Research Article |
Corresponding author: ShuaiFei Chen ( shuaifei.chen@gmail.com ) Academic editor: Rungtiwa Phookamsak
© 2020 FeiFei Liu, Seonju Marincowitz, ShuaiFei Chen, Michael Mbenoun, Panaghiotis Tsopelas, Nikoleta Soulioti, Michael J. Wingfield.
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 F, Marincowitz S, Chen S, Mbenoun M, Tsopelas P, Soulioti N, Wingfield MJ (2020) Novel species of Huntiella from naturally-occurring forest trees in Greece and South Africa. MycoKeys 69: 33-52. https://doi.org/10.3897/mycokeys.69.53205
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Huntiella species are wood-infecting, filamentous ascomycetes that occur in fresh wounds on a wide variety of tree species. These fungi are mainly known as saprobes although some have been associated with disease symptoms. Six fungal isolates with typical culture characteristics of Huntiella spp. were collected from wounds on native forest trees in Greece and South Africa. The aim of this study was to identify these isolates, using morphological characters and multigene phylogenies of the rRNA internal transcribed spacer (ITS) region, portions of the β-tubulin (BT1) and translation elongation factor 1α (TEF-1α) genes. The mating strategies of these fungi were also determined through PCR amplification of mating type genes. The study revealed two new species; one from Platanus orientalis in Greece and one from Colophospermum mopane and Senegalia nigrescens in South Africa. These novel taxa have been provided with the names, H. hellenica sp. nov. and H. krugeri sp. nov., respectively. The former species was found to have a homothallic and the latter a heterothallic mating system.
Ceratocystidaceae, Ceratocystis moniliformis Complex, Colophospermum mopane, Huntiella, Platanus orientalis, saprobes, Senegalia nigrescens
Huntiella species are members of the family Ceratocystidaceae (Microascales, Sordariomycetes) as defined by
Many species in the Ceratocystidaceae are important pathogens of woody plants, including agricultural, fruit and forest tree crops (
Huntiella species are most commonly isolated from freshly-made wounds on trees, to which they are vectored by insects, especially sap-feeding beetles in the Nitidulidae (
Huntiella spp. are particularly interesting in terms of their mating biology. Huntiella fecunda and H. moniliformis were, for example, shown to exhibit a unisexual mating system, unlike the many heterothallic species found in this genus (
Huntiella species are most commonly found in tropical and sub-tropical regions of the world (
The objective of this study was to identify two fungal isolates collected from Platanus orientalis L. in Greece and four isolates from Colophospermum mopane (Benth.) J. Léonard and Senegalia nigrescens (Oliv.) P. Hurter in South Africa. These fungi displayed typical culture characteristics of Huntiella spp., including rapid growth on agar medium, white fluffy mycelia when young, as well as the production of fruity aroma. Identification was accomplished, based on morphology and multigene phylogenies for the ITS, BT1 and TEF-1α gene regions. Furthermore, we considered the mating biology of these isolates in order to complement our taxonomic studies.
Three South African Huntiella isolates were collected from fresh wounds of Colophospermum mopane in Kruger National Park in April 2009 and another one of the South African isolates was obtained from a broken branch of a Senegalia nigrescens tree damaged by elephants in Kruger National Park in June 2010. The isolates from Greece were obtained from the stump of a Platanus orientalis tree that was cut about two months before sampling, in a natural forest along the banks of the Spercheios River in Phthiotis Regional Unit during November 2018. Isolation from wood samples was performed using a trapping technique originally described by
Isolates from Greece were made by transferring ascospore masses from the tips of the ascomata on the surface of Platanus twig baits, formed on infected wood surface, to 2% malt extract agar (MEA: 20 g Biolab malt extract, 20 g Difco agar, 1 litre water), using a sterile needle under a dissection microscope (Carl Zeiss Co. Ltd., Oberkochen, Germany). The South African isolate was obtained by transferring mycelial strands from infected wood on to MEA. Primary isolations were incubated for 3–7 d at 25 °C. From these isolations, purified cultures from single hyphal tips were prepared for morphological characterisation, phylogenetic analyses and mating-type studies. All purified isolates were deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa and the living culture collection (PPRI) of the South African National Collection of Fungi (NCF), Roodeplaat, Pretoria, South Africa. The dried-down type specimens were deposited in the National Collection of Fungi (PREM), Roodeplaat, Pretoria, South Africa.
