KJ462785 KJ463133 KJ463017 N/A Lombard et al. 2015
Ca. arbusta CBS 136079 Soil in Eucalyptus plantation GuangXi, China X. Zhou & G. Zhao KJ462787 KJ463135 KJ463018 KJ462904 Lombard et al. 2015
Ca. asiatica CBS 114073 Leaf litter Thailand N.L. Hywel-Jones AY725705 AY725658 AY725741 AY725616 Crous et al. 2004
CBS 112711 Leaf litter Thailand N.L. Hywel-Jones AY725702 AY725655 AY725738 AY725613 Crous et al. 2004
Ca. australiensis CBS 112954 Ficus pleurocarpa Australia C. Pearce & B. Paulu GQ267293 DQ190699 GQ267363 DQ190596 Crous et al. 2006
Ca. brassicicola CBS 112841 Brassica sp. Indonesia M.J. Wingfield KX784689 N/A KX784561 KX784619 Lombard et al. 2016
Ca. canadiana CBS 110817 Picea sp. Canada S. Greifenhagen GQ267297 AF348228 AY725743 AF348212 Lombard et al. 2010b
Ca. chinensis CBS 114827 Soil Hong Kong E.C.Y. Liew AY725710 AY725661 AY725747 AY725619 Lombard et al. 2010b
CBS 112744 Soil Hong Kong E.C.Y. Liew AY725709 AY725660 AY725746 AY725618 Lombard et al. 2010b
Ca. colhounii CBS 293.79 Camellia sinensis Indonesia N/A GQ267301 DQ190639 GQ267373 DQ190564 Lombard et al. 2010b
CBS 114704 Arachis pintoi Australia D. Hutton GQ267300 DQ190638 GQ267372 DQ190563 Lombard et al. 2010b
Ca. colombiensis CBS 112220 Eucalyptus grandis Colombia M.J. Wingfield AY725711 AY725662 AY725748 GQ267207 Lombard et al. 2010b
CBS 112221 E. grandis Colombia M.J. Wingfield AY725712 AY725663 AY725749 AY725620 Lombard et al. 2010b
Ca. crousiana CBS 127198 E. grandis FuJian, China M.J. Wingfield HQ285822 HQ285808 MF527084 HQ285794 Chen et al. 2011; This study
CBS 127199 E. grandis FuJian, China M.J. Wingfield HQ285823 HQ285809 MF527085 HQ285795 Chen et al. 2011; This study
Ca. curvispora CBS 116159 Soil Madagascar P.W. Crous GQ267302 AY725664 GQ267374 AF333394 Lombard et al. 2010b
Ca. eucalypti CBS 125275 E. grandis Sumatra Utara M.J. Wingfield GQ267338 GQ267267 GQ267430 GQ267218 Lombard et al. 2010b
CBS 125276 E. grandis Sumatra Utara M.J. Wingfield GQ267339 GQ267268 GQ267431 GQ267219 Lombard et al. 2010b
Ca. expansa CBS 136247 Soil in Eucalyptus plantation Guangxi, China X. Zhou & G. Zhao KJ462798 KJ463146 KJ463029 KJ462914 Lombard et al. 2015
CBS 136078 Soil in Eucalyptus plantation Guangdong, China X. Zhou & G. Zhao KJ462797 KJ463145 KJ463028 KJ462913 Lombard et al. 2015
Ca. fujianensis CBS 127201 E. grandis FuJian, China M.J. Wingfield HQ285820 HQ285806 MF527089 HQ285792 Chen et al. 2011; This study
CBS 127200 E. grandis FuJian, China M.J. Wingfield HQ285819 HQ285805 MF527088 HQ285791 Chen et al. 2011; This study
Ca. guangxiensis CBS 136092 Soil in Eucalyptus plantation Guangxi, China X. Mou & R. Chang KJ462803 KJ463151 KJ463034 KJ462919 Lombard et al. 2015
CBS 136094 Soil in Eucalyptus plantation Guangxi, China X. Mou & R. Chang KJ462804 N/A KJ463035 KJ462920 Lombard et al. 2015
Ca. hainanensis CBS 136248 Soil in Eucalyptus plantation Hainan, China X. Mou & S.F. Chen KJ462805 KJ463152 KJ463036 N/A Lombard et al. 2015
Ca. hongkongensis CBS 114828 Soil Hong Kong E.C.Y. Liew AY725717 AY725667 AY725755 AY725622 Lombard et al. 2010b
CBS 114711 Soil Hong Kong M.J. Wingfield AY725716 AY725666 AY725754 AY725621 Lombard et al. 2010b
Ca. ilicicola CBS 190.50 Solanum tuberosum Indonesia K.B. Boedijn &J. Reitsma AY725726 AY725676 AY725764 AY725631 Lombard et al. 2010b
CBS 112215 A. hypogaea U.S.A. Beute AY725726 AY725684 AY725765 AY725639 Crous et al. 2004
Ca. indonesiae CBS 112823 Syzygium aromaticum Indonesia M.J. Wingfield AY725718 AY725668 AY725756 AY725623 Lombard et al. 2010b
CBS 112840 S. aromaticum Indonesia M.J. Wingfield AY725720 AY725670 AY725758 AY725625 Lombard et al. 2010b
C. indonesiana CBS 112936 Soil Indonesia M.J. Wingfield KX784701 N/A KX784573 KX784631 Lombard et al. 2016
Ca. indusiata CBS 144.36 N/A N/A N/A GQ267332 GQ267262 GQ267453 GQ267239 Lombard et al. 2010b
CBS 114684 Rhododendron sp. U.S.A. N.E. El-Gholl GQ267333 DQ190653 GQ267454 AF232862 Lombard et al. 2010b
Ca. kyotensis CBS 170.77 Idesia polycarpa New Zealand N/A GQ267308 GQ267249 GQ267380 GQ267209 Lombard et al. 2010b
CBS 413.67 Paphiopedilum callosum Celle, Germany W. Gerlach GQ267307 GQ267248 GQ267379 GQ267208 Lombard et al. 2010b
Ca. lateralis CBS 136629 Soil in Eucalyptus plantation Guangxi,China X. Zhou & G. Zhao KJ462840 KJ463186 KJ463070 KJ462955 Lombard et al. 2015
Species Isolate no. tef1
433 435 436 437 438 441 443 444 446 447 448 450 452 453 457 473 483
Species Isolate no. His3 cmdA
45 234 272 293 344 353 368 169 204 205 210 238 244 266 293 325 334 411 429 432 474
Ca. lichi CERC 8866 Soil Central China S.F. Chen MF527039 MF527055 MF527071 MF527097 This study
CERC 8841 Soil Central China S.F. Chen MF527036 MF527052 MF527068 MF527094 This study
CERC 8848 Soil Central China S.F. Chen MF527037 MF527053 MF527069 MF527095 This study
CERC 8850 Soil Central China S.F. Chen MF527038 MF527054 MF527070 MF527096 This study
CERC 8871 Soil Central China S.F. Chen MF527040 MF527056 MF527072 MF527098 This study
CERC 8890 Soil Central China S.F. Chen MF527041 MF527057 MF527073 MF527099 This study
CERC 8900 Soil Central China S.F. Chen MF527042 MF527058 MF527074 MF527100 This study
CERC 8906 Soil Central China S.F. Chen MF527043 MF527059 MF527075 MF527101 This study
CERC 8928 Soil Central China S.F. Chen MF527044 MF527060 MF527076 MF527102 This study
