Corresponding author: ShuaiFei Chen (
Academic editor: R. Phookamsak
Liu FF, Marincowitz S, Chen SF, Mbenoun M, Tsopelas P, Soulioti N, Wingfield MJ (2020) Novel species of
Many species in the
The objective of this study was to identify two fungal isolates collected from
Three South African
Isolates from Greece were made by transferring ascospore masses from the tips of the ascomata on the surface of
All the isolates obtained in this study were used for DNA sequence-based characterisation. Total genomic DNA was extracted from the mycelium of isolates grown on 2% MEA for 3–4 d at 25 °C, using Prepman Ultra Sample Preparation Reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocols. Three gene regions were amplified for sequencing and phylogenetic analyses. These included the Internal Transcribed Spacer (
A total volume of 25 μl PCR reaction mixture contained 1 μl of DNA template, 0.5 μl (10 pM) of each primer (Forward and Reverse), 5 μl MyTaq PCR buffer (Bioline GmbH, Germany) and 0.3 μl of MyTaq DNA Polymerase (Bioline GmbH, Germany). The PCR reactions were conducted using an Applied Biosystems ProFlex PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The PCR programme for amplification of the
The programme Geneious v. 7.0 was used to edit and assemble raw sequence reads into contigs (
Single gene sequence datasets of the
Morphological features were studied on the isolates grown on 2% MEA. The fruiting structures were initially mounted in water and this was later replaced with 85% lactic acid and in which measurements were made and images captured. Nikon microscopes (Eclipse N
A study of growth in culture was conducted at temperatures from 5–35 °C at 5 °C intervals on the 90 mm Petri dishes containing 2% MEA. A mycelial plug (5 mm diam.) taken from an actively-growing colony was placed at the centres of Petri dishes. Four replicates per isolate were used to study growth rate and the experiment was repeated once. Colony diameters were assessed by taking two measurements perpendicular to each other for all isolates daily and growth rates were calculated. Colony characteristics were described on the same medium used for the growth studies and colours were assessed using the colour charts of
The mating type (
Six isolates resembling
List of
Speciesa | CMW No.b | Other No.b | GenBank accession No.c | Hosts (or substrate) | Origin | Reference | ||
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CMW 43029 | CERC 2170; |
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China |
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CBS 139654 | ||||||||
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CMW 44684 | CERC 2827; |
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China |
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CBS 143283 | ||||||||
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CMW 44686 | CERC 2829; |
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China |
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CBS 143282 | ||||||||
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CMW 49312 | CERC 2854; |
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China |
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CBS 143286 | ||||||||
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CMW 49314 | CERC 2862; |
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China |
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CBS 143285 | ||||||||
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CMW 8242 | CBS 112907 |
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Bhutan |
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CMW 8217 | CBS 114289 |
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Bhutan |
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CMW 15245 | CBS 122299 |
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Malawi |
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CMW 15248 | CBS 122300 |
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Malawi |
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CMW 24658 | CBS 127185 |
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China |
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CMW 24661 | CBS 127186 |
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China |
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CMW 36932 | CBS 131674 |
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Cameroon |
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CMW 37102 | CBS 131675 |
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Cameroon |
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CMW 43452 | CERC 2158; |
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China |
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CBS 143577 | ||||||||
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CMW 43453 | CERC 2162; |
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China |
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CBS 143288 | ||||||||
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CMW 36826 | CBS 131277 |
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South Africa |
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CMW 36828 | CBS 131279 |
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South Africa |
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CMW 25918 | CBS 129735 |
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South Africa |
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CMW 25914 | CBS 129737 |
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South Africa |
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CMW 44692 | CERC 2840; |
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China |
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CBS 143291 | ||||||||
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CMW 44693 | CERC 2841; |
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China |
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CBS 143290 | ||||||||
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CMW 49307 | CERC 2753; |
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China |
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CBS 143294 | ||||||||
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CMW 49309 | CERC 2763; |
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China |
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CBS143292 | ||||||||
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CMW 49302 | CERC 2449; |
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China |
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CBS 143296 | ||||||||
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CMW 49303 | CERC 2451a; |
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China |
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CBS 143295 | ||||||||
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CMW 43436 | CERC 2132; |
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China |
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CBS 143298 | ||||||||
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CMW 49299 | CERC 2133; |
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China |
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CBS 143297 | ||||||||
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CMW 44372 | CERC 2740; |
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China |
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CBS 143300 | ||||||||
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CMW 49306 | CERC 2749; |
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China |
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CBS 143299 | ||||||||
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CMW 21106 |
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Indonesia |
