Research Article |
Corresponding author: Jiwen Xia ( zhenjunxue@126.com ) Academic editor: Pedro Crous
© 2021 Zhaoxue Zhang, Taichang Mu, Shubin Liu, Rongyu Liu, Xiuguo Zhang, Jiwen Xia.
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:
Zhang Z, Mu T, Liu S, Liu R, Zhang X, Xia J (2021) Morphological and phylogenetic analyses reveal a new genus and two new species of Tubakiaceae from China. MycoKeys 84: 185-201. https://doi.org/10.3897/mycokeys.84.73940
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Species of Tubakiaceae have often been reported as plant pathogens or endophytes, commonly isolated from a wide range of plant hosts. The isolated fungi were studied through a complete examination, based on multilocus phylogenies from combined datasets of ITS/LSU/rpb2 and ITS/tef1/tub2, in conjunction with morphological characteristics. Five strains isolated from Lithocarpus fohaiensis and Quercus palustris in China represented a new genus of Tubakiaceae, Obovoideisporodochium and three species, viz. Obovoideisporodochium lithocarpi sp. nov., Tubakia lushanensis sp. nov. and T. dryinoides.
multigene phylogeny, new genus, new species, taxonomy, Tubakia
Diaporthales represents an important order in Sordariomycetes containing taxa that are mainly isolated as endophytes, saprobes or plant pathogens on various hosts (
Tubakia, the type genus of Tubakiaceae, was introduced by
During field trips to collect plant pathogens causing leaf spots symptoms in China, several specimens associated with typical diaporthalean symptoms were collected from various tree hosts, i.e. Betula dahurica (Betulaceae), Juglans regia (Juglandaceae), Prunus davidiana (Rosaceae), Lithocarpus fohaiensis, Quercus mongolica and Q. palustris (Fagaceae). Based on morphological analyses as well as phylogenetic data, this study presents a new genus of Tubakiaceae, Obovoideisporodochium and three species, viz. Obovoideisporodochium lithocarpi sp. nov., Tubakia lushanensis sp. nov. and T. dryinoides from diseased leaves of L. fohaiensis or Q. palustris.
The samples were collected from the Shandong and Yunnan Provinces, China. The strains were isolated from diseased leaves of Lithocarpus fohaiensis and Quercus palustris using tissue isolation methods. Tissue fragments (5 mm × 5 mm) were taken from the margin of leaf lesions and surface-sterilised by consecutively immersing in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s and then rinsing in sterile distilled water for 1 min. The pieces were dried with sterilised paper towels and placed on potato dextrose agar (PDA). All the PDA plates were incubated in a biochemical incubator at 25°C for 2–4 days. The colonies from the periphery were picked out and inoculated on to new PDA plates. Colony photos after 7 days and 15 days were taken by a digital camera (Canon Powershot G7X). Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope, all fitted with Olympus DP80 high definition colour digital cameras to photo-document fungal structures. All fungal strains were stored in 10% sterilised glycerine at 4°C for further studies. The holotype specimens are deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Ex-type cultures are deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).
Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in
The PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μl reaction volume, which contained 12.5 μl Green Taq Mix (Vazyme, Nanjing, China), 1 μl of each forward and reverse primer (10 μM stock) (Biosune, Shanghai, China) and 1 μl template genomic DNA in amplifier, adjusted with distilled deionised water to a total volume of 25 μl. The PCR parameters were as follows: 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at a suitable temperature for 50 s, extension at 72°C for 1 min and a final elongation step at 72°C for 10 min. The annealing temperatures for the genes were 55°C for ITS, 52°C for LSU, 53°C for tub2, 48°C for tef1 and 56°C for rpb2. The PCR products were separated with the 1% agarose gel, with added GelRed and UV light used to visualise the fragments. Sequencing was done bi-directionally, conducted by the Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA v. 7.0 (
Species and GenBank accession numbers of DNA sequences used in this study. New sequences in bold.
