Research Article |
Corresponding author: ShuaiFei Chen ( shuaifei.chen@gmail.com ) Academic editor: George Mugambi
© 2017 QianLi Liu, ShuaiFei Chen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Liu QL, Chen SF (2017) Two novel species of Calonectria isolated from soil in a natural forest in China. MycoKeys 26: 25-60. https://doi.org/10.3897/mycokeys.26.14688
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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.
C ylindrocladium , pathogen, phylogeny, taxonomy
Calonectria species include many notorious plant pathogens and are widely distributed in tropical and subtropical areas of the world (
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 (
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 (
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 (
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
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.
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
Based on previous research (
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
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 (
Species | Isolate No.†,‡ | Substrate | Sampling site | Collector | GenBank accession No.§,| | Reference | ||||||||||||||||
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tef1 | his3 | cmdA | tub2 | |||||||||||||||||||
Calonectria acicola | CBS 114813 | Pinus radiata | New Zealand | H. Pearson | GQ267292 | DQ190693 | GQ267360 | DQ190591 |
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CBS 114812 | P. radiata | New Zealand | H. Pearson | GQ267291 | DQ190692 | GQ267359 | DQ190590 |
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Ca. aconidialis | CBS 136086 | Soil in Eucalyptus plantation | HaiNan, China | X. Mou & S.F. Chen | KJ462785 | KJ463133 | KJ463017 | N/A¶ |
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Ca. arbusta | CBS 136079 | Soil in Eucalyptus plantation | GuangXi, China | X. Zhou & G. Zhao | KJ462787 | KJ463135 | KJ463018 | KJ462904 |
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Ca. asiatica | CBS 114073 | Leaf litter | Thailand | N.L. Hywel-Jones | AY725705 | AY725658 | AY725741 | AY725616 |
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CBS 112711 | Leaf litter | Thailand | N.L. Hywel-Jones | AY725702 | AY725655 | AY725738 | AY725613 |
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Ca. australiensis | CBS 112954 | Ficus pleurocarpa | Australia | C. Pearce & B. Paulu | GQ267293 | DQ190699 | GQ267363 | DQ190596 |
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Ca. brassicicola | CBS 112841 | Brassica sp. | Indonesia | M.J. Wingfield | KX784689 | N/A | KX784561 | KX784619 |
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Ca. canadiana | CBS 110817 | Picea sp. | Canada | S. Greifenhagen | GQ267297 | AF348228 | AY725743 | AF348212 |
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Ca. chinensis | CBS 114827 | Soil | Hong Kong | E.C.Y. Liew | AY725710 | AY725661 | AY725747 | AY725619 |
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CBS 112744 | Soil | Hong Kong | E.C.Y. Liew | AY725709 | AY725660 | AY725746 | AY725618 |
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Ca. colhounii | CBS 293.79 | Camellia sinensis | Indonesia | N/A | GQ267301 | DQ190639 | GQ267373 | DQ190564 |
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CBS 114704 | Arachis pintoi | Australia | D. Hutton | GQ267300 | DQ190638 | GQ267372 | DQ190563 |
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Ca. colombiensis | CBS 112220 | Eucalyptus grandis | Colombia | M.J. Wingfield | AY725711 | AY725662 | AY725748 | GQ267207 |
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CBS 112221 | E. grandis | Colombia | M.J. Wingfield | AY725712 | AY725663 | AY725749 | AY725620 |
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Ca. crousiana | CBS 127198 | E. grandis | FuJian, China | M.J. Wingfield | HQ285822 | HQ285808 | MF527084 | HQ285794 |
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CBS 127199 | E. grandis | FuJian, China | M.J. Wingfield | HQ285823 | HQ285809 | MF527085 | HQ285795 |
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Ca. curvispora | CBS 116159 | Soil | Madagascar | P.W. Crous | GQ267302 | AY725664 | GQ267374 | AF333394 |
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Ca. eucalypti | CBS 125275 | E. grandis | Sumatra Utara | M.J. Wingfield | GQ267338 | GQ267267 | GQ267430 | GQ267218 |
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CBS 125276 | E. grandis | Sumatra Utara | M.J. Wingfield | GQ267339 | GQ267268 | GQ267431 | GQ267219 |
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Ca. expansa | CBS 136247 | Soil in Eucalyptus plantation | Guangxi, China | X. Zhou & G. Zhao | KJ462798 | KJ463146 | KJ463029 | KJ462914 |