Ca. macroconidialis CBS 114880 E. grandis South Africa P.W. Crous GQ267313 DQ190655 GQ267393 AF232855 Lombard et al. 2010b
Ca. magnispora CBS 136249 Soil in Eucalyptus plantation Guangxi, China X. Mou & R. Chang KJ462841 KJ463187 KJ463071 KJ462956 Lombard et al. 2015
Ca. malesiana CBS 112752 Soil Indonesia M.J. Wingfield AY725722 AY725672 AY725760 AY725627 Lombard et al. 2010b
CBS 112710 Debris Thailand N.L. Hywel-Jones AY725721 AY725671 AY725759 AY725626 Lombard et al. 2010b
Ca. microconidialis CBS 136638 E. urophylla × E. grandis clone seedling leaf Guangdong, China G. Zhao KJ462845 KJ463191 KJ463075 KJ462960 Lombard et al. 2015
CBS 136633 E. urophylla × E. grandis clone seedling leaf Guangdong, China G. Zhao KJ462842 KJ463188 KJ463072 KJ462957 Lombard et al. 2015
Ca. montana CERC 8952 Soil Central China S.F. Chen MF527049 MF527065 MF527081 MF527107 This study
CERC 8930 Soil Central China S.F. Chen MF527045 MF527061 MF527077 MF527103 This study
CERC 8932 Soil Central China S.F. Chen MF527046 MF527062 MF527078 MF527104 This study
CERC 8936 Soil Central China S.F. Chen MF527047 MF527063 MF527079 MF527105 This study
CERC 8938 Soil Central China S.F. Chen MF527048 MF527064 MF527080 MF527106 This study
CERC 8957 Soil Central China S.F. Chen MF527050 MF527066 MF527082 MF527108 This study
CERC 8966 Soil Central China S.F. Chen MF527051 MF527067 MF527083 MF527109 This study
Ca. monticola CPC 28835 Soil Thailand P.W. Crous KT964773 N/A KT964771 KT964769 Crous et al. 2015
CPC 28836 Soil Thailand P.W. Crous KT964774 N/A KT964772 KT964770 Crous et al. 2015
Ca. multiseptata CBS 112682 Eucalyptus sp. Indonesia M.J. Wingfield FJ918535 DQ190659 GQ267397 DQ190573 Lombard et al. 2010b
Ca. nymphaeae CBS 131802 Nymphaea tetragona Guiyang, Guizhou S.Y. Qin KC555273 N/A N/A JN984864 Xu et al. 2012
HGUP 100004 N. tetragona Guiyang, Guizhou Y. Wang KC555274 N/A N/A JN984865 Xu et al. 2012
Ca. pacifica CBS 109063 Araucaria heterophylla Hawaii, USA M. Aragaki AY725724 GQ267255 AY725762 GQ267213 Lombard et al. 2010b
CBS 114038 Ipomoea aquatica New Zealand C.F. Hill GQ267320 AY725675 GQ267402 AY725630 Lombard et al. 2010b
Ca. paracolhounii CBS 114679 N/A USA A.Y. Rossman KX784714 N/A KX784582 KX784644 Lombard et al. 2016
CBS 114705 Annona reticulata Australia D. Hutton KX784715 N/A N/A KX784645 Lombard et al. 2016
Ca. parakyotensis CBS 136085 Soil in Eucalyptus plantation Guangdong, China X. Mou & R. Chang KJ462851 KJ463197 KJ463081 N/A Lombard et al. 2015
CBS 136095 Soil in Eucalyptus plantation Guangxi, China X. Mou & R. Chang KJ462852 KJ463198 KJ463082 N/A Lombard et al. 2015
Ca. parva CBS 110798 Eucalyptus grandis roots South Africa P.W. Crous KX784716 N/A KX784583 KX784646 Lombard et al. 2016
Ca. pauciramosa CMW 5683 E. grandis South Africa P.W. Crous FJ918565 FJ918531 GQ267405 FJ918514 Lombard et al. 2010b
CMW 30823 E. grandis South Africa P.W. Crous FJ918566 FJ918532 GQ267404 FJ918515 Lombard et al. 2010b
Ca. penicilloides CBS 174.55 Prunus sp. Japan Tubaki GQ267322 GQ267257 GQ267406 AF333414 Lombard et al. 2010b
Ca. pluriramosa CBS 136976 Soil in Eucalyptus plantation Guangxi, China X. Zhou & G. Zhao KJ462882 KJ463228 KJ463112 KJ462995 Lombard et al. 2015
Ca. pseudokyotensis CBS 137332 Soil in Eucalyptus plantation Guangxi,China X. Zhou & G. Zhao KJ462881 KJ463227 KJ463111 KJ462994 Lombard et al. 2015
Ca. pseudocolhounii CBS 127195 E. dunnii FuJian, China M.J. Wingfield HQ285816 HQ285802 MF527091 HQ285788 Chen et al. 2011; This study
CBS 127196 E. dunnii FuJian, China M.J. Wingfield HQ285817 HQ285803 MF527092 HQ285789 Chen et al. 2011; This study
Ca. pseudoreteaudii CBS 123694 E. urophylla × E. grandis cutting Guangdong, China M.J. Wingfield FJ918541 FJ918519 GQ267411 FJ918504 Lombard et al. 2010b
CBS 123696 E. urophylla × E. grandis cutting Guangdong, China M.J. Wingfield FJ918542 FJ918520 GQ267410 FJ918505 Lombard et al. 2010b
Ca. queenslandica CBS 112146 E. urophylla Australia B. Brown FJ918543 FJ918521 GQ267415 AF389835 Lombard et al. 2010b
CBS 112155 E. pellita Australia K.M. Old FJ918544 DQ190667 GQ267416 AF389834 Lombard et al. 2010b
Ca. reteaudii CBS 112144 E. camaldulensis Vietnam M.J. Dudzinski FJ918537 DQ190661 GQ267417 AF389833 Lombard et al. 2010b
CBS 112143 E. camaldulensis Vietnam M.J. Dudzinski FJ918536 DQ190660 GQ267418 GQ240642 Lombard et al. 2010b
Ca. sphaeropendunculata CBS 136081 Soil in Eucalyptus plantation Guangxi, China X. Zhou & G. Zhao KJ462890 KJ463236 KJ463120 KJ463003 Lombard et al. 2015
Ca. sumatrensis CBS 112829 Soil Indonesia M.J. Wingfield AY725733 AY725696 AY725771 AY725649 Lombard et al. 2010b
CBS 112934 Soil Indonesia M.J. Wingfield AY725735 AY725698 AY725773 AY725651 Lombard et al. 2010b
Ca. syzygiicola CBS 112831 Soil Indonesia M.J. Wingfield KX784736 N/A N/A KX784663 Lombard et al. 2016
Ca. terrae-reginae CBS 112151 E. urophylla Australia C. Hanwood FJ918545 FJ918522 GQ267451 FJ918506 Lombard et al. 2010b
CBS 112634 Xanthorrhoea australis Australia T. Baigent FJ918546 DQ190668 GQ267452 FJ918507 Lombard et al. 2010b
Ca. turangicola CBS 136077 Soil in Eucalyptus plantation Guangxi, China X. Zhou & G. Zhao KJ462900 KJ463246 N/A KJ463013 Lombard et al. 2015
CBS 136093 Soil in Eucalyptus plantation Guangxi, China X. Mou & R. Chang KJ462901 KJ463247 KJ463130 KJ463014 Lombard et al. 2015