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 (ITS) regions 1 and 2, including the 5.8S rRNA, a partial β-tubulin 1 gene (BT1) and a partial Translation Elongation factor-1α gene (TEF-1α), amplified using the set of primers as described by
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 ITS, BT1 and TEF1-α gene regions was as follows: an initial denaturation step at 95 °C for 5 min followed by 35 cycles of 30 s at 95 °C, 45 s at 56 °C and 60 s at 72 °C and a final extension step at 72 °C for 10 min. Amplified fragments were purified using ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA) to remove excess primers and dNTPs. Amplicons were sequenced in both directions using an ABI PRISM™ 3100 DNA sequencer (Applied Biosystems, USA) at the Sequencing Facility of the Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, South Africa.
The programme Geneious v. 7.0 was used to edit and assemble raw sequence reads into contigs (
Single gene sequence datasets of the ITS, BT1 and TEF-1α and the combined dataset of the three gene regions were analysed using Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI). The appropriate substitution model for each dataset was obtained using the software package jModeltest v. 2.1.5 (
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 Ni, SMZ 18, Nikon, Tokyo, Japan) mounted with a camera (Nikon DS Ri-2) were used for all observations. Fifty measurements of each relevant microscopic structure were made when available and these are presented as minimum–maximum and average ± standard deviation.
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 (MAT) of the studied Huntiella spp. was determined, based on the results of the mating type PCR reactions (
Six isolates resembling Huntiella spp. were included in this study. Two isolates had ascomata with long necks, conical spines on the ascomatal bases and hat-shaped ascospores and four isolates had only thielaviopsis-like asexual state (
Speciesa | CMW No.b | Other No.b | GenBank accession No.c | Hosts (or substrate) | Origin | Reference | ||
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ITS | BT1 | TEF-1α | ||||||
Ceratocystis cercfabiensis | CMW 43029 | CERC 2170; | KP727592 | KP727618 | KP727643 | Eucalyptus sp. | China |
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CBS 139654 | ||||||||
Huntiella ani | CMW 44684 | CERC 2827; | MH118602 | MH118635 | MH118668 | Eucalyptus sp. | China |
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CBS 143283 | ||||||||
H. ani | CMW 44686 | CERC 2829; | MH118603 | MH118636 | MH118669 | Eucalyptus sp. | China |
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CBS 143282 | ||||||||
H. bellula | CMW 49312 | CERC 2854; | MH118607 | MH118640 | MH118673 | Eucalyptus sp. | China |
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CBS 143286 | ||||||||
H. bellula | CMW 49314 | CERC 2862; | MH118610 | MH118643 | MH118676 | Eucalyptus sp. | China |
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CBS 143285 | ||||||||
H. bhutanensis | CMW 8242 | CBS 112907 | AY528951 | AY528956 | AY528961 | Picea spinulosa | Bhutan |
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H. bhutanensis | CMW 8217 | CBS 114289 | AY528957 | AY528962 | AY528952 | P. spinulosa | Bhutan |
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H. ceramica | CMW 15245 | CBS 122299 | EU245022 | EU244994 | EU244926 | Eucalyptus grandis | Malawi |
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H. ceramica | CMW 15248 | CBS 122300 | EU245024 | EU244996 | EU244928 | E. grandis | Malawi |
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H. chinaeucensis | CMW 24658 | CBS 127185 | JQ862729 | JQ862717 | JQ862741 | Eucalyptus sp. | China |
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H. chinaeucensis | CMW 24661 | CBS 127186 | JQ862731 | JQ862719 | JQ862743 | Eucalyptus sp. | China |
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H. chlamydoformis | CMW 36932 | CBS 131674 | KF769087 | KF769109 | KF769098 | Theobroma cacao | Cameroon |
