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Research Article
Morphological and phylogenetic analyses reveal two new Alternaria species (Pleosporales, Pleosporaceae) in Alternaria section from Cucurbitaceae plants in China
expand article infoSein Lai Lai Aung‡, Feng-Yin Liu‡, Ya-Nan Gou‡, Zin Mar Nwe‡, Zhi-He Yu‡, Jian-Xin Deng‡
‡ Yangtze University, Jingzhou, China

Corresponding author: Jian-Xin Deng ( djxin555@yangtzeu.edu.cn )

Academic editor: Rungtiwa Phookamsak
© 2024 Sein Lai Lai Aung, Feng-Yin Liu, Ya-Nan Gou, Zin Mar Nwe, Zhi-He Yu, Jian-Xin Deng.
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: Aung SLL, Liu F-Y, Gou Y-N, Nwe ZM, Yu Z-H, Deng J-X (2024) Morphological and phylogenetic analyses reveal two new Alternaria species (Pleosporales, Pleosporaceae) in Alternaria section from Cucurbitaceae plants in China. MycoKeys 107: 125-139. https://doi.org/10.3897/mycokeys.107.124814
Open Access

Abstract

Alternaria species are commonly found as saprophytes, endophytes and plant pathogens. During a survey of small-spored Alternaria in China, two new species were discovered from Cucurbitaceae plants collected in Hubei and Sichuan provinces. This study identified two new species of Alternaria using seven genes (ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG, and OPA10-2) for phylogenetic analyses and morphological characteristics. The two new species A. jingzhouensis and A. momordicae were described and illustrated. Alternaria jingzhouensis sp. nov., associated with Citrullus lanatus, is characterized by producing muriform, ellipsoidal, flask-shaped, rostrate, and beaked conidia. It differs from A. koreana, A. ovoidea, and A. baoshanensis by bearing conidia in a simple conidiogenous locus with occasionally longer beaks in a chain, and from A. momordicae sp. nov. by having shorter beaks. Alternaria momordicae sp. nov. from Momordica charantia was distinct from A. koreana, A. ovoidea, and A. baoshanensis by producing muriform, long ellipsoid or ovoid to obclavate, sometimes inverted club-shaped conidia on a single conidiogenous locus with a wider body and longer beak in a chain, and distinct from A. jingzhouensis sp. nov. by a longer beak conidia. These two species were clearly distinguished from other species in the section Alternaria based on DNA based phylogeny and morphological characteristics. The morphological features were discussed and compared to relevant species in the present paper.

Key words

Morphology, novel species, phylogeny, small-spored Alternaria, taxonomy

Introduction

The Cucurbitaceae, also called cucurbits or the gourd family, consists of approximately 975 species belonging to 98 genera (Xu and Chang 2017). There are 35 genera with 151 species in China (Raven and Wu 2022). This family includes highly nutritious vegetables with significant economic value, such as cucumber, pumpkin, and so on. Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is a popular fruit worldwide, and its seeds contain high levels of proteins, lipids and medicinal properties (Wani et al. 2011, Maoto et al. 2019). China is the world’s leading producer of watermelons (Qiang et al. 2024). Bitter gourd (Momordica charantia L.) is normally cultivated in China for its fruit as a popular vegetable and traditional medicine (Sun et al. 2023). Alternaria-like leaf blight can severely affect the crop production of Cucurbitaceae (Maheswari and Sankaralingam 2010; Ma et al. 2021). Many Alternaria species have been reported to be associated with cucurbit plants, including A. alternata (Fr.) Keissl. (Chen et al. 1993; Zhao et al. 2016a, 2016b; Ma et al. 2021), A. baoshanensis J.F. Li, Phookamsak & Jeewon (Li et al. 2023), A. brassicae (Berk.) Sacc. (Simmons 2007), A. brassicae var. nigrescens (Peglion) Sacc. & Traverso (Simmons 2007), A. caudata Cooke & Ellis (Simmons 2007), A. cucumericola E.G. Simmons & C.F. Hill (Simmons 2007), A. cucumerina (Ellis & Everh.) J.A. Elliott (Chen et al. 1993; Zhang 2003; Simmons 2007; Ma et al. 2021), A. cylindrorostra T.Y. Zhang (Zhang 2003; Simmons 2007), A. gaisen Nagano ex Bokura (Ma et al. 2021), A. granulosa (Bubák) E.G. Simmons (Simmons 2007), A. hydrangeae D. F. Pei & J. X. Deng (Liu et al. 2022), A. infecotria E.G. Simmons (Ma et al. 2021), A. loofahae E.G. Simmons & Aragaki (Simmons 2007), A. nigrescens (Peglion) Neerg. (Simmons 2007), A. peponicola (Rabenh.) E.G. Simmons (Zhang 2003; Simmons 2007), A. peponis Yatel (Simmons 2007), and A. tenuissima (Kunze) Wiltshire (Chen et al. 1993; Zhao et al. 2016a, 2016b; Ma et al. 2021).