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CMW 21107 | CBS 124009 |
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Indonesia |
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CMW 44374 | CERC 2742; |
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China |
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CBS 143302 | ||||||||
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CMW 44376 | CERC 2746; |
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China |
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CBS 143301 | ||||||||
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CMW 21117 | CBS 124013 |
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Indonesia |
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CMW 21115 | CBS 124015 |
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Indonesia |
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CMW 9590 | CBS 116452 |
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South Africa |
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CMW 4114 | CBS 118151 |
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Ecuador |
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CMW 9986 | CBS 109441 |
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Australia | Yuan & Mohammed 2002 |
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CMW 10214 | CBS 115792 |
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Australia | Yuan & Mohammed 2002 |
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CMW 23803 | CBS 122291 |
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South Africa |
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CMW 23802 |
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South Africa |
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CMW 11048 | CBS 115787 |
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Oman |
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CMW 3800 | CBS 117839 |
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Oman |
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CMW 36916 | CBS 131672 |
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Cameroon |
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CMW 36910 |
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Cameroon |
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CMW 25911 | CBS 129733 |
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South Africa |
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CMW 30703 | CBS 129734 |
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South Africa |
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CMW 17300 | CBS 121151 |
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South Africa |
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CMW 17297 |
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South Africa |
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CMW 22449 | CBS 122517 |
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Ecuador |
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CMW 22444 | CBS 122518 |
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Ecuador |
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CMW 21109 | CBS 124011 |
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Indonesia |
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CMW 21111 | CBS 124012 |
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Indonesia |
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CMW 13011 | CBS 115867 |
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Indonesia |
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CMW 13012 | CBS 118242 |
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Indonesia |
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CMW 28917 |
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Australia |
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CMW 28920 |
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Australia |
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a Species indicated in bold are newly described in this study. b CBS = Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CERC = Culture collection of China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), ZhanJiang, GuangDong Province, China; CMW = Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; PPRI = The living culture collection (PPRI) of the South African National Collection of Fungi (NCF), Roodeplaat, Pretoria, South Africa. c GenBank accession numbers indicated in bold are generated in this study.
All six isolates, included in this study, were successfully sequenced at all three selected gene regions for phylogenetic analyses, resulting in DNA sequence data of approximately 614, 574 and 830 bp for the
The three tree topologies resulting from
The name refers to the country, Greece where this fungus was collected.
Homothallic, with sexually complementary isolates having both the MAT1-1-1 and MAT1-2-1 genes.
Cultures on 2% MEA in dark in 8 d showing circular growth with even edge, mycelium flat, superficial, medium dense and texture becoming pelt-like with age, colour above not uniform, salmon (11f’) to ochreous (15b’) with inner half irregularly umber (13m), below ochreous (15b’) with inner half irregularly umber (13i’) at centre. Optimum growth temperatures at 30 °C at 9.6 mm/d, followed by at 25 °C (7.6 mm/d), 35 °C (7.2 mm/d), 20 °C (4.7 mm/d), 15 °C (3.2 mm/d), 10 °C (1.1 mm/d) and 5 °C (0.2 mm/d).
Greece, Phthiotis, near the village Kastri, occurring on freshly-cut stumps of
Micrographs of
The sexual state of
The name refers to the Kruger National Park in South Africa, where this fungus was collected.
Heterothallic with isolates having either a MAT1-1-1 gene or a MAT1-2-1 gene.
Micrographs of
Cultures on 2% MEA in dark in 8 d showing circular growth with even edge, mycelium superficial, flat, dense, colour above uniformly white, below luteous (19). Optimum growth temperatures were at 30 °C at 9 mm/d, followed by at 25 °C (8.2 mm/d), 35 °C (6.2 mm/d), 20 °C (6 mm/d), 15 °C (3.4 mm/d), 10 °C (0.9 mm/d) and 5 °C (0.3 mm/d).
South Africa, Mpumalanga, Kruger National Park, Satara rest camp,
South Africa, Mpumalanga, Kruger National Park, Punda Maria,
This study led to the discovery of two novel
The stump of
The novel species described in this study showed typical characteristics of
Comparison of DNA sequence data for multiple gene regions is essential when seeking to identify species in
Primers, developed to identify the mating type idiomorphs in
The two new species of
This study was initiated through the bilateral agreement between the Governments of South Africa and China and supported by The National Key R&D Program of China (China-South Africa Forestry Joint Research Centre Project; project No. 2018YFE0120900), the National Ten-thousand Talents Program (Project No. W03070115) and the GuangDong Top Young Talents Program (Project No. 20171172). We acknowledge members of Tree Protection and Cooperation Programme (TPCP) and the National Research Foundation (NRF), South Africa for financial support.
Figure S1.
phylogenetic tree
Sequences generated from this study are printed in bold type. Bold branches indicate posterior probabilities values ≥ 0.9. Bootstrap values and posterior probabilities value are presented above branches as
Figure S2.
phylogenetic tree
Sequences generated from this study are printed in bold type. Bold branches indicate posterior probabilities values ≥ 0.9. Bootstrap values and posterior probabilities values are presented above branches as
Figure S3.
phylogenetic tree
Sequences generated from this study are printed in bold type. Bold branches indicate posterior probabilities values ≥ 0.9. Bootstrap values and posterior probabilities values are presented above branches as