Research Article |
Corresponding author: Jian Xin Deng ( djxin555@yangtzeu.edu.cn ) Corresponding author: Qi Li Li ( 65615384@qq.com ) Academic editor: Cecile Gueidan
© 2021 Lin He, Hong Cheng, Lin Zhao, Aye Aye Htun, Zhi He Yu, Jian Xin Deng, Qi Li Li.
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:
He L, Cheng H, Zhao L, Htun AA, Yu ZH, Deng JX, Li QL (2021) Morphological and molecular identification of two new Alternaria species (Ascomycota, Pleosporaceae) in section Radicina from China. MycoKeys 78: 187-198. https://doi.org/10.3897/mycokeys.78.64853
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The fungal genus Alternaria was distributed widely and found in different habitats such as plant or indoor environment. During an investigation into this genus in China, two new Alternaria species, Alternaria vulgarae and A. divaricatae were respectively isolated from diseased leaves of Foeniculum vulgare and Saposhnikovia divaricata, which both belonged to Umbelliferae. Phylogenetically, they were determined as new species belonging in the section Radicina of Alternaria based on the combined four gene fragments of ITS, TEF1, GAPDH and RPB2. Morphologically, the two species were illustrated and compared with other relevant Alternaria species in section Radicina.
Alternaria, new taxon, phylogeny, Pleosporaceae, taxonomy
Alternaria Nees (1816) was typified by Alternaria tenuis (the synonym of A. alternata), a species with muriform and catenulate conidia. Since then, hundreds of new species were proposed in the genus. Meanwhile, because of unstable taxonomic standards (morphological characteristics, host and growing environment, etc.), the controversies about species boundary started and never stopped (
On the other hand, multigene phylogenetic analyses have provided strong support for the re-definition of the Alternaria genus. Many sequences of gene regions such as the internal transcribed spacer region of rDNA (ITS), large subunit ribosomal DNA (LSU), mitochondrial small subunit (mtSSU), Alternaria major allergen (ALT), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), RNA polymerase second largest subunit (RPB2), and ATPase etc. were applied to delimit the genus (
During the investigation into Alternaria species in China, two new taxa were isolated from umbelliferous plants, Foeniculum vulgare and Saposhnikovia divaricata. The study was designed to determine them based on a polyphasic approach including morphology and phylogenetic analyses.
Leaves of Foeniculum vulgare and Saposhnikovia divaricata with necrotic spots were respectively collected from Wenjiang district (Chengdu, Sichuan in June, 2015) and Badong county (Yichang, Hubei in July, 2016) in China. For fungal isolation, the samples were stored in sterile plastic bags and transported to the laboratory. The tissues were cut into small segments and placed on moist filter papers within Petri dishes then incubated at 25 °C to stimulate sporulation. After 24 h, the samples were examined under a stereomicroscope. Alternaria-like spores were picked up and inoculated to potato dextrose agar (PDA: Difco, Montreal, Canada) using sterilized glass needles. All isolated pure cultures were inoculated to test-tube slants and stored at 4 °C. Dried cultures from the single spore and ex-type strains were deposited in the Fungi Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China.
To determine colonial characteristics (size, color and texture of colony), the strains were cultured on PDA at 25 °C for 7 days in darkness. To analyze the morphological features of conidia (conidial size, shape, sporulation, etc.), fresh mycelia were transferred on potato carrot agar (PCA) and V8 juice agar (V8A) then incubated at 22 °C under an 8 hour photoperiod for 7 days (
Genomic DNA was extracted from fresh mycelia growing on PDA after 3–5 days of growth following the CTAB method described in
Alternaria strains and their accession numbers used in the phylogenetic analysis.