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CBS 136078 | Soil in Eucalyptus plantation | Guangdong, China | X. Zhou & G. Zhao | KJ462797 | KJ463145 | KJ463028 | KJ462913 |
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Ca. fujianensis | CBS 127201 | E. grandis | FuJian, China | M.J. Wingfield | HQ285820 | HQ285806 | MF527089 | HQ285792 |
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CBS 127200 | E. grandis | FuJian, China | M.J. Wingfield | HQ285819 | HQ285805 | MF527088 | HQ285791 |
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Ca. guangxiensis | CBS 136092 | Soil in Eucalyptus plantation | Guangxi, China | X. Mou & R. Chang | KJ462803 | KJ463151 | KJ463034 | KJ462919 |
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CBS 136094 | Soil in Eucalyptus plantation | Guangxi, China | X. Mou & R. Chang | KJ462804 | N/A | KJ463035 | KJ462920 |
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Ca. hainanensis | CBS 136248 | Soil in Eucalyptus plantation | Hainan, China | X. Mou & S.F. Chen | KJ462805 | KJ463152 | KJ463036 | N/A |
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Ca. hongkongensis | CBS 114828 | Soil | Hong Kong | E.C.Y. Liew | AY725717 | AY725667 | AY725755 | AY725622 |
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CBS 114711 | Soil | Hong Kong | M.J. Wingfield | AY725716 | AY725666 | AY725754 | AY725621 |
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Ca. ilicicola | CBS 190.50 | Solanum tuberosum | Indonesia | K.B. Boedijn &J. Reitsma | AY725726 | AY725676 | AY725764 | AY725631 |
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CBS 112215 | A. hypogaea | U.S.A. | Beute | AY725726 | AY725684 | AY725765 | AY725639 |
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Ca. indonesiae | CBS 112823 | Syzygium aromaticum | Indonesia | M.J. Wingfield | AY725718 | AY725668 | AY725756 | AY725623 |
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CBS 112840 | S. aromaticum | Indonesia | M.J. Wingfield | AY725720 | AY725670 | AY725758 | AY725625 |
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C. indonesiana | CBS 112936 | Soil | Indonesia | M.J. Wingfield | KX784701 | N/A | KX784573 | KX784631 |
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Ca. indusiata | CBS 144.36 | N/A | N/A | N/A | GQ267332 | GQ267262 | GQ267453 | GQ267239 |
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CBS 114684 | Rhododendron sp. | U.S.A. | N.E. El-Gholl | GQ267333 | DQ190653 | GQ267454 | AF232862 |
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Ca. kyotensis | CBS 170.77 | Idesia polycarpa | New Zealand | N/A | GQ267308 | GQ267249 | GQ267380 | GQ267209 |
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CBS 413.67 | Paphiopedilum callosum | Celle, Germany | W. Gerlach | GQ267307 | GQ267248 | GQ267379 | GQ267208 |
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Ca. lateralis | CBS 136629 | Soil in Eucalyptus plantation | Guangxi,China | X. Zhou & G. Zhao | KJ462840 | KJ463186 | KJ463070 | KJ462955 |
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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 |
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Ca. magnispora | CBS 136249 | Soil in Eucalyptus plantation | Guangxi, China | X. Mou & R. Chang | KJ462841 | KJ463187 | KJ463071 | KJ462956 |
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Ca. malesiana | CBS 112752 | Soil | Indonesia | M.J. Wingfield | AY725722 | AY725672 | AY725760 | AY725627 |
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CBS 112710 | Debris | Thailand | N.L. Hywel-Jones | AY725721 | AY725671 | AY725759 | AY725626 |
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Ca. microconidialis | CBS 136638 | E. urophylla × E. grandis clone seedling leaf | Guangdong, China | G. Zhao | KJ462845 | KJ463191 | KJ463075 | KJ462960 |
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CBS 136633 | E. urophylla × E. grandis clone seedling leaf | Guangdong, China | G. Zhao | KJ462842 | KJ463188 | KJ463072 | KJ462957 |
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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 |
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CPC 28836 | Soil | Thailand | P.W. Crous | KT964774 | N/A | KT964772 | KT964770 |
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Ca. multiseptata | CBS 112682 | Eucalyptus sp. | Indonesia | M.J. Wingfield | FJ918535 | DQ190659 | GQ267397 | DQ190573 |
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Ca. nymphaeae | CBS 131802 | Nymphaea tetragona | Guiyang, Guizhou | S.Y. Qin | KC555273 | N/A | N/A | JN984864 |
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HGUP 100004 | N. tetragona | Guiyang, Guizhou | Y. Wang | KC555274 | N/A | N/A | JN984865 |