Species | Voucher1 | Host/Substrate | Country | GenBank accession number | ||||
ITS | LSU | tef1 | tub2 | rpb2 | ||||
Greeneria uvicola | FI12007 | ‒ | Uruguay | HQ586009 | GQ870619 | ‒ | ‒ | ‒ |
Involutiscutellula rubra | CBS 192.71* | Quercus phillyraeoides | Japan | MG591899 | MG591993 | MG592086 | MG592180 | MG976476 |
MUCC 2303 | Quercus phillyraeoides | Japan | MG591900 | MG591994 | MG592087 | MG592181 | MG976477 | |
ATCC 22473 | Quercus phillyraeoides | Japan | MG591901 | MG591995 | MG592088 | ‒ | MG976478 | |
Oblongisporothyrium castanopsidis | CBS 124732 | Castanopsis cuspidata | Japan | MG591849 | MG591942 | MG592037 | MG592131 | MG976453 |
CBS 189.71* | Castanopsis cuspidata | Japan | MG591850 | MG591943 | MG592038 | MG592132 | MG976454 | |
Obovoideisporodochium lithocarpi | SAUCC 0748* | Lithocarpus fohaiensis | China | MW820279 | MW821346 | MZ996876 | MZ962157 | MZ962155 |
SAUCC 0745 | Lithocarpus fohaiensis | China | MW820280 | MW821347 | MZ996877 | MZ962158 | MZ962156 | |
Paratubakia subglobosa | CBS 124733 | Quercus glauca | Japan | MG591913 | MG592008 | MG592102 | MG592194 | MG976489 |
CBS 193.71* | Quercus glauca | Japan | MG591914 | MG592009 | MG592103 | MG592195 | MG976490 | |
Paratubakia subglobosoides | MUCC 2293* | Quercus glauca | Japan | MG591915 | MG592010 | MG592104 | MG592196 | MG976491 |
Racheliella wingfieldiana | CBS 143669* | Syzigium guineense | Africa | MG591911 | MG592006 | MG592100 | MG592192 | MG976487 |
Sphaerosporithyrium mexicanum | CPC 32258 | Quercus eduardi | Mexico | MG591895 | MG591989 | MG592082 | MG592176 | ‒ |
CPC 33021* | Quercus eduardi | Mexico | MG591896 | MG591990 | MG592083 | MG592177 | MG976473 | |
Tubakia americana | CBS 129014 | Quercus macrocarpa | USA | MG591873 | MG591966 | MG592058 | MG592152 | MG976449 |
Tubakia californica | CPC 31505* | Quercus kelloggii | USA | MG591835 | MG591928 | MG592023 | MG592117 | MG976451 |
Tubakia dryina | CBS 112097* | Quercus robur | Italy | MG591851 | MG591944 | MG592039 | MG592133 | MG976455 |
Tubakia dryinoides | SAUCC 1924 | Quercus palustris | China | MW784842 | MW784852 | MW842260 | MW842263 | MW842266 |
CBS 329.75 | Quercus sp. | France | MG591874 | MG591967 | MG592059 | MG592153 | MG976458 | |
MUCC2290 | Castanea crenata | Japan | MG591876 | MG591968 | MG592061 | MG592155 | MG976459 | |
MUCC2291 | Castanea crenata | Japan | MG591877 | MG591969 | MG592062 | MG592156 | MG976460 | |
MUCC2292* | Quercus phillyraeoides | Japan | MG591878 | MG591970 | MG592063 | MG592157 | MG976461 | |
Tubakia hallii | CBS 129013 | Quercus stellata | USA | MG591880 | MG591972 | MG592065 | MG592159 | MG976462 |
Tubakia iowensis | CBS 129012* | Quercus macrocarpa | USA | MG591879 | MG591971 | MG592064 | MG592158 | ‒ |
Tubakia japonica | ATCC 22472* | Castanea crenata | Japan | MG591886 | MG591978 | MG592071 | MG592165 | MG976465 |
Tubakia koreana | KCTC46072 | Quercus mongolica | South Korea | KP886837 | ‒ | ‒ | ‒ | ‒ |
Tubakia liquidambaris | CBS 139744 | Liquidambar styraciflua | USA | MG605068 | MG605077 | MG603578 | ‒ | ‒ |
Tubakia lushanensis | SAUCC 1921 | Quercus palustris | China | MW784677 | MW784850 | MW842262 | MW842265 | MW842268 |
SAUCC 1923* | Quercus palustris | China | MW784678 | MW784851 | MW842261 | MW842264 | MW842267 | |
Tubakia melnikiana | CPC 32255* | Quercus canbyi | Mexico | MG591893 | MG591987 | MG592080 | MG592174 | MG976472 |
Tubakia oblongispora | MUCC 2295* | Quercus serrata | Japan | MG591897 | MG591991 | MG592084 | MG592178 | MG976474 |