Section | Species | Strain | Host/Substrate | Country | GenBank accession numbers | |||
ITS | GAPDH | TEF1 | RPB2 | |||||
Alternaria | A. alternata | CBS 916.96 T | Arachis hypogaea | India | AF347031 | AY278808 | KC584634 | KC584375 |
A. tenuissima | CBS 918.96 R | Dianthus sp. | UK | AF347032 | AY278809 | KC584693 | KC584435 | |
Althernantherae | A. alternantherae | CBS 124392 | Solanum melongena | China | KC584179 | KC584096 | KC584633 | KC584374 |
A. perpunctulata | CBS 115267 T | Alternanthera philoxeroides | USA | KC584210 | KC584129 | KC584676 | KC584418 | |
Gypsophilae | A. gypsophilae | CBS 107.41 T | Gypsophila elegans | USA | KC584199 | KC584118 | KC584660 | KC584401 |
A. nobilis | CBS 116490 R | Dianthus caryophyllus | New Zealand | KC584208 | KC584127 | KC584673 | KC584415 | |
A. vaccariae | CBS 116533 R | Vaccaria hispanica | USA | KC584223 | KC584146 | KC584696 | KC584438 | |
A. vaccariicola | CBS 118714 T | Vaccaria hispanica | USA | KC584224 | KC584147 | KC584697 | KC584439 | |
Radicina | A. carotiincultae | CBS 109381 T | Daucus carota | USA | KC584188 | KC584106 | KC584645 | KC584386 |
A. chlamydosporifera | FMR 17360 T | Rabbit dung | Spain | LR133924 | LR133927 | LR133929 | LR133926 | |
A. divaricatae sp. nov. | YZU 151055 T | Saposhnikovia divaricata | China | MW541932 | – | MW579314 | MW579316 | |
YZU 151059 | Saposhnikovia divaricata | China | MW541933 | – | MW579315 | MW579317 | ||
A. glehniae | YZU 161149 T | Glehnia littoralis | China | MK279385 | – | MK279392 | MK279394 | |
A. petroselini | CBS 112.41 T | Petroselinum sativum | Unknown | KC584211 | KC584130 | KC584677 | KC584419 | |
A. radicina | CBS 245.67 T | Daucus carota | USA | KC584213 | KC584133 | KC584681 | KC584423 | |
A. selini | CBS 109382 T | Petroselinum crispum | Saudi Arabia | AF229455 | AY278800 | KC584684 | KC584426 | |
A. smyrnii | CBS 109380 R | Smyrnium olusatrum | UK | AF229456 | KC584138 | KC584687 | KC584429 | |
A. vulgarae sp. nov. | YZU 161234 T | Foeniculum vulgare | China | MW541936 | MW579308 | MW579310 | MW579312 | |
YZU 161235 | Foeniculum vulgare | China | MW541937 | MW579309 | MW579311 | MW579313 | ||
Porri | A. dauci | CBS 117097 R | Daucus carota | USA | KC584192 | KC584111 | KC584651 | KC584392 |
A. porri | CBS 116698 R | Allium cepa | USA | DQ323700 | KC584132 | KC584679 | KC584421 | |
Sonchi | A. cinerariae | CBS 116495 R | Ligularia sp. | USA | KC584190 | KC584109 | KC584648 | KC584389 |
A. sonchi | CBS 119675 R | Sonchus asper | Canada | KC584220 | KC584142 | KC584691 | KC584433 | |
Out-group | Stemphylium herbarum | CBS 191.86 T | Medicago sativa | India | KC584239 | AF443884 | KC584731 | KC584471 |
Preliminary BLAST searches in GenBank with ITS and TEF1 sequences of the present isolates indicated that they had a close phylogenetic relationship with species from section Radicina of Alternaria. Subsequently, sequence data of 19 Alternaria species and Stemphylium herbarum CBS 191.86 (outgroup) were retrieved from National Center for Biotechnology Information (NCBI), mostly published in
The combined dataset of twenty-four strains (including 20 references and present four strains) had a length of 2166 characters with gaps after alignment, 536 characters for ITS, 247 for TEF1, 537 for GAPDH and 846 for RPB2. Of these characters, 1555 were constant and 198 were variable and parsimony-uninformative. MP analysis of the remaining 413 parsimony-informative characters resulted in one parsimonious tree of 995 lengths (CI = 0.739, RI = 0.815, RC = 0.602); Tree topologies computed from the MP, BI, and ML analysis were similar and the ML tree was shown in Fig.