The genus Alternaria Nees von Esenbeck (1816) is categorized according to its morphological characteristics, typified by A. alternata with muriform and catenulate conidia (Simmons 2007). Simmons (1992) applied standard criteria to achieve solid taxonomic outcomes for Alternaria species, primarily relying on the sporulation patterns and developmental morphology of conidia. In 2007, Simmons illustrated approximately 276 species (148 large-spored species and 128 small-spored species) and provided a final summary of morphological taxonomy on Alternaria. The small-spored species fall into 10 subsections containing the type species of A. alternata (Simmons 2007). In 2003, Zhang identified approximately 80 small-spored species associated with specific host plant families in China.

To date, the utilization of multigene phylogenetic analyses has played a crucial role in understanding the Alternaria genus (Pryor and Gilbertson 2000; Pryor and Bigelow 2003; Hong et al. 2005; Runa et al. 2009; Woudenberg et al. 2013, 2014; Lawrence et al. 2013, 2014, 2016; Poursafar et al. 2018). The genus contains 24 internal clades (sections) and six monotypic lineages (Woudenberg et al. 2013) using type or referenced strains collected by Simmons (2007), which has recently been updated to 29 sections (Li et al. 2023). Small-spored Alternaria species are also frequently isolated from Cucurbitaceae in China (Ma et al. 2021). Woudenberg et al. (2015) provided a clear and stable species classification of section Alternaria based on the genomic and multi-loci analyses, from which the species commonly produce concatenated conidia (Norphanphoun et al. 2021; Li et al. 2022; Gou et al. 2022). Consequently, the combination of morphology and molecular techniques provides a better understanding of species in section Alternaria (Aung et al. 2020).

During the investigation of small-spored Alternaria species in China, two new taxa were isolated from gourd plants of Citrullus lanatus and Momordica charantia. The aim of this study was to characterize and differentiate both taxa using morphology and multigene sequence analyses. This research sought to enhance understanding of Alternaria species diversity within the Cucurbitaceae family, offering crucial taxonomic information for species conservation efforts.

Materials and methods

Isolation

Leaves of Citrullus lanatus and Momordica charantia with necrotic spots were collected from Jingzhou, Hubei in 2022 and Deyang City, Sichuan Province in 2016 China, respectively. To facilitate isolation, the specimens were carefully enclosed in sterile plastic bags and transported to the laboratory. Subsequently, the tissues were accurately divided into small segments, arranged on moist filter papers within Petri dishes, and incubated at 25 °C to promote spore production. After sporulation, spores of Alternaria were individually collected using sterilized glass needles under a stereo microscope (Shunyu SZM series) and transferred onto potato dextrose agar (PDA) plates. Each distinct culture was purified and preserved in test-tube slants maintained at 4 °C. Additionally, dried cultures derived from individual spores and reference strains were stored in the Fungi Herbarium of Yangtze University (YZU), located in Jingzhou, Hubei, China.

Morphology

To study the features of colonies, the strains were grown on PDA at 25 °C for 7 days without light. To examine the characteristics of the conidia (size, shape, sporulation, etc.), fresh mycelia were transferred to potato carrot agar (PCA) and V8 juice agar (V8A) plates and then placed in an incubator at 22 °C with an 8-hour light cycle for 7 days (Simmons 2007). A total of 50 conidia were randomly selected and photographed for the morphological determination after mounting the conidia into lactophenol picric acid under an ECLIPSE Ni-U microscope system (Nikon, Japan). The sporulation patterns and morphological characteristics were also recorded.