Statistics resulting from phylogenetic analyses.

Dataset Phylogenetic group No. of taxa No. of bp Maximum parsimony
PIC No. of trees Tree length CI§ RI| RC HI#
tef1 Prolate 45 515 210 8 448 0.7054 0.8847 0.6240 0.2946
his3 Prolate 38 449 140 6 340 0.6941 0.9176 0.6369 0.3059
cmdA Prolate 42 476 152 792 245 0.7591 0.9295 0.7056 0.2408
tub2 Prolate 45 579 204 18 350 0.8085 0.9395 0.7597 0.1914
tef1/his3/cmdA/tub2 Prolate 45 2019 706 1 1484 0.6880 0.8940 0.6150 0.3120
tef1 Sphaero-Naviculate 51 522 159 33 330 0.7030 0.9056 0.6367 0.2969
his3 Sphaero-Naviculate 47 455 138 11 386 0.6632 0.9110 0.6042 0.3367
cmdA Sphaero-Naviculate 49 473 138 48 228 0.7763 0.9406 0.7302 0.2236
tub2 Sphaero-Naviculate 47 534 174 4 401 0.7107 0.9216 0.6550 0.2892
tef1/his3/cmdA/tub2 Sphaero-Naviculate 51 1984 609 1350 1535 0.6190 0.8790 0.6047 0.3810
Dataset Phylogenetic group Maximum likelihood
Subst. model†† NST‡‡ Rate matrix Rates
tef1 Prolate TIM2+G 6 1.6588 2.3553 1.6588 1.0000 4.4652 Gamma
his3 Prolate GTR+G 6 1.8190 7.5654 4.6281 1.4320 15.6259 Gamma
cmdA Prolate HKY+G 2 Gamma
tub2 Prolate TPM3uf+G 6 1.5151 4.2112 1.0000 1.5151 4.2112 Gamma
tef1/his3/cmdA/tub2 Prolate TIM2+I+G 6 1.3725 3.6221 1.3725 1.0000 5.1226 Gamma
tef1 Sphaero-Naviculate GTR+G 6 2.3612 2.5155 0.6227 0.7074 5.0226 Gamma
his3 Sphaero-Naviculate HKY+I+G 2 Gamma
cmdA Sphaero-Naviculate TrN+G 6 1.0000 3.8308 1.0000 1.0000 6.4755 Gamma
tub2 Sphaero-Naviculate TPM3uf+G 6 1.5714 4.6055 1.0000 1.5714 4.6055 Gamma
tef1/his3/cmdA/tub2 Sphaero-Naviculate GTR+I+G 6 1.6318 3.8130 1.0888 1.1609 5.2579 Gamma

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, Walldorf, 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).

Results

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 self-sterile (heterothallic) or had retained the ability to recombine to produce fertile progeny.

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).