Tubakia paradryinoides | MUCC 2294* | Quercus acutissima | Japan | MG591898 | MG591992 | MG592085 | MG592179 | MG976475 |
Tubakia seoraksanensis | CBS 127490* | Quercus mongolica | South Korea | MG591907 | KP260499 | MG592094 | MG592186 | ‒ |
CBS 127491 | Quercus mongolica | South Korea | HM991735 | KP260500 | MG592095 | MG592187 | MG976484 | |
Tubakia sierrafriensis | CPC 33020 | Quercus eduardi | Mexico | MG591910 | MG592005 | MG592099 | MG592191 | MG976486 |
Tubakia sp. | CBS 115011 | Quercus robur | Netherlands | MG591912 | MG592007 | MG592101 | MG592193 | MG976488 |
Tubakia suttoniana | CBS 639.93 | Quercus sp. | Italy | MG591921 | MG592016 | MG592110 | MG592202 | MG976493 |
The generated consensus sequences for each gene were subjected to megablast searches to identify closely-related sequences in the NCBI’s GenBank nucleotide database (
Phylogenetic analyses were based on Maximum Likelihood (ML) and Bayesian Inference (BI) for the multilocus analyses. For BI, the best evolutionary model for each partition was determined using MrModelTest v. 2.3 (
The alignment contained 37 isolates representing Tubakia and allied taxa and a strain of Greeneria uvicola (FI12007) was used as outgroup. The final alignment contained a total of 2459 characters used for the phylogenetic analyses, including alignment gaps, viz. ITS: 1–676, LSU: 677–1545, rpb2: 1546–2459. Of these characters, 1858 were constant, 115 were variable and parsimony-uninformative and 486 were parsimony-informative. MrModelTest recommended that the Bayesian analysis should use Dirichlet base frequencies for the ITS, LSU and rpb2. The GTR+I+G model was proposed for ITS, LSU and rpb2. The MCMC analysis of the three concatenated genes, run for 700,000 generations, resulted in 14,001 trees. The initial 3500 trees, representative of the analysis burn-in phase, were discarded, while the remaining trees were used to calculate posterior probabilities in the majority rule consensus trees (Fig.
Phylogram of Tubakiaceae, based on the concatenated ITS, LSU and rpb2 sequence alignment. The BI and ML bootstrap support values above 0.74 and 74% are shown at the first and second position, respectively. The tree is rooted to Greeneria uvicola (culture FI12007) and ex-type cultures are indicated in bold face. Strains from the current study are in red. Some branches were shortened for layout purposes – these are indicated by two diagonal lines with the number of times a branch was shortened indicated next to the lines.
The alignment contained 37 isolates representing Tubakia and allied taxa and a strain of Greeneria uvicola (FI12007) was used as outgroup. The final alignment contained a total of 1939 characters used for the phylogenetic analyses, including alignment gaps, viz. ITS: 1–676, tef1: 677–1358, tub2: 1359–1939. Of these characters, 1077 were constant, 136 were variable and parsimony-uninformative and 726 were parsimony-informative. MrModelTest recommended that the Bayesian analysis should use Dirichlet base frequencies for the ITS, tef1 and tub2 data partitions. The GTR+I+G model was proposed for ITS and HKY+I+G for tef1 and tub2. The MCMC analysis of the three concatenated genes, run for 170,000 generations resulted in 3401 trees. The initial 850 trees, representative of the analysis burn-in phase, were discarded, while the remaining trees were used to calculate posterior probabilities in the majority rule consensus trees (Fig.