DNA extraction, PCR amplification and sequencing

Fresh mycelia growing on PDA were used to extract genomic DNA with the CTAB method, as described by Watanabe et al. (2010). To amplify multigene fragments, including the internal transcribed spacer rDNA region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), RNA polymerase second largest subunit (RPB2), Alternaria major allergen gene (Alt a 1), endopolygalacturonase gene (EndoPG), and an anonymous gene region (OPA10-2), primer pairs were employed including ITS5/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F/RPB2-7cR (Liu et al. 1999), Alt-for/Alt-rev (Hong et al. 2005), PG3/PG2b (Andrew et al. 2009) and OPA10-2L/OPA10-2R (Andrew et al. 2009), respectively. The PCR reaction mixture was 25 μL, including 21 μL of 1.1×Taq PCR Star Mix from TSINGKE, 2 μL of template DNA, and 1 μL of each primer. The amplification process was carried out in an Eppendorf Mastercycler, following the protocols outlined by Woudenberg et al. (2015). After a successful amplification, the PCR products were purified and sequenced by TSINGKE company (Beijing, China). The obtained sequences were assembled using BioEdit v. 7.2.3 (Hall 1999) and primarily aligned with PHYDIT v.3.2 (Chun 1995) then deposited into GenBank (https://www.ncbi.nlm.nih.gov/) (Table 1).

Table 1.
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Alternaria strains used in this study and their GenBank accession numbers.