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H. chlamydoformis | CMW 37102 | CBS 131675 | KF769088 | KF769110 | KF769099 | Terminalia superba | Cameroon |
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H. confusa | CMW 43452 | CERC 2158; | MH118583 | MH118616 | MH118649 | Acacia confusa | China |
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CBS 143577 | ||||||||
H. confusa | CMW 43453 | CERC 2162; | MH118584 | MH118617 | MH118650 | A. confusa | China |
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CBS 143288 | ||||||||
H. cryptoformis | CMW 36826 | CBS 131277 | KC691462 | KC691486 | KC691510 | Terminalia sericea | South Africa |
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H. cryptoformis | CMW 36828 | CBS 131279 | KC691464 | KC691488 | KC691512 | Ziziphus mucronata | South Africa |
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H. decipiens | CMW 25918 | CBS 129735 | HQ203218 | HQ203235 | HQ236437 | E. cloeziana | South Africa |
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H. decipiens | CMW 25914 | CBS 129737 | HQ203219 | HQ203236 | HQ236438 | E. maculata | South Africa |
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H. eucalypti | CMW 44692 | CERC 2840; | MH118605 | MH118638 | MH118671 | Eucalyptus sp. | China |
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CBS 143291 | ||||||||
H. eucalypti | CMW 44693 | CERC 2841; | MH118606 | MH118639 | MH118672 | Eucalyptus sp. | China |
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CBS 143290 | ||||||||
H. fabiensis | CMW 49307 | CERC 2753; | MH118596 | MH118629 | MH118662 | Eucalyptus sp. | China |
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CBS 143294 | ||||||||
H. fabiensis | CMW 49309 | CERC 2763; | MH118599 | MH118632 | MH118665 | Eucalyptus sp. | China |
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CBS143292 | ||||||||
H. fecunda | CMW 49302 | CERC 2449; | MH118586 | MH118619 | MH118652 | Eucalyptus sp. | China |
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CBS 143296 | ||||||||
H. fecunda | CMW 49303 | CERC 2451a; | MH118587 | MH118620 | MH118653 | Eucalyptus sp. | China |
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CBS 143295 | ||||||||
H. glaber | CMW 43436 | CERC 2132; | MH118580 | MH118613 | MH118646 | E. exserta | China |
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CBS 143298 | ||||||||
H. glaber | CMW 49299 | CERC 2133; | MH118581 | MH118614 | MH118647 | E. exserta | China |
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CBS 143297 | ||||||||
H. hellenica | CMW 54800 | PPRI 27982 | MT524073 | MT513125 | MT513131 | Platanus orientalis | Greece | Present study |
H. hellenica | CMW 54801 | PPRI 27983 | MT524072 | MT513124 | MT513130 | P. orientalis | Greece | Present study |
H. inaequabilis | CMW 44372 | CERC 2740; | MH118590 | MH118623 | MH118656 | Eucalyptus sp. | China |
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CBS 143300 | ||||||||
H. inaequabilis | CMW 49306 | CERC 2749; | MH118595 | MH118628 | MH118661 | Eucalyptus sp. | China |
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CBS 143299 | ||||||||
H. inquinana | CMW 21106 | EU588587 | EU588666 | EU588674 | Acacia mangium | Indonesia |
|
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H. inquinana | CMW 21107 | CBS 124009 | EU588588 | EU588667 | EU588675 | A. mangium | Indonesia |
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H. krugeri | CMW 36849 | CBS 131676 PPRI 27952 | MT524068 | MT513120 | MT513126 | A. nigrescens | South Africa | Present study |
H. krugeri | CMW 55933 | MT524069 | MT513121 | MT513127 | Colophospermum mopane | South Africa | Present study | |
H. krugeri | CMW 55934 | MT524070 | MT513122 | MT513128 | C. mopane | South Africa | Present study | |
H. krugeri | CMW 55935 | MT524071 | MT513123 | MT513129 | C. mopane | South Africa | Present study | |
H. meiensis | CMW 44374 | CERC 2742; | MH118591 | MH118624 | MH118657 | Eucalyptus sp. | China |
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CBS 143302 | ||||||||
H. meiensis | CMW 44376 | CERC 2746; | MH118594 | MH118627 | MH118660 | Eucalyptus sp. | China |
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CBS 143301 | ||||||||
H. microbasis | CMW 21117 | CBS 124013 | EU588593 | EU588672 | EU588680 | A. mangium | Indonesia |
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H. microbasis | CMW 21115 | CBS 124015 | EU588592 | EU588671 | EU588679 | A. mangium | Indonesia |
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H. moniliformis | CMW 9590 | CBS 116452 | AY431101 | AY528985 | AY529006 | E. grandis | South Africa |
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H. moniliformis | CMW 4114 | CBS 118151 | AY528997 | AY528986 | AY529007 | Shizolobium parahyba | Ecuador |
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H. moniliformopsis | CMW 9986 | CBS 109441 | AY528998 | AY528987 | AY529008 | E. obliqua | Australia | Yuan & Mohammed 2002 |
H. moniliformopsis | CMW 10214 | CBS 115792 | AY528999 | AY528988 | AY529009 | E. sieberi | Australia | Yuan & Mohammed 2002 |
H. oblonga | CMW 23803 | CBS 122291 | EU245019 | EU244991 | EU244951 | A. mearnsii | South Africa |
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H. oblonga | CMW 23802 | EU245020 | EU244992 | EU244952 | A. mearnsii | South Africa |
|
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H. omanensis | CMW 11048 | CBS 115787 | DQ074742 | DQ074732 | DQ074737 | Mangifera indica | Oman |
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H. omanensis | CMW 3800 | CBS 117839 | DQ074743 | DQ074733 | DQ074738 | M. indica | Oman |
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H. pycnanthi | CMW 36916 | CBS 131672 | KF769096 | KF769118 | KF769107 | The. cacao | Cameroon |
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H. pycnanthi | CMW 36910 | KF769095 | KF769117 | KF769106 | The. cacao | Cameroon |
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H. salinaria | CMW 25911 | CBS 129733 | HQ203213 | HQ203230 | HQ236432 | E. maculata | South Africa |
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H. salinaria | CMW 30703 | CBS 129734 | HQ203214 | HQ203231 | HQ236433 | E. saligna | South Africa |
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H. savannae | CMW 17300 | CBS 121151 | EF408551 | EF408565 | EF408572 | A. nigrescens | South Africa |
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H. savannae | CMW 17297 | EF408552 | EF408566 | EF408573 | Combretum zeyheri | South Africa |
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H. sublaevis | CMW 22449 | CBS 122517 | FJ151431 | FJ151465 | FJ151487 | Terminalia ivorensis | Ecuador |
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H. sublaevis | CMW 22444 | CBS 122518 | FJ151430 | FJ151464 | FJ151486 | T. ivorensis | Ecuador |
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H. sumatrana | CMW 21109 | CBS 124011 | EU588589 | EU588668 | EU588676 | A. mangium | Indonesia |
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H. sumatrana | CMW 21111 | CBS 124012 | EU588590 | EU588669 | EU588677 | A. mangium | Indonesia |
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H. tribiliformis | CMW 13011 | CBS 115867 | AY528991 | AY529001 | AY529012 | Pinus merkusii | Indonesia |
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H. tribiliformis | CMW 13012 | CBS 118242 | AY528992 | AY529002 | AY529013 | P. merkusii | Indonesia |
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H. tyalla | CMW 28917 | HM071899 | HM071909 | HQ236448 | E. grandis | Australia |
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H. tyalla | CMW 28920 | HM071896 | HM071910 | HQ236449 | E. grandis | Australia |
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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 ITS, BT1 and TEF-1α gene regions, respectively. These newly-generated sequences were deposited in GenBank (Table
The three tree topologies resulting from ML, MP and BI were concordant and showed similar phylogenetic relationships amongst taxa (Fig.