China, Sichuan Province, Chengdu City, Wenjiang District, Herb Garden of Chengdu University of Traditional Chinese Medicine, from leaf spot of Saposhnikovia divaricata. 17 June, 2015, J.X Deng, (YZU-H-0029, holotype), ex-type culture YZU 151055.
In reference to the host species name, divaricata.
Colonies on PDA (Fig.
China, Sichuan Province, Chengdu City, Wenjiang District, Herb Garden of Chengdu University of Traditional Chinese Medicine, from leaf spot of Saposhnikovia divaricata. 17 June, 2015, L He, living culture YZU 151059.
Phylogenetically, Alternaria divaricatae forms a distinct clade in section Radicina, which appears to be sister to a clade including A. petroselini, A. selini and A. vulgarae (Fig.
Morphological comparison of the present species and other Altenraria species in section Radicina
Species | Conidia | Conidia per chain | Medium | ||
---|---|---|---|---|---|
Shape | Size (μm) | Septa | |||
A. atrocariis | Ovoid, ellipsoid | 50–100×25–38 | 3–12 | 1–2 | Hosta |
A. divaricatae sp. nov. | Short-ovoid,subglobose, ellipsoid | 21–38×12–26 | 1–4 | 1 | PCAd |
22–39×13–24 | 1–4 | V8Ad | |||
A. carotiincultae | Long ovoid or ellipsoid | 40–80×15–23 | 5–7 (–11) | 1–3 | PCA a |
A. chlamydosporifera | ellipsoidal or ovoid, occasionally, subglobose | 12–41×7–20 | 1–3(–4) | 1, occasionally 2 | PCA b |
A. glehniae | Long ovoid, ellipsoid | 20‒40 (–48)×10‒20 | 3–7 | 1, occasionally 2 | PCA c |
A. petroselini | Short-ovoid to subsphaeroid | 35–62(‒66)×20–26 | 6–8 | 1, rarely to 2 | PCA a |
A. radicina | Short-broad or long-narrow ellipsoid and ovoid | 42–63×15–20 | 4–8 | 1, seldom up to 2 | PCA a |
A. selini | Short-ovoid | 32–42(–50)×22–27 | 3–5 | 1–3 | PCA a |
Long-ellipsoid | 48–65(–50)×15–20 | Up to 7 | |||
A. smyrnii | Ovoid, obovoid | 40–58×18–22 | 7–8(–10) | 1–2 | PCA a |
Narrower ellipsoid | 67–96×13–16 | ||||
A. vulgarae sp. nov. | Short-ovoid, ovoid or long-ellipsoid | 25–50 (–70)×16–27 | 1–5 | 1 | PCAd |
24–55 (–77)×13–26 | 1–8 | V8Ad |
China, Hubei Province, Yichang city, Badong county on infected leaves of Foeniculum vulgare. 19 July, 2016, J.X Deng, (YZU-H-0040, holotype), ex-type culture YZU 161234.
In reference to the host species name, vulgare.
Colonies on PDA (Fig.
China, Hubei Province, Yichang city, Badong county on infected leaves of Foeniculum vulgare. 19 July, 2016, L He, living culture YZU 161235.
Phylogenetic analysis based on combining four gene fragments indicated that Alternaria vulgarae fell in an individual branch in section Radicina of Alternaria and displayed a close relationship with A. petroselini and A. selini with high supported values (Fig.
Morphologically, Alternaria radicina species-group was one of the 10 subsections (A–1) and comprised 8 species described by
In the current study, two new Alternaria species belonged to the section Radicina based on morphological and phylogenetic analysis. Alternaria divaricatae was identified as a novel species based on unique morphological and well-supported phylogenetic analysis (Fig.
The financial support of the work was given by the National Natural Science Foundation of China (31400014) and Guangxi Key Laboratory of Biology for Crop Disease and Insect Pests (2019-KF-01).