Species Strain Host/Substrate Country GenBank accession numbers
ITS GAPDH TEF1 RPB2 Alt a 1 EndoPG OPA10-2
A. alternantherae CBS 124392 Solanum melongena China KC584179 KC584096 KC584633 KC584374 KP123846 np np
A. alternata CBS 916.96T Arachis hypogaea India AF347031 AY278808 KC584634 KC584375 AY563301 JQ811978 KP124632
CBS 106.34T Linum usitatissimum Unknown Y17071 JQ646308 KP125078 KP124771 KP123853 KP124000 KP124608
CBS 102596T Citrus jambhiri USA KP124328 KP124183 KP125104 KP124796 KP123877 KP124030 KP124637
CBS 121336T Allium sp. USA KJ862254 KJ862255 KP125141 KP124833 KJ862259 KP124067 KP124676
CBS 121547T Pyrus bretschneideri China KP124372 KP124224 KP125150 KP124842 KP123920 KP124076 KP124685
CBS 119543T Citrus paradisi USA KP124363 KP124215 KP125139 KP124831 KP123911 KP124065 KP124674
CBS 918.96R Dianthus chinensis UK AF347032 AY278809 KC584693 KC584435 AY563302 KP124026 KP124633
CBS 127671T Stanleya pinnata USA KP124381 KP124233 KP125159 KP124851 KP123929 KP124085 KP124694
CBS 121455T Broussonetia papyrifera China KP124368 KP124220 KP125146 KP124838 KP123916 KP124072 KP124681
CBS 117.44T Godetia sp. Denmark KP124303 KP124160 KP125079 KP124772 KP123854 KP124001 KP124609
CBS 127672T Astragalus bisulcatus USA KP124382 KP124234 KP125160 KP124852 KP123930 KP124086 KP124695
CBS 102.47R Citrus sinensis USA KP124304 KP124161 KP125080 KP124773 KP123855 KP124002 KP124610
CBS 102599T Minneola tangelo Turkey KP124330 KP124185 KP125106 KP124798 KP123879 KP124032 KP124639
CBS 102595T Citrus jambhiri USA FJ266476 AY562411 KC584666 KC584408 AY563306 KP124029 KP124636
CBS 103.33T Soil Egypt KP124302 KP124159 KP125077 KP124770 KP123852 KP123999 KP124607
A. arborescens CBS 126.60 Wook UK KP124397 KP124249 KP125175 KP124867 JQ646390 KP124101 KP124710
CBS 119545T Senecio skirrhodon New Zealand KP124409 KP124260 KP125187 KP124879 KP123956 KP124113 KP124723
CBS 101.13T Peat soil Switzerland KP124392 KP124244 KP125170 KP124862 KP123940 KP124096 KP124705
CBS 105.24 Solanum tuberosum Unknown KP124393 KP124245 KP125171 KP124863 KP123941 KP124097 KP124706
CBS 119544T Avena sativa New Zealand KP124408 JQ646321 KP125186 KP124878 KP123955 KP124112 KP124722
CBS 105.49 Contaminant blood culture Italy KP124396 KP124248 KP125174 KP124866 KP123944 KP124100 KP124709
CBS 112749 Malus domestica South Africa KP124401 KP124253 KP125179 KP124871 KP123948 KP124105 KP124715
A. baoshanensis MFLU 21-0124T Curcubita moschata China MZ622003 OK236706 OK236613 OK236659 OK236760 np np
MFLU 21-0296 C. moschata China MZ622004 OK236707 OK236612 OK236660 OK236759 np np
A. breviconidiophora MFLUCC 21-0786T Digitalis sp. Italy MZ621997 OK236698 OK236604 OK236651 OK236751 np np
A. burnsii CBS 118817T Tinospora cordifolia India KP124424 KP124274 KP125202 KP124893 KP123971 KP124128 KP124738
CBS 118816T Rhizophora mucronata India KP124423 KP124273 KP125201 KP124892 KP123970 KP124127 KP124737
A. ellipsoidialis MFLUCC 21-0132T Brassica sp. Italy MZ621989 OK236690 OK236596 OK236643 OK236743 np np
A. eupatoriicola MFLUCC 21-0122T Eupatorium cannabinum Italy MZ621982 OK236683 OK236589 OK236636 OK236736 np np
A. falcata MFLUCC 21-0123T Atriplex sp. Italy MZ621992 OK236693 OK236599 OK236649 OK236746 np np
A. gaisen CBS 632.93R Pyrus pyrifolia Japan KC584197 KC584116 KC584658 KC584399 KP123974 AY295033 KP124742
CBS 118488R P. pyrifolia Japan KP124427 KP124278 KP125206 KP124897 KP123975 KP124132 KP124743
A. gossypina CBS 102601T Minneola tangelo Colombia KP124433 KP124282 KP125212 KP124903 KP123979 KP124138 KP124749
CBS 104.32T Gossypium sp. Zimbabwe KP124430 JQ646312 KP125209 KP124900 JQ646395 KP124135 KP124746
A. jacinthicola CBS 878.95 Arachis hypogaea Mauritius KP124437 KP124286 KP125216 KP124907 KP123983 KP124142 KP124753
CBS 133751T Eichhornia crassipes Mali KP124438 KP124287 KP125217 KP124908 KP123984 KP124143 KP124754
A. jingzhouensis sp. nov. YZU 221144T Citrullus lanatus China OR883772 OR887690 OR887686 OR887688 OR887694 OR887692 OR887684
YZU 221145 C. lanatus China OR901948 OR914170 OR914166 OR914168 OR914174 OR914172 OR914176
A. koreana SPL2-1T Atractylodes ovata Korea LC621613 LC621647 LC621715 LC621681 LC631831 LC631844 LC631857
SPL2-4 A. ovata Korea LC621615 LC621649 LC621717 LC621683 LC631832 LC631845 LC631858
A. longipes CBS 121333R Nicotiana tabacum USA KP124444 KP124293 KP125223 KP124914 KP123990 KP124150 KP124761
CBS 540.94R N. tabacum USA AY278835 AY278811 KC584667 KC584409 AY563304 KP124147 KP124758
A. minimispora MFLUCC 21-0127T Citrullus lanatus Thailand MZ621980 OK236705 OK236587 OK236634 OK236734 np np
A. momordicae sp. nov. YZU 161378T Momordica charantia China OR883774 OR887691 OR887687 OR887689 OR887695 OR887693 OR887685
YZU 161379 M. charantia China OR901949 OR914171 OR914167 OR914169 OR914175 OR914173 OR914177
A. muriformispora MFLUCC 21-0784T Plantago sp. Italy MZ621976 OK236677 OK236583 OK236630 OK236730 np np
A. obpyriconidia MFLUCC 21-0121T Vicia faba Italy MZ621978 OK236680 OK236585 OK236633 OK236732 np np
A. ovoidea MFLUCC 0782T Dactylis glomerata Italy MZ622005 OK236708 OK236614 OK236661 OK236761 np np
MFLU 21- 0298 D. glomerata Italy MZ622006 OK236709 OK236615 OK236662 OK236762 np np
A. orobanches MFLUCC 21-0137T Orobanche sp. Italy MZ622007 OK236710 np np OK236763 np np
MFLU 21-0303 Orobanche sp. Italy MZ622008 OK236711 np np OK236764 np np
A. phragmiticola MFLUCC 21-0125T Phragmites sp. Italy MZ621994 OK236696 OK236602 OK236649 OK236749 np np
A. rostroconidia MFLUCC 21-0136T Arabis sp. Italy MZ621969 OK236670 OK236576 OK236623 OK236723 np np
A. salicicola MFLUCC 22-0072T Salix alba Russia MZ621999 OK236700 OK236606 OK236653 OK236753 np np
A. tomato CBS 103.30 Solanum lycopersicum Unknown KP124445 KP124294 KP125224 KP124915 KP123991 KP124151 KP124762
CBS 114.35 S. lycopersicum Unknown KP124446 KP124295 KP125225 KP124916 KP123992 KP124152 KP124763
A. torilis MFLUCC 14-0433T Torilis arvensis Italy MZ621988 OK236688 OK236594 OK236641 OK236741 np np