ML tree of Huntiella species generated from the combined DNA sequence data of ITS, BT1 and TEF-1α DNA. 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 ML/MP/BI. Bootstrap value < 50% or probabilities values < 0.9 are marked with *. Nodes lacking the support value are marked with -. Ceratocystis cercfabiensis (CMW 43029) represents the outgroup.
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.
Sexual state. Ascomata produced in 2% MEA in a week, perithecial; ascomatal bases mostly embedded in thick or loose mycelial mat, globose to ellipsoidal or obpyriform, pale brown when young, becoming dark brown with age, 173–377 µm long (avg. 238.8 µm), 157–493 µm wide (avg. 218.2 µm), ornamented with spine-like structures, dark brown, conical, 12–29 µm long, 4–9 µm wide at base becoming attenuated; ostiolar necks upright, straight, occasionally situated at off-centre of base, darker than base when young, 344–616 µm long (avg. 515.5 µm), 34–60 µm wide (avg. 46.6 µm) at base, gradually tapering towards apex; ostiolar hyphae hyaline, straight to divergent, 15–39 µm long, 1–3 µm wide, tapering towards apex. Asci evanescent. Ascospores hyaline, subglobose, aseptate, covered with sheath giving a hat-like feature in side view, 4–5.5 × 3–4.5 µm (5 ± 0.23 × 4 ± 0.28 µm) excluding sheath.
Asexual state. Thielaviopsis-like Conidiophores macronematous, simple or branched; when branched radiating from basal cell once, often reduced to conidiogenous cells. Conidiogenus cells endoblastic, hyaline, varying from lageniform to cylindrical depending spore shape; in case of thick barrel-shaped conidia, apex often becoming wider than base. Conidia hyaline, 1-celled, in two recognisable shapes; majority ellipsoidal to barrel-shaped (side swollen, ends round), typical fat barrel-shaped 5–8 × 4.5–7.5 µm (5.9 ± 0.61 × 5.3 ± 0.55 µm), width of some barrel-shaped ranging 2.5–4 µm wide; rectangular-shaped (side straight, ends truncated), not commonly found, 5–9 × 1–3 µm (6.9 ± 1.18 × 2.3 ± 0.38 µm). Aleurioconidia not observed.
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 Platanus orientalis in a natural forest along the banks of the Spercheios River, Nov. 2018, P. Tsopelas & N. Soulioti, PREM 62889, holotype (dried culture of CMW 54800), culture ex-holotype CMW 54800 = PPRI 27982, other cultures CMW 54801 = PPRI 27983.
Micrographs of Huntiella hellenica sp. nov. (ex-holotype CMW 54800 = PPRI 27982) A culture grown on 2% MEA at 30 °C (optimum growth temperature) in the dark for 34 d B, C colony with ascomatal base embedded in mycelia with ascospore mass at the tip of ostiolar neck D–F young ascoma showing development of ostiolar neck and less-pigmented base G, H mature ascoma ornamented with spines I close-up of ascomatal wall showing spines J–L close up of ornament (spin-like) M, N Ostiolar hyphae O Ascospores P Ascospores covered with sheath appearing like a hat Q, R Germinating ascospores S Lageniform conidiogenous cell T Cylindrical-shape conidiogenous cell U Conidia in various shapes from diverse barrel-shaped to rectangular-shaped V rectangular-shaped conidia W chains of conidia. Scale bars: 1 mm (B, C); 50 µm (D–H); 10 µm (I–W).
The sexual state of H. hellenica developed at temperatures over 25 °C. Cultures incubated at 20 °C and below produced only the asexual state. Huntiella hellenica is closely related to H. savannae (
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.
Sexual state. Not observed.
Asexual state . Produced on 2% MEA in 3 weeks. Thielaviopsis-like. Conidiophores macronematous, upright, simple or branched in one tier, 29–37 µm in length, often reduced to conidiogenous cells; Conidiogenous cells enteroblastic, lageniform, 10–20 µm long, 1.5–3 µm wide, tapering towards apex. Conidia hyaline, rectangular-shaped, usually straight, with top-end conidium often club-shaped, 4–11 × 1–2 µm (avg. 6.2 × 1.7 µm). Aleurioconidia hyaline, holoblastic, mostly terminal, ellipsoidal to subglobose with an extended tube-like base, club-shaped, 4–7 × 2–3 µm (5.6 ± 0.76 × 2.5 ± 0.24 µm).
Micrographs of Huntiella krugeri sp. nov. (ex-holotype CMW 36849 = CBS 131676 = PPRI 27952). A Culture grown on 2% MEA in the dark for 34 d B, C Conidiogenous cell D Conidia in various shapes E Chain of conidia in different shapes F Chain of rectangular-shaped conidia with top-end of club-shaped G, H Aleurioconidia. Scale bars: 10 µm (B–H).
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, Senegalia nigrescens, June 2010, M. Mbenoun, PREM 62883, holotype (dried culture of CMW 36849), culture ex-holotype CMW 36849 = CBS 131676 = PPRI 27952.
South Africa, Mpumalanga, Kruger National Park, Punda Maria, Colophospermum mopane, April 2009, M. Mbenoun, CMW 55933, CMW 55934, CMW 55935.
Huntiella krugeri is closely related to H. hellenica described in the present study, H. cryptoformis (
This study led to the discovery of two novel Huntiella species isolated from Platanus orientalis in Greece, Colophospermum mopane and Senegalia nigrescens in the Kruger National Park of South Africa. These two species, provided with the name H. hellenica and H. krugeri, respectively, were shown to reside in the African Clade of Huntiella (
The stump of P. orientalis, from which H. hellenica emerged, was sampled approximately two months after tree felling and it was also infected by the pathogen Ceratocystis platani, which causes a devastating disease in natural stands of P. orientalis in Greece. Colonszation of the stump with H. hellenica could have occurred on the freshly-cut surface with a contaminated tool as occurs for C. platani (
The novel species described in this study showed typical characteristics of Huntiella spp. They grew rapidly in culture; their mycelium was white when young and turned dark with age. The one species that displayed a sexual state - H. hellenica, produced hat-shaped ascospores and had short conical spines on the ascomatal bases. Temperature is known to influence the ability of Huntiella spp. to produce a sexual state (
Comparison of DNA sequence data for multiple gene regions is essential when seeking to identify species in Huntiella (
Primers, developed to identify the mating type idiomorphs in Huntiella spp. (
The two new species of Huntiella, discovered in this study, bring the total number of species in the genus to 31. These are found in many different regions of the world and on a wide variety of woody substrates (
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. ML tree of Huntiella species generated from the ITS DNA sequence data
Data type: phylogenetic tree
Explanation note: 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 ML/MP/BI. Bootstrap value < 50% or probabilities values < 0.9 are marked with *. Nodes lacking the support value are marked with -. Ceratocystis cercfabiensis (CMW 43029) represents the outgroup.
Figure S2. ML tree of Huntiella species generated from the BT1 DNA sequence data
Data type: phylogenetic tree
Explanation note: 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 ML/MP/BI. Bootstrap value < 50% or probabilities values < 0.9 are marked with *. Nodes lacking the support value are marked with -. Ceratocystis cercfabiensis (CMW 43029) represents the outgroup.
Figure S3. ML tree of Huntiella species generated from the TEF-1α DNA sequence data
Data type: phylogenetic tree
Explanation note: 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 ML/MP/BI. Bootstrap value < 50% or probabilities values < 0.9 are marked with *. Nodes lacking the support value are marked with -. Ceratocystis cercfabiensis (CMW 43029) represents the outgroup.