Phylogram of Tubakiaceae, based on the concatenated ITS, tef1 and tub2 sequence alignment. The BI and ML bootstrap support values above 0.74 and 74% are shown at the first and second position, respectively. The tree is rooted to Greeneria uvicola (culture FI12007) and ex-type cultures are indicated in bold face. Strains from the current study are in red. Some branches were shortened for layout purposes – these are indicated by two diagonal lines with the number of times a branch was shortened indicated next to the lines.
Based on phylogenetic data (Figs
Obovoideisporodochium lithocarpi Z. X. Zhang, J. W. Xia & X. G. Zhang
Composed of “obovoideisporo-” (obovoid spores) and “-dochium” (referring to the conidioma, i.e. sporodochium).
Genus of Tubakiaceae. Living as endophyte in leaves and causing leaf spots. Asexual morph: mycelium consisting of septate, smooth and hyaline hyphae, thin-walled. Conidiomata sporodochial, appeared within 20 days or longer, formed on agar surface, slimy, pale bluish-green, semi-submerged. Sporodochial conidiophores densely and irregularly branched, bearing apical whorls of 2–3 phialides; sporodochial phialides monophialidic, subulate to subcylindrical, smooth, thin-walled, tapering towards apex, swelling at base. Conidia formed singly, obovoid to ellipsoid, smooth, thin walled, apex obtuse, base with inconspicuous to conspicuous hilum. Sexual morph: unknown.
In the two phylogenetic trees (Figs
China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Lithocarpus fohaiensis (Fagaceae), 11 Sep 2020, Z. X. Zhang, (holotype HSAUP0748, ex-type living culture SAUCC 0748).
Name refers to the genus of the host plant Lithocarpus fohaiensis.
Asexual morph: mycelium consisting of septate, smooth and hyaline hyphae, thin-walled, 1.0–2.0 μm. Colonies on PDA incubated at 25°C in the dark with an average radial growth rate of 5–6 mm/d and reaching 75–80 mm diam. in 14 d, formed some conspicuous concentric circles, aerial mycelium cottony, white initially, then becoming greyish-sepia. Conidiomata sporodochial, appeared within 20 days or longer, formed on agar surface, slimy, pale bluish-green, semi-submerged. Sporodochial conidiophores densely and irregularly branched, 12.0–26.5 × 1.5–3.0 μm, bearing apical whorls of 2–3 phialides; sporodochial phialides monophialidic, subulate to subcylindrical, 9.5–20.0 × 1.5–3.0 μm, smooth, thin-walled, tapering towards apex, swelling at base. Conidia formed singly, obovoid to ellipsoid, 5.5–8.0 × 2.5–4.0 μm, length/width ratio 1.7–3.1, hyaline, smooth, thin walled, apex obtuse, base with inconspicuous to conspicuous hilum, 0.4–0.9 μm diam. Sexual morph: unknown.
Cultures incubated on MEA at 25°C in darkness, attaining 52.0–58.0 mm diam. after 14 d (growth rate 3.5–4.0 mm diam./d), grey-white to creamy white with irregular margin, spread like petals from the inside and outside, reverse dark to light brown, distributed in an irregular circle. Conidial formation not observed.
China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Lithocarpus fohaiensis (Fagaceae), 11 Sep 2020, Z. X. Zhang, HSAUP0745; living culture SAUCC 0745.
In the two phylogenetic trees (Figs
China, Shandong Province: Zibo Lushan National Forest Park, on diseased leaves of Quercus palustris Münchh (Fagaceae), 20 Sep 2020, Z. X. Zhang, (holotype HSAUP1923, ex-type living culture SAUCC 1923).
. Named after the type locality, Lushan National Forest Park.
Asexual morph: Leaf spots irregular, occurring on leaf veins and at leaf edges. Colonies on PDA incubated at 25°C in the dark with an average radial growth rate of 5–7 mm/d and occupying an entire 90 mm Petri dish in 14 d, forming some conspicuous concentric circles, aerial mycelium cottony, white initially, then becoming greyish-sepia. Conidiomata pycnidial, usually globose or subglobose when viewed from above, formed on agar surface, black, semi-submerged, up to 200 μm diam. Pycnidial wall composed of an outer layer of yellow-brown, thick-walled textura angularis and an inner layer with hyaline, thin-walled cells. Conidiophores reduced to conidiogenous cells lining the inner cavity, ampulliform or flask-shaped, smooth, hyaline, 9.0–15.0 μm × 2.0–4.0 μm. Conidia solitary, globose to irregular globose, ellipsoid to broad ellipsoid, 10.0–18.0 μm × 7.5–16.0 μm, length/width ratio 1.0–1.7, slightly lighter and wall thin when immature, slightly darker and wall thickened when ripening, smooth, apex rounded, base with peg-like hila, 1.3–2.3 μm diam. Microconidia not observed. Sexual morph not observed.
Cultures incubated on MEA at 25°C in darkness, attaining 52.0–56.0 mm diam. after 14 d (growth rate 3.7–4.0 mm diam./d), creamy white to pale brown with regular margin, grey near the centre and hyphae clusters, reverse brown to dark brown rings, heterogeneous colour, with creamy-white edge. Conidial formation not observed.
China, Shandong Province: Zibo Lushan National Forest Park, on diseased leaves of Quercus palustris Münchh. (Fagaceae), 20 Sep 2020, Z. X. Zhang, HSAUP1921; living culture SAUCC 1921.
The phylogenetic analysis of a combined three-gene alignment (ITS, tef1 and tub2) showed that T. lushanensis formed an independent clade and is phylogenetically distinct from its closest sister species T. seoraksanensis. This species can be distinguished from T. seoraksanensis by 65 different nucleotides in the concatenated alignment (21/628 in the ITS, 31/581 in the tef1 and 13/521 in the tub2). Morphologically, T. lushanensis differs from T. seoraksanensis in having smaller conidia (10.0–18.0 μm × 7.5–16.0 μm vs. 13.0–25.0 μm × 10.0–15.0 μm) (
Asexual morph: Living as endophyte in leaves, forming distinct leaf lesions, shape and size variable, subcircular to angular-irregular, pale brown to brown. Colonies on PDA incubated at 25°C in the dark with an average radial growth rate of 5–7 mm/d and occupying an entire 90 mm Petri dish in 14 d, forming some conspicuous concentric circles, aerial mycelium cottony, white initially, then becoming greyish-sepia. Conidiomata sporodochial, appeared within 14 days or longer, formed on agar surface, slimy, black, semi-submerged. Sporodochial conidiophores densely and irregularly branched, 11.0–24.0 μm × 1.5–5.0 μm, bearing apical whorls of 2–3 phialides; sporodochial phialides monophialidic, subulate to subcylindrical, 9.0–16.0 μm × 1.5–5.0 μm, smooth, thin-walled, apex obtuse to truncate, sometimes forming indistinct periclinal thickenings. Conidia solitary, ellipsoid to obovoid, 6.5–14.0 μm × 4.0–6.0 μm, wall thin, up to 1.0 μm, hyaline to subhyaline, smooth, apex and base broadly rounded, with inconspicuous to conspicuous basal hilum (frill), occasionally somewhat peg-like and truncate when conspicuous. Microconidia not observed. Sexual morph not observed.
Cultures incubated on MEA at 25°C in darkness, attaining 38.0–42.0 mm diam. after 14 d (growth rate 2.7–3.0 mm diam./d), margin scalloped, at first creamy white, grey near the centre, reverse light brown to dark, with olivaceous edge. Conidial formation not observed.
China, Shandong Province: Zibo Lushan National Forest Park, on diseased leaves of Quercus palustris (Fagaceae), 20 Sep 2020, Z. X. Zhang, HSAUP1924, living culture SAUCC 1924.
In the study of the phylogenetic affinity and position of Tubakia in the Ascomycota hierarchical system,
The present study found two new species, one of which represents a novel genus in Tubakiaceae. In order to support the validity of the new species, we followed the guidelines of
The centre of genetic diversity of Tubakia appears to be in East Asia, where Quercus and other genera of Fagaceae are the most common hosts (
This work was supported by the National Natural Science Foundation of China (no. 31900014, 31750001 and 31770016).