Notes: Novel species proposed in this study are marked in bold. Ex-type strains are marked ‘T’. Representative strains are marked ‘R’. No products are ‘np’.

Phylogenetic analyses

Preliminary BLAST searches on the National Center for Biotechnology Information (NCBI) website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) indicated that the current species are highly similar to species within the Alternaria genus. Subsequently, sequence data of 57 Alternaria strains and A. alternantherae Holcomb & Antonop. CBS 124392 (outgroup) were retrieved from the GenBank database and referenced from relevant publications (Woudenberg et al. 2015; Li et al. 2022; Romain et al. 2022) (Table 1). The gene sequences were concatenated and edited manually with equal weight in MEGA v.11.0.13 (Tamura et al. 2021), and gaps were treated as missing data. Bayesian inference (BI) analysis was carried out using MrBayes v. 3.2.6 (Ronquist et al. 2012). This analysis employed a Markov Chain Monte Carlo (MCMC) algorithm to estimate Bayesian posterior probabilities. The best-fit evolutionary model (GTR+I+G) was determined using MrModeltest v. 2.3 (Nylander 2004, Posada and Crandall 1998) with the Akaike Information Criterion (AIC). In MrModeltest, the file "MrModelblock″ was executed in the PAUP path (Swofford 2002) and the MrMt path (Nylander 2004). Bayesian analyses included two parallel runs for 10,000,000 generations (ngen) with the stop rule option and a sampling frequency set to every 100 generations (samplefreq=100). The run was stopped when the standard deviation of split frequencies reached a value below 0.01. The first 25% of sampled trees were discarded as burn-in. Additionally, a maximum likelihood (ML) analysis was performed using RAxML v.7.0.3 (Stamatakis et al. 2008). The GTRGAMMAI model was implemented using ML+ rapid bootstrap setting with 1000 replications to assess branch support. The tree was visualized with FigTree v1.4.3 (Rambaut 2016). Nodes in the phylogram displayed branch support values equal to or above 0.60/60% for posterior probability (PP)/bootstrap (BS) values.

Results

Phylogenetic analyses

The dataset includes a total of 58 Alternaria strains with 3627 characters in total after alignment. The dataset consists of 533 characters for ITS, 574 for GAPDH, 216 for TEF1, 757 for RPB2, 444 for EndoPG, 469 for Alt a 1, and 634 for OPA10-2. Both Bayesian inference (BI) and maximum likelihood (ML) analyses yielded similar topologies. The ML tree was selected for discussing the placement of our new species (Fig. 1). The results indicated that all Alternaria strains in the present study fell into Alternaria section with PP values of 1.0. The present four strains separated into two individual clades sister to A. koreana O. Hassan, B.B.N.D. Romain, J.S. Kim & T. Chang, A. ovoidea J.F. Li, Camporesi, Bhat & Phookamsak, A. baoshanensis, and A. orobanches J.F. Li, Camporesi, Phookamsak & Jeewon (Bayesian posterior probability (BI-BPP)/Maximum-Likelihood bootstrap proportions (ML-BS) = 0.64/74%).