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Ca. pacifica | CBS 109063 | Araucaria heterophylla | Hawaii, USA | M. Aragaki | AY725724 | GQ267255 | AY725762 | GQ267213 |
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CBS 114038 | Ipomoea aquatica | New Zealand | C.F. Hill | GQ267320 | AY725675 | GQ267402 | AY725630 |
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Ca. paracolhounii | CBS 114679 | N/A | USA | A.Y. Rossman | KX784714 | N/A | KX784582 | KX784644 |
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CBS 114705 | Annona reticulata | Australia | D. Hutton | KX784715 | N/A | N/A | KX784645 |
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Ca. parakyotensis | CBS 136085 | Soil in Eucalyptus plantation | Guangdong, China | X. Mou & R. Chang | KJ462851 | KJ463197 | KJ463081 | N/A |
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CBS 136095 | Soil in Eucalyptus plantation | Guangxi, China | X. Mou & R. Chang | KJ462852 | KJ463198 | KJ463082 | N/A |
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Ca. parva | CBS 110798 | Eucalyptus grandis roots | South Africa | P.W. Crous | KX784716 | N/A | KX784583 | KX784646 |
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Ca. pauciramosa | CMW 5683 | E. grandis | South Africa | P.W. Crous | FJ918565 | FJ918531 | GQ267405 | FJ918514 |
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CMW 30823 | E. grandis | South Africa | P.W. Crous | FJ918566 | FJ918532 | GQ267404 | FJ918515 |
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Ca. penicilloides | CBS 174.55 | Prunus sp. | Japan | Tubaki | GQ267322 | GQ267257 | GQ267406 | AF333414 |
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Ca. pluriramosa | CBS 136976 | Soil in Eucalyptus plantation | Guangxi, China | X. Zhou & G. Zhao | KJ462882 | KJ463228 | KJ463112 | KJ462995 |
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Ca. pseudokyotensis | CBS 137332 | Soil in Eucalyptus plantation | Guangxi,China | X. Zhou & G. Zhao | KJ462881 | KJ463227 | KJ463111 | KJ462994 |
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Ca. pseudocolhounii | CBS 127195 | E. dunnii | FuJian, China | M.J. Wingfield | HQ285816 | HQ285802 | MF527091 | HQ285788 |
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CBS 127196 | E. dunnii | FuJian, China | M.J. Wingfield | HQ285817 | HQ285803 | MF527092 | HQ285789 |
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Ca. pseudoreteaudii | CBS 123694 | E. urophylla × E. grandis cutting | Guangdong, China | M.J. Wingfield | FJ918541 | FJ918519 | GQ267411 | FJ918504 |
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CBS 123696 | E. urophylla × E. grandis cutting | Guangdong, China | M.J. Wingfield | FJ918542 | FJ918520 | GQ267410 | FJ918505 |
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Ca. queenslandica | CBS 112146 | E. urophylla | Australia | B. Brown | FJ918543 | FJ918521 | GQ267415 | AF389835 |
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CBS 112155 | E. pellita | Australia | K.M. Old | FJ918544 | DQ190667 | GQ267416 | AF389834 |
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Ca. reteaudii | CBS 112144 | E. camaldulensis | Vietnam | M.J. Dudzinski | FJ918537 | DQ190661 | GQ267417 | AF389833 |
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CBS 112143 | E. camaldulensis | Vietnam | M.J. Dudzinski | FJ918536 | DQ190660 | GQ267418 | GQ240642 |
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Ca. sphaeropendunculata | CBS 136081 | Soil in Eucalyptus plantation | Guangxi, China | X. Zhou & G. Zhao | KJ462890 | KJ463236 | KJ463120 | KJ463003 |
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Ca. sumatrensis | CBS 112829 | Soil | Indonesia | M.J. Wingfield | AY725733 | AY725696 | AY725771 | AY725649 |
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CBS 112934 | Soil | Indonesia | M.J. Wingfield | AY725735 | AY725698 | AY725773 | AY725651 |
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Ca. syzygiicola | CBS 112831 | Soil | Indonesia | M.J. Wingfield | KX784736 | N/A | N/A | KX784663 |
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Ca. terrae-reginae | CBS 112151 | E. urophylla | Australia | C. Hanwood | FJ918545 | FJ918522 | GQ267451 | FJ918506 |
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CBS 112634 | Xanthorrhoea australis | Australia | T. Baigent | FJ918546 | DQ190668 | GQ267452 | FJ918507 |
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Ca. turangicola | CBS 136077 | Soil in Eucalyptus plantation | Guangxi, China | X. Zhou & G. Zhao | KJ462900 | KJ463246 | N/A | KJ463013 |
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CBS 136093 | Soil in Eucalyptus plantation | Guangxi, China | X. Mou & R. Chang | KJ462901 | KJ463247 | KJ463130 | KJ463014 |
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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 |
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 (
ML analyses were performed using PHYML v. 3.0 (
Based on the morphological characteristics, datasets were separated into two groups: the Prolate Group and the Sphaero-Naviculate Group (
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;
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;
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
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
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
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
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
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
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.
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) |
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 |
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
lichi, which is Calonectria in Chinese.
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.
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.
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.
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.
Soil in a natural forest.
Central China.
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.
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 (
montis, meaning mountain in Latin, referring to the location where this fungus was collected.
Calonectria montana can be distinguished from the phylogenetically closely related species Ca. canadiana by the size of macroconidia.
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.
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.
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.
Soil under the natural forest.
Central China.
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.
Calonectria montana is a new addition to the Ca. kyotensis complex and is phylogenetically closely related to Ca. canadiana (
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 |
|
Ca. eucalypti | (66–)69–75(–80) × (5–)6 | 72 × 6 | 3 | 4–6 | broadly clavate |
|
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 |
|
Ca. nymphaeae | 55–63 × 5.3–6.3 | 61 × 5.9 | 3–4 | 3–5 | clavate |
|
Ca. paracolhounii | (37–)39–43(–45) × 4–5 | 41 × 5 | 3 | 3–5 | narrowly clavate |
|
Ca. pseudocolhounii | (49–)55–65(–74) × (3.5–)4–5(–5.5) | 60 × 4.5 | (1–)3 | (3.5–)4–5(–6) | clavate |
|
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 |
|
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 (
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 (
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,
Accurate diagnosis of plant diseases and identification of their casual agents provide the foundation for developing effective disease management strategies (
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.
Phylogenetic tree of Calonectria species in the Prolate group based on maximum likelihood (ML) analysis of tef1 gene sequences
Data type: molecular data
Explanation note: 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).
Phylogenetic tree of Calonectria species in the Prolate group based on maximum likelihood (ML) analysis of his3 gene sequences
Data type: molecular data
Explanation note: 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).
Phylogenetic tree of Calonectria species in the Prolate group based on maximum likelihood (ML) analysis of cmdA gene sequences
Data type: molecular data
Explanation note: 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).
Phylogenetic tree of Calonectria species in the Prolate group based on maximum likelihood (ML) analysis of tub2 gene sequences
Data type: molecular data
Explanation note: 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).
Phylogenetic tree of Calonectria species in the Sphaero-Naviculate group based on maximum likelihood (ML) analysis of tef1 gene sequences
Data type: molecular data
Explanation note: 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).
Phylogenetic tree of Calonectria species in the Sphaero-Naviculate group based on maximum likelihood (ML) analysis of his3 gene sequences
Data type: molecular data
Explanation note: 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).
Phylogenetic tree of Calonectria species in the Sphaero-Naviculate group based on maximum likelihood (ML) analysis of cmdA gene sequences
Data type: molecular data
Explanation note: 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).
Phylogenetic tree of Calonectria species in the Sphaero-Naviculate group based on maximum likelihood (ML) analysis of tub2 gene sequences
Data type: molecular data
Explanation note: 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).