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

Species Isolate no. tef1
28 81 89 90 91 92 93 100 120 121 124 184 185 186 243 418 425 432
Ca. lichi CERC 8866§ A A| C T T A A C A
CERC 8841 A A C T T A A C A
CERC 8848 A A C T T A A C A
CERC 8850 A A C T T A A C A
CERC 8871 A A C T T A A C A
CERC 8890 A A C T T A A C A
CERC 8900 A A C T T A A C A
CERC 8906 A A C T T A A C A
CERC 8928 A A C T T A A C A
Ca. colhounii CBS 293.79 C T A C A A C C A G C A
CBS 114704 C T A C A A C C A G C A
Ca. eucalypti CBS 125275 A T C T A A C A
CBS 125276 A T C T A A C A
Ca. fujianensis CBS 127201 A T C T G A A A A C A
CBS 127200 A T C T G A A A A C A
Ca. nymphaeae CBS 131802 A T C T G A C A
HGUP 100004 A T C T G A C A
Ca. paracolhounii CBS 114679 A T T T T A A G C C
Ca. pseudocolhounii CBS 127195 A T C T A A C A
CBS 127196 A T C T A A C A
Species Isolate no. tef1
433 435 436 437 438 441 443 444 446 447 448 450 452 453 457 473 483
Ca. lichi CERC 8866§ T T C T C T T A C T A C T T T G
CERC 8841 T T C T C T T A C T A C T T T G
CERC 8848 T T C T C T T A C T A C T T T G
CERC 8850 T T C T C T T A C T A C T T T G
CERC 8871 T T C T C T T A C T A C T T T G
CERC 8890 T T C T C T T A C T A C T T T G
CERC 8900 T T C T C T T A C T A C T T T G
CERC 8906 T T C T C T T A C T A C T T T G
CERC 8928 T T C T C T T A C T A C T T T G
Ca. colhounii CBS 293.79 T T C C C C T A C T A C C T C G C
CBS 114704 T T C C C C T A C T A C C T C G C
Ca. eucalypti CBS 125275 T T C T C T T A C T A C T T T G C
CBS 125276 T T C T C T T A C T A C T T T G C
Ca. fujianensis CBS 127201 T T C T C T T A C T A C T T T G
CBS 127200 T T C T C T T A C T A C T T T G
Ca. nymphaeae CBS 131802 T T C T C T T A C T A C T T N/A
HGUP 100004 T T C T C T T A C T A C T T N/A
Ca. paracolhounii CBS 114679 A G T T C T G G G T G G N/A N/A N/A
Ca. pseudocolhounii CBS 127195 T T C T C T T A C T A C T T T G
CBS 127196 T T C T C T T A C T A C T T T G
Species Isolate no. His3 cmdA
45 234 272 293 344 353 368 169 204 205 210 238 244 266 293 325 334 411 429 432 474
Ca. lichi CERC 8866§ A T A C C C A G A C C G G G A G G C C C T
CERC 8841 A T A C C C A G A C C G G G A G G C C C T
CERC 8848 A T A C C C A G A C C G G G A G G C C C T
CERC 8850 A T A C C C A G A C C G G G A G G C C C T
CERC 8871 A T A C C C A G A C C G G G A G G C C C T
CERC 8890 A T A C C C A G A C C G G G A G G C C C T
CERC 8900 A T A C C C A G A C C G G G A G G C C C T
CERC 8906 A T A C C C A G A C C G G G A G G C C C T
CERC 8928 A T A C C C A G A C C G G G A G G C C C T
Ca. colhounii CBS 293.79 A T A T C T C G A C C G G G A G G C T C C
CBS 114704 A T A T C T C G A C C G G G A G G C T C C
Ca. eucalypti CBS 125275 T T T T T C G A C C G G G A G G C C T C
CBS 125276 T T T T T C G A C C G G G A G G C C T C
Ca. fujianensis CBS 127201 A T A C C C A G A C C G G G A G G C C C T
CBS 127200 A T A C C C A G A C C G G G A G G C C C T
Ca. nymphaeae CBS 131802 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
HGUP 100004 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
Ca. paracolhounii CBS 114679 N/A N/A N/A N/A N/A N/A N/A T T G A C C A T C A G C C N/A
Ca. pseudocolhounii CBS 127195 A C A T C T C G A C C G G G A G G C C T T
CBS 127196 A C A T C T C G A C C G G G A G G C C T T
Species Isolate no. tub2
24 28 33 68 98 103 427 442 446 455 534 535 536 537 541 547 550 551
Ca. lichi CERC 8866§ C G C C A C C C T A G T G C T C T C
CERC 8841 C G C C A C C C T A G T G C T C T C
CERC 8848 C G C C A C C C T A G T G C T C T C
CERC 8850 C G C C A C C C T A G T G C T C T C
CERC 8871 C G C C A C C C T A G T G C T C T C
CERC 8890 C G C C A C C C T A G T G C T C T C
CERC 8900 C G C C A C C C T A G T G C T C T C
CERC 8906 C G C C A C C C T A G T G C T C T C
CERC 8928 C G C C A C C C T A G T G C T C T C
Ca. colhounii CBS 293.79 N/A N/A N/A C C C C C T A G T G C T C T C
CBS 114704 N/A N/A N/A C C C C C T A G T G C T C T C
Ca. eucalypti CBS 125275 T A T C C C C C C G G T G C T C T C
CBS 125276 T A T C C C C C C G G T G C T C T C
Ca. fujianensis CBS 127201 C A C C C T C C T A G T G C T C T C
CBS 127200 C A C C C T C C T A G T G C T C T C
Ca. nymphaeae CBS 131802 C A C C A C C C T A T C G
HGUP 100004 C A C C A C C C T A T C G
Ca. paracolhounii CBS 114679 N/A N/A N/A A C C C G T A G T G C T C T C
Ca. pseudocolhounii CBS 127195 T A T C C C T C T A G T G C T C T C
CBS 127196 T A T C C C T C T A G T G C T C T C

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

Ca. colhounii Ca. eucalypti Ca. fujianensis Ca. nymphaeae Ca. paracolhounii Ca. pseudocolhounii
Ca. lichi 22(16/3/2/1) 19(4/6/2/7) 10(6/0/0/4) 14(5/NA/NA/9) 34(19/NA/11/4) 13(3/4/1/5)
Ca. colhounii 19(12/3/2/2) 24(18/3/2/1) 24(15/NA/NA/9) 42(28/NA/12/2) 18(13/1/3/1)
Ca. eucalypti 22(6/6/2/8) 18(4/NA/NA/14) 45(26/NA/12/7) 11(1/4/1/5)
Ca. fujianensis 16(5/NA/NA/11) 37(23/NA/11/3) 15(5/4/1/5)
Ca. nymphaeae 32(20/NA/NA/12) 16(4/NA/NA/12)
Ca. paracolhounii 36(20/NA/12/4)
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).

Single nucleotide polymorphism comparisons in four gene regions between Calonectria montana and Ca. canadiana.

Species Isolate no. tef1 his3
50 497 28 29 34 47 49 50 58 64 92 110 123 138 157 159 177 180 188 196
Ca. montana CERC 8952 § C C C C G C G C C C C C C G A T G
CERC 8930 C C C C G C G C C C C C C G A T G
CERC 8932 C C C C G C G C C C C C C G A T G
CERC 8936 C C C C G C G C C C C C C G A T G
CERC 8938 C C C C G C G C C C C C C G A T G
CERC 8957 C C C C G C G C C C C C C G A T G
CERC 8966 C C C C G C G C C C C C C G A T G
Ca. canadiana CBS 110817 T G G T A T G A T T G T A A A C T A T
Species Isolate no. his3
199 202 205 212 213 220 227 229 257 300 321 336 339 372 378 397 400 403 418 421
Ca. montana CERC 8952 C C C C T C C C C C C C T C C C C T C
CERC 8930 C C C C T C C C C C C C T C C C C T C
CERC 8932 C C C C T C C C C C C C T C C C C T C
CERC 8936 C C C C T C C C C C C C T C C C C T C
CERC 8938 C C C C T C C C C C C C T C C C C T C
CERC 8957 C C C C T C C C C C C C T C C C C T C
CERC 8966 C C C C T C C C C C C C T C C C C T C
Ca. canadiana CBS 110817 T A T G G A G T G G A A C G G T T G A
Species Isolate no. cmdA tub2
470 3 4 7 10 174 181 336 403 439 502
Ca. montana CERC 8952 T G T C T C T C C T C
CERC 8930 T G T C T C T C C T C
CERC 8932 T G T C T C T C C T C
CERC 8936 T G T C T C T C C T C
CERC 8938 T G T C T C T C C T C
CERC 8957 T G T C T C T C C T C
CERC 8966 T G T C T C T C C T C
Ca. canadiana CBS 110817 C A C G C T C T A C T

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 No: 821348
Figure 3

Etymology

lichi, which is Calonectria in Chinese.

Diagnosis

Calonectria lichi differs from the phylogenetically closely related species Ca. colhounii, Ca. eucalypti, Ca. fujianensis, Ca. nymphaeae, Ca. paracolhounii and Ca. pseudocolhounii with respect to the macroconidia dimensions.

Type

CHINA. From soil under a natural forest in central China, 07 April 2016, ShuaiFei Chen, CSFF 2019 – holotype, CERC 8866 = CGMCC 3.18733 – ex-type culture.

Description

Sexual morph unknown. Macroconidiophores consisting of a stipe, a suite of penicillate arranged fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, (39.5–)78.5–160.5(–206.5) × (4.5–)5.5–7.5(–8.5) µm; stipe extension septate, straight to flexuous, (124–)139.5–187.5(–218) µm long, 2.5–5 µm wide at the apical septum, terminating in a clavate vesicle, (3.5–)4–5(–5.5) µm diam, lateral stipe extensions (90° to main axis) absent. Conidiogenous apparatus (44–)56–92(–108.5) µm long, (35–)52–82.5(–94) µm wide; primary branches aseptate to 1–septate, (12–)16.5–33.5(–46.5) × (4–)4.5–6.5(–9) µm; secondary branches aseptate, (7–)9.5–16(–21) × (3–)3.5–5(–6) µm; tertiary branches aseptate, (7.5–)9–12.5(–14.5) × (3–)3.5–4.5(–6) µm; additional branches (–5), aseptate, (5.5–)8.5–12.5(–14) × (2.5–)3.5–4.5(–5.5) µm; each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, (6–)8–12(–14.5) × (2.5–)3–4(–5) µm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (53–)60.5–70.5(–79) × (5–)5.5–6.5(–7) µm (av. = 65.7 × 6 µm), 3–septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime. Megaconidia and microconidia not observed.

Culture characteristics

Colonies forming abundant white aerial mycelium on MEA at 25 °C after seven days, with feathery, irregular margins at the edges, moderate sporulation. Surface with white to buff outer margins, and salmon (13’d) inner region, becoming ochreous (44) towards the center, reverse sienna (8) to umber (9) with abundant chlamydospores throughout the medium, forming microsclerotia. Optimal growth temperature at 25 °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 21.9 mm, 30.8 mm, 41.5 mm, 54.4 mm and 37.2 mm, respectively.

Substratum

Soil in a natural forest.

Distribution

Central China.

Other specimens examined

CHINA. From soil in a natural forest in central China, 07 April 2016, ShuaiFei Chen, CSFF 2020, culture CERC 8850 = CGMCC 3.18732; CHINA. From soil under a natural forest in central China, 07 April 2016, ShuaiFei Chen, CSFF 2021, culture CERC 8890 = CGMCC 3.18734; CHINA. From soil in a natural forest in central China, 07 April 2016, ShuaiFei Chen, culture CERC 8841, CERC 8848, CERC 8871, CERC 8900, CERC 8906 and CERC 8928.

Notes

Calonectria lichi is a new species in the Ca. colhounii complex and is closely related to Ca. colhounii, Ca. eucalypti, Ca. fujianensis, Ca. nymphaeae, Ca. paracolhounii and Ca. pseudocolhounii (Crous 2002, Lombard et al. 2010b, 2016, Chen et al. 2011, Xu et al. 2012, Crous et al. 2015). The macroconidia of Ca. lichi (av. 65.7 × 6.0 µm) are longer and wider than those of Ca. colhounii (av. 65 × 5 µm), Ca. fujianensis (av. 52.5 × 4 µm), Ca. nymphaeae (av. 61 × 5.9 µm), Ca. paracolhounii (av. 41 × 5 µm) and Ca. pseudocolhounii (av. 60 ×4.5 µm), but narrower than those of Ca. eucalypti (av. 72 × 6µm).

Calonectria montana Q.L. Liu & S.F. Chen, sp. nov.

MycoBank No: 821349
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.

Type

CHINA. From soil under a natural forest in central China, 07 April 2016, ShuaiFei Chen, holotype CSFF 2022, ex-type culture CERC 8952 = CGMCC 3.18735.

Description

Sexual morph unknown. Macroconidiophores consisting of a stipe, a suite of penicillate arranged fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, (30–)52–91(–123.5) × (4–)5.5–8(–9.5) µm; stipe extension septate, straight to flexuous (76.5–)107–168(–211.5) µm long, (2.5–)3–4.5(–5.5) µm wide at the apical septum, terminating in a pyriform to sphaeropedunculate vesicle, (4–)7–11(–12.5) µm diam, lateral stipe extensions (90° to main axis) absent. Conidiogenous apparatus (40–)49–87.5(–102.5) µm long, (44–)62–91(–104) µm wide; primary branches aseptate to 1–septate, (14.5–)19.5–34(–55.5) × (4–)4.5–6(–7) µm; secondary branches aseptate, (11–)13.5–23(–33) × (3–)4–5(–6) µm; tertiary branches aseptate, (9–)11–15(–16.5) × (3.5–)3.5–4.5(–5) µm; each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, (8–)10.5–12.5(–15.5) × (2.5–)3.5–4.5(–5) µm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (37.5–)40.5–45.5(–51.5) × 4–5(–5.5) µm (av. = 43.2 × 4.6 µm), 1–septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime. Megaconidia and microconidia not observed.

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.

Substratum

Soil under the natural forest.

Distribution

Central China.

Other specimens examined

CHINA. From soil in a natural forest in central China, 07 April 2016, ShuaiFei Chen, CSFF 2023, culture CERC 8957 = CGMCC 3.18736; From soil in a natural forest in central China, 07 April 2016, ShuaiFei Chen, CSFF 2024, culture CERC 8966 = CGMCC 3.18737; From soil in a natural forest in central China, 07 April 2016, ShuaiFei Chen, culture CERC 8930, CERC 8932, CERC 8936 and CERC 8938.

Notes

Calonectria montana is a new addition to the Ca. kyotensis complex and is phylogenetically closely related to Ca. canadiana (Crous 2002, Crous et al. 2004, Lombard et al. 2015, 2016). The macroconidia of Ca. montana (av. 43.2 × 4.6 µm) are shorter and wider than those of Ca. canadiana (av. 50 × 4 µm).

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

Ca. canadiana
Ca. montana 51(2/38/1/10)

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

Speices Macroconidia (L × W)†,‡ Macroconidia average (L × W)†,‡ Macroconidia septation Vesicle (Min. – Max.)†,§ Vesicle shape Reference
Ca. lichi | (53–)60.5–70.5(–79) × (5–)5.5–6.5(–7) 65.7 × 6 3 (3.5–)4–5(–5.5) clavate This study
Ca. colhounii (45–)60–70(–80) × (4–)5(–6) 65 × 5 (1–)3 3–4 clavate Crous 2002
Ca. eucalypti (66–)69–75(–80) × (5–)6 72 × 6 3 4–6 broadly clavate Lombard et al. 2010b
Ca. fujianensis (48–)50–55(–60) × (2.5–)3.5–4.5(–5) 52.5 × 4 (1–)3 (3–)3.5–4.5(–5) clavate Chen et al. 2011
Ca. nymphaeae 55–63 × 5.3–6.3 61 × 5.9 3–4 3–5 clavate Xu et al. 2012
Ca. paracolhounii (37–)39–43(–45) × 4–5 41 × 5 3 3–5 narrowly clavate Lombard et al. 2016
Ca. pseudocolhounii (49–)55–65(–74) × (3.5–)4–5(–5.5) 60 × 4.5 (1–)3 (3.5–)4–5(–6) clavate Chen et al. 2011
Ca. montana (37.5–)40.5–45.5(–51.5) × 4–5(–5.5) 43.2 × 4.6 1 (4–)7–11(–12.5) sphaeropedunculate This study
Ca. canadiana (38–)48–55(–65) × 4(–5) 50 × 4 1 6–10 pyriform to sphaeropedunculate Kang et al. 2001; Lechat et al. 2010
Figure 3. 

Calonectria lichi. a–c Macroconidiophore d–f Clavate vesicles g–i Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides j–k Macroconidia Scale bars: a–c = 50 μm; d–f = 5 μm; g–k = 10 μm.

Figure 4. 

Calonectria montana. a–c Macroconidiophores d–f Sphaeropedunculate vesicles g–h Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides i–j Macroconidia Scale bars: a–c = 20 μm; d–j = 10 μm.

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. nymphaeae, 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.

Acknowledgments

This study was supported by the Fundamental Research Funds for the Central Non-Profit Research Institution of CAF (Project No. CAFYBB2014MA018) and the National Natural Science Foundation of China (NSFC) (Project numbers: 31622019 and 31400546). We thank LetPub (www.letpub.com) for linguistic assistance during the preparation of this manuscript.

References

  • Aiello D, Cirvilleri G, Polizzi G, Vitale A (2013) Effects of fungicide treatments for the control of epidemic and exotic Calonectria diseases in Italy. Plant Disease 97: 37–43. https://doi.org/10.1094/PDIS-03-12-0266-RE
  • Alfenas RF, Pereira OL, Jorge VL, Crous PW, Alfenas AC (2013a) A new species of Calonectria causing leaf blight and cutting rot of three forest tree species in Brazil. Tropical Plant Pathology 38: 513–521. https://doi.org/10.1590/S1982-56762013000600007
  • Alfenas RF, Pereira OL, Ferreira MA, Jorge VL, Crous PW, Alfenas AC (2013b) Calonectria metrosideri, a highly aggressive pathogen causing leaf blight, root rot, and wilt of Metrosideros spp. in Brazil. Forest Pathology 43: 257–265. https://doi.org/10.1111/efp.12035
  • Alfenas RF, Lombard L, Pereira OL, Alfenas AC, Crous PW (2015) Diversity and potential impact of Calonectria species in Eucalyptus plantations in Brazil. Studies in Mycology 80: 89–130. https://doi.org/10.1016/j.simyco.2014.11.002
  • Booth TH, Jovanovic T, Old KM, Dudzinski MJ (2000) Climatic mapping to identify high-risk areas for Cylindrocladium quinqueseptatum leaf blight on eucalypts in mainland South East Asia and around the world. Environmental Pollution 108: 365–372. https://doi.org/10.1016/S0269-7491(99)00215-8
  • Chen SF, Lombard L, Roux J, Xie YJ, Wingfield MJ, Zhou XD (2011) Novel species of Calonectria associated with Eucalyptus leaf blight in Southeast China. Persoonia 26: 1–12. https://doi.org/10.3767/003158511X555236
  • Crous PW, Phillips AJL, Wingfield MJ (1991) The genera Cylindrocladium and Cylindrocladiella in South Africa, with special reference to forest nurseries. South African Forestry Journal 157: 69–85. https://doi.org/10.1080/00382167.1991.9629103
  • Crous PW (2002) Taxonomy and pathology of Cylindrocladium (Calonectria) and allied genera. APS Press, St. Paul, Minnesota, USA.
  • Crous PW, Groenewald JZ, Risède JM, Simoneau P, Hywel-Jones NL (2004) Calonectria species and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in Mycology 50: 415–430.
  • Crous PW, Groenewald JZ, Risède JM, Simoneau P, Hyde KD (2006) Calonectria species and their Cylindrocladium anamorphs: species with clavate vesicles. Studies in Mycology 55: 213–226. https://doi.org/10.3114/sim.55.1.213
  • Ferreira FA (1989) Patologia florestal. Principais doenças florestais no Brasil. Viçosa, Sociedade de Investigaçoes Florestais, Viçosa, MG, Brazil, 570 pp.
  • Gadgil PD, Dick MA (2004) Fungi silvicolae novazelandae: 5. New Zealand Journal of Forestry Science 34: 316–323.
  • Gai Y, Deng Q, Pan R, Chen X, Deng M (2012) First Report of Cylindrocladium Black Rot of Peanut Caused by Cylindrocladium parasiticum (Teleomorph Calonectria ilicicola) in Jiangxi Province, China. Plant Disease 96: 586. https://doi.org/10.1094/PDIS-11-11-1010
  • Gehesquière B, Crouch JA, Marra RE, Van Poucke K, Rys F, et al (2015) Characterization and taxonomic reassessment of the box blight pathogen Calonectria pseudonaviculata, introducing Calonectria henricotiae sp. nov. Plant Pathology 65: 37–52. https://doi.org/10.1111/ppa.12401
  • Groenewald JZ, Nakashima C, Nishikawa J, Shin HD, Park JH, Jama AN, Groenewald M, Braun U, Crous PW (2013) Species concepts in Cercospora: spotting the weeds among the roses. Studies in Mycology 75: 115–170. https://doi.org/10.3114/sim0012
  • Guan M, Pan R, Gao X, Xu D, Deng Q, Deng M (2010) First report of red crown rot caused by Cylindrocladium parasiticum on soybean in Guangdong, southern China. Plant Disease 94: 485. https://doi.org/10.1094/PDIS-94-4-0485B
  • Guerber JC, Correll JC (2001) Characterization of Glomerella acutata, the teleomorph of Colletotrichum acutatum. Mycologia 93: 216–229. https://doi.org/10.2307/3761619
  • Kang J, Crous PW, Schoch CL (2001) Species concepts in the Cylindrocladium floridanum and Cy. Spathiphylli complexes (Hypocreaceae) based on multi-allelic sequence data, sexual compatibility and morphology. Systematic and Applied Microbiology 24: 206–217. https://doi.org/10.1078/0723-2020-00026
  • Li N, Zhao X, Liu AX, Liu H (2010) Brown spot disease of tree peony caused by Cylindrocladium canadense in China. Journal of General Plant Pathology 76: 295–298. https://doi.org/10.1007/s10327-010-0245-2
  • Li GQ, Chen SF, Wu ZH, Zhou XD, Xie YJ (2014a) Preliminary Analyses on Diversity and Pathogenicity of Calonectria spp. on Eucalyptus in China. Chinese Journal of Tropical Crops 35: 1183–1191. [In Chinese]
  • Li GQ, Li JQ, Liu FF, Li TH, Chen SF (2014b) Preliminary Analyses on Pathogenicity of Twelve Calonectria spp. on Ten Eucalyptus Clones in China. Eucalypt Science & Technology 31: 1–7. https://doi.org/10.13987/j.cnki.askj.2014.04.001 [In Chinese]
  • Lombard L, Crous PW, Wingfield BD, Wingfield MJ (2010c) Multigene phylogeny and mating tests reveal three cryptic species related to Calonectria pauciramosa. Studies in Mycology 66: 15–30. https://doi.org/10.3114/sim.2010.66.02
  • Lombard L, Polizzi G, Guarnaccia V, Vitale A, Crous PW (2011) Calonectria spp. causing leaf spot, crown and root rot of ornamental plants in Tunisia. Persoonia 27: 73–79. https://doi.org/10.3767/003158511X615086
  • Lombard L, Chen SF, Mou X, Zhou XD, Crous PW, Wingfield MJ (2015) New species, hyper-diversity and potential importance of Calonectria spp. from Eucalyptus in South China. Studies in Mycology 80: 151–188. https://doi.org/10.1016/j.simyco.2014.11.003
  • Luo JS, Wang MS, Lin XX, Zhang YY (2009) Pathogenic identification of Phoenix canariensis leaf spot disease. Chinese journal of tropical crops 30: 104–107. [In Chinese]
  • Mirabolfathy M, Ahangaran Y, Lombard L, Crous PW (2013) Leaf blight of Buxus sempervirens in northern forests of Iran caused by Calonectria pseudonaviculata. Studies in Mycology 85: 159–198. http://dx.doi.org/10.1094/PDIS-03-13-0237-PDN
  • Nirenburg HI (1981) A simplified method for identifying Fusarium spp. occurring on wheat. Canadian Journal of Botany 59: 1599–1609. https://doi.org/10.1139/b81-217
  • Old KM, Pham QT, Dudzinski MJ, Gibbs RJ (1999) Eucalyptus pathology in Vietnam. In: Proceedings of the workshop on eucalypt diseases, ACIAR, Ho Chi Minh City, Vietnam. CSIRO Forestry and Forest Products, Canberra and Forest Science Institute of Vietnam, Hanoi, 5.
  • Old KM, Wingfield MJ, Yuan ZQ (2003) A manual of diseases of eucalypts in South-East Asia. Centre for International Forestry Research, Indonesia.
  • Pan R, Deng Q, Xu D, Ji C, Deng M, Chen W (2012) First Report of Peanut Cylindrocladium Black Rot Caused by Cylindrocladium parasiticum in Fujian Province, Eastern China. Plant Disease 99: 890. http://dx.doi.org/10.1094/PDIS11110982
  • Peerally A (1991) The classification and phytopathology of Cylindrocladium species. Mycotaxon 40: 323–366.
  • Pei WH, Cao JF, Yang MY, Zhao ZJ, Xue SM (2015) First report of black rot of Medicago sativa caused by Cylindrocladium parasiticum (teleomorph Calonectria ilicicola) in Yunnan Province, China. Plant Disease 99: 890. http://dx.doi.org/10.1094/PDIS11141171PDN
  • Polizzi G, Catara V (2001) First report of leaf spot caused by Cylindrocladium pauciramosum on Acacia retinodes, Arbutus unedo, Feijoa sellowiana and Dodonaea viscosa in southern Italy. Plant Disease 85: 803. https://doi.org/10.1094/PDIS.2001.85.7.803C
  • Polizzi G, Vitale A, Aiello D, Castello I, Guarnaccia V, Parlavecchio G (2009) First record of crown and root rot caused by Cylindrocladium pauciramosum on brush cherry in Italy. Plant Disease 93: 547. http://dx.doi.org/10.1094/PDIS9350547A
  • Rayner RW (1970) A mycological colour chart. Commonwealth Mycological Institute and British Mycological Society. Kew, Surrey, UK.
  • Rodas CA, Lombard L, Gryzenhoinf M, Slippers B, Wingfield MJ (2005) Cylindrocladium blight of Eucalyptus grandis in Colombia. Australasian Plant Pathology 34: 143–149. https://doi.org/10.1071/AP05012
  • Saracchi M, Rocchi F, Pizzatti C, Cortesi P (2008) Box blight, a new disease of Buxus in Italy caused by Cylindrocladium buxicola. Journal of plant pathology 90: 581–584.
  • Serrato-Diaz LM, Latoni-Brailowsky EI, Rivera-Vargas LI, Goenaga R, Crous PW, French-Monar RD (2013) First Report of Calonectria hongkongensis Causing Fruit Rot of Rambutan (Nephelium lappaceum). Plant Disease 97: 1117. http://dx.doi.org/10.1094/PDIS01130008PDN
  • Sobers EK, Littrell RH (1974) Pathogenicity of three species of Cylindrocladium to select hosts. Plant Disease Reporter 58: 1017–1019.
  • Swofford DL (2003) PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). V. 4.0b10. Sinauer Associates, Sunderland, Massachusetts, USA.
  • Tamura K, Stecher G, Peterson D, Filipski A, Sudhir Kumar (2013) MEGA6: Molecular evolutionary genetics analysis v. 6.0. Molecular Biology and Evolution 30: 2725–2729. https://doi.org/10.1093/molbev/mst197
  • Vitale A, Aiello D, Castello I, Dimartino MA, Parlavecchio G, Polizzi G (2009) Severe outbreak of crown rot and root rot caused by Cylindrocladium pauciramosum on strawberry tree in Italy. Plant Disease 93: 842. http://dx.doi.org/10.1094/PDIS9380842B
  • Vitale A, Crous PW, Lombard L, Polizzi G (2013) Calonectria diseases on ornamental plants in Europe and the Mediterranean basin: an overview. Journal of Plant Pathology 95: 463–476. http: //doi .org/10.4454/JPP.V95I3.007
  • Xu JJ, Qin SY, Hao YY, Ren J, Tan P, Bahkali AH, Hyde KD, Wang Y (2012) A new species of Calonectria causing leaf disease of water lily in China. Mycotaxon 122: 177–185. https://doi.org/10.5248/122.177
  • Yang W, Zheng L, Wang C, Xie CP (2014) The First Report of Calonectria pteridis causing a Leaf Spot Disease on Serenoa repens in China. Plant Disease 98: 854–855. https://doi.org/10.1094/PDIS11131167PDN
  • Zhou XD, Xie YJ, Chen SF, Wingfield MJ (2008) Diseases of eucalypt plantations in China: challenges and opportunities. Fungal Diversity 32: 1–7.