Figure 1. 

Phylogenetic tree of the Alternaria species most related to the new taxa based on maximum likelihood analysis using the combined gene sequences of ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG and OPA10-2 which rooted with Alternaria alternantherae (CBS 124392) from sect. Alternantherae. The Bayesian posterior probabilities >0.60 (PP) and bootstrap support values >60 (BS) are given at the nodes (PP/BS). The novel species are highlighted in bold. Ex-type isolates are marked with a superscript T and Representative isolates are marked with a superscript R.

The clade containing YZU 161378 and YZU 161379 was closely related to A. baoshanensis, A. koreana, A. ovoidea, and forming a distinct branch. While another clade, YZU 221144 and YZU 221145 was found to be independent with a posterior probability (PP) of 1.00 and bootstrap (BS) values of 68%, and it was closely related to A. orobanches. These results suggest that the present strains represent two new taxa.

Taxonomy

Alternaria jingzhouensis S.L.L. Aung & J.X. Deng, sp. nov.

MycoBank No: MycoBank No: 851272
Fig. 2

Type

China, Hubei Province, Jingzhou city, Yangtze University (west campus) on infected leaves of Citrullus lanatus 2022, F.Y Liu, (YZU-H-2022030, holotype), ex-type culture YZU 221144.

Etymology

Named after the collecting locality, Jingzhou (Hubei, China)

Description

Colonies on PDA (7 d at 25 °C) pale luteous to amber in the center, white at the edges, light to moderate rosy buff or pale saffron in reverse, cottony surface and 49–52 mm in diam., at 25 °C for 7 days (Fig. 2A, B). On PCA (7 d at 22 °C), conidiophores arising from substrate, simple, straight or flexuous, light to olivaceous buff, 41–99 (–151) × 3.5–5 μm (x̄ = 73 × 4.4 µm, n = 20), conidiogenous cells 5–11 × 3–6 µm (x̄ = 8 × 4 µm, n = 20), mono- to polytretic, terminal, determinate, cylindrical, olivaceous buff, smooth, thin-walled, apically doliiform, with 1 conidiogenous locus cicatrized on conidial secession, sometimes swollen near conidiogenous loci; conidia 3–5 units per chain, arising from the apex or near the apex of the conidiophores or terminal hyphae, muriform, ellipsoidal, flask-shaped, rostrate, beaked, 28–51 × 11–21 μm (x̄ = 38 × 16.4, n = 50), with 1–4 transverse septa with 0–2 branching (Fig. 2C, E); On V8A (7 d at 22 °C), conidiophores 40–94 × 4–7 μm (x̄ = 58 × 5, n = 20), simple, straight or flexuous, light to olivaceous buff; conidiogenous cells 5–13 × 3–6 µm (x̄ = 8 × 4 µm, n = 20), mono- to polytretic, terminal, determinate, cylindrical, olivaceous buff, smooth, thin-walled, apically doliiform, with 1 conidiogenous locus, sometimes swollen near conidiogenous loci cicatrized on conidial secession; conidia 3–5 units per chain, arising from the apex or near the apex of the conidiophores or terminal hyphae, muriform, ellipsoidal, flask-shaped, rostrate, beaked, 22–51 × 3–16 μm (x̄ = 33.9×13.2, n = 50), 1–6 transverse septa with 0–2 branching (Fig. 2D, F).

Figure 2. 

Alternaria jingzhouensis sp. nov. (ex-type YZU 221144) A, B seven-day-old culture on PDA C, D conidiophores and conidia on PCA and V8A, respectively E, F conidia on PCA and V8A, respectively. Scale bars: 25 μm (E, F); 50 μm (C, D).

Additional isolate examined

China, Hubei Province, Jingzhou city, Yangtze University (west campus) on infected leaves of Citrullus lanatus 2022, F.Y Liu, living culture YZU 221145.

Notes

Phylogenetically, A. jingzhouensis sp. nov. is different from its sister species A. baoshanensis, A. koreana, A. momordicae sp. nov., A. orobanches and A. ovoidea based on sequences derived from seven genes (Fig. 1). After conducting a nucleotide pairwise comparison as recommended by Jeewon and Hyde (2016), the present species can be readily distinguished from the closet species A. koreana, A. momordicae sp. nov. and A. orobanches constructed on any of the ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG, and OPA10-2 genes, which has 1 bp difference in the ITS region, 1 bp in GAPDH, 1 bp in TEF1, 7 pb in RPB2, 9 bp in Alt a1, 10 bp in EndoPG, and 4 bp in OPA10-2 when compared with A. koreana, 1 bp in GAPDH, 4 bp in RPB2, and 11 bp in OPA10-2 when compared with A. momordicae sp. nov. and 49 bp differences in the ITS region when compared with sister species A. orobanches. Morphologically, the species is distinct from A. baoshanensis, A. koreana, and A. ovoidea as it produces conidia on a simple conidiogenous locus with occasionally longer beaks in a chain of 3–5 units, and from A. momordicae sp. nov. by having shorter beaks (Table 2).

Table 2.
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Conidial features of the novel Alternaria species proposed here and their closest relatives in section Alternaria.

Species Conidia Conidia per chain Medium Reference
Shape Body (µm) Beak (µm) Septa
A. baoshanensis Subglobose to ellipsoidal, or subcylindrical to obpyriform 25–60 × 12–22 Short beak 3–6 1–3 PCA Li et al. (2023)
A. jingzhouensis sp. nov. Ellipsoidal, flask-shaped, rostrate, beaked 28–51 × 11–21 2–7(–15) 1–4 3–5 PCA Present study
22–51 × 3–16 3–7 1–6 3–5 V8A Present study
A. koreana Obovate to long ellipsoid 12.9–61.2×8.6–20.7 4.5–9.1 2–8 1–2 SNA Romain et al. (2022)
A. momordicae sp. nov. Obclavate, inverted club-shaped 6–42 × 4–34 2–19.5 1–5 3–4 PCA Present study
24–61 × 10–17 3–25.5 1–5 3–4 V8A Present study
A. orobanches Obclavate to ovoid 20–50 × 10–20 – 3–6 1–2 PCA Li et al. (2023)
A. ovoidea Ovoid 48–65 × 15.5–30 – 1–3 1 PDA Li et al. (2022)

Alternaria momordicae S.L.L. Aung & J.X. Deng, sp. nov.

MycoBank No: MycoBank No: 851270
Fig. 3

Type

China, Sichuan Province, Deyang city infected leaves of Momordica charantia. 2016, J.X Deng, (YZU-H-2016001, holotype), ex-type culture YZU 161378.

Etymology

Refers to the host genus, Momordica.

Description

Colonies on PDA (7 d at 25 °C) greyish yellow-green, light white at the edge, buff to salmon in reverse, surface compact, 50–55 mm in diam. (Fig. 3A, B). On PCA (7 d at 22 °C), conidiophores arising from substrate, simple, straight or flexuous, septate, olivaceous buff to olivaceous, 26.5–93 × 3–4 μm (x̄ = 59.5× 3.8 μm, n = 20); conidiogenous cells 5–10 × 3–5 µm (x̄ = 7 × 4 µm, n = 20), mono- to polytretic, terminal, determinate, cylindrical, olivaceous buff to olivaceous, smooth, thin-walled, apically doliiform, with 1 conidiogenous locus cicatrized on conidial secession, sometimes swollen near conidiogenous loci; conidia 3–4 units per chain, arising from the apex or near the apex of the conidiophores or terminal hyphae, muriform, long ellipsoid or ovoid to obclavate, sometime inverted club-shaped, 6–42 × 4–34 μm (x̄ = 32.8 × 13.5 μm, n = 50), 1–5 transverse septa, apical beak 2–19.5 μm long and 1–2 septa (Fig. 3C, E); On V8A(7 d at 22 °C), conidiophores straight or curved, smooth-walled, olivaceous buff 23–63(–208) × 3–5 μm (x̄ = 64.9 × 4.2 μm, n = 20); conidiogenous cells 5–13 × 3–4 µm (x̄ = 7 × 4 µm, n = 20), mono- to polytretic, terminal, determinate, cylindrical, olivaceous buff, smooth, thin-walled, apically doliiform, with 1 conidiogenous locus cicatrized on conidial secession, sometimes swollen near conidiogenous loci; conidia 3–4 units per chain, muriform, long ellipsoid or ovoid to obclavate, inverted club-shaped, 24–61×10–17 μm (x̄ = 39 × 14.3 μm, n = 50), 1–5 transverse septa with apical beak 3–25.5 μm long and 1–2 septa (Fig. 3D, F).

Figure 3. 

Alternaria momordicae sp. nov. (ex-type YZU 161378) A, B seven-day-old culture on PDA C, D conidiophores and conidia on PCA and V8A, respectively E, F conidia on PCA and V8A, respectively. Scale bars: 25 μm (E, F); 50 μm (C, D).

Additional isolate examined

China, Sichuan Province, Deyang city infected leaves of Momordica charantia. 2016, J.X Deng, living culture YZU 161379.

Notes

After the combined dataset of ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG and OPA10-2 gene fragments, A. momordicae sp. nov. is readily distinguished from its sister species A. baoshanensis, A. jingzhouensis sp. nov., A. koreana, and A. ovoidea, (Fig. 1). After a nucleotide pairwise comparison as suggested by Jeewon and Hyde (2016), the present species can be readily distinguished from the closet species A. koreana and others related a novel species based on any of the ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG, and OPA10-2 genes, which has 1 bp difference in the ITS region, 1 bp in GAPDH, 1 bp in TEF1, 4 bp in RPB2, 8 bp in Alt a1 and 10 bp in EndoPG when compared with A. koreana and 1 bp in GAPDH, 4 bp in RPB2, and 11 bp in OPA10-2 when compared with A. jingzhouensis sp. nov.. Morphologically, A. momordicae sp. nov. produces conidia on PCA that are significantly shorter than those on V8A. It can be distinguished from A. baoshanensis, A. koreana, and A. ovoidea by producing conidia on a single conidiogenous locus with a wider body and longer beak in a chain of 3–4 units. Additionally, it differs from A. jingzhouensis sp. nov. by having a longer beak (Table 2).

Discussion

Most of the Alternaria species published before the year 2000s relied on morphology to characterize the species status (Simmons 2007). In this study, two new Alternaria species, A. jingzhouensis and A. momordicae, have been identified and illustrated using the morphological method of Simmons (2007) and phylogenetic analysis of seven gene loci. Both resemble the type small-spored species of A. alternata in morphology but are easily distinguished by short chains, which also differentiate them from each other and their phylogenetically closely related species of A. baoshanensis, A. koreana, A. ovoidea and A. orobanches by the chain formation of sporulation patterns (Table 2). In recent publications, the Alternaria species descriptions have not followed the morphological standard created by Simmons (2007) (Romain et al. 2022; Li et al. 2022). Simmons (2007) classified the genus Alternaria into small-spored and large-spored taxa based on morphology. Andrew et al. (2009) noted that phylogenetic studies have confirmed a distinct separation between large- and small-spored Alternaria species. Woudenberg et al. (2015) identified 35 morphospecies as synonyms of A. alternata, but their relationships remain unclear due to inconsistencies and lack of detailed morphological information. Accurate identification and classification of species within these small-spored Alternaria species require strong identification through multigene sequence analysis (Kgatle et al. 2018). Li et al. (2023) described that recent studies using combined multi-locus phylogeny suggest that certain A. alternata species classified under section Alternaria may not constitute a monophyletic group in DNA sequence-based phylogenies. To reduce potential misidentification of morphological characteristics within this section, this study utilized PCA and V8A media for 7 days at 22 °C to identify Alternaria species, following Simmons’ (2007) recommendations. These media effectively promote typical morphological characteristics. Hence, it is strongly recommended to use the standard of morphological identification for further describing small-spored and large-spored Alternaria in order to reduce taxonomic ambiguity caused by different temperatures and substrates.

With the development of molecular studies, the species-group was re-defined and the section Alternaria was introduced and updated (Pryor and Gilbertson 2000; Lawrence et al. 2013; Woudenberg et al. 2013; Li et al. 2023). The section Alternaria is one of the small-spored Alternaria species groups and comprises 11 phylogenetic species and one species complex (Woudenberg et al. 2015). The two new Alternaria species are identified as members of section Alternaria according to the multigene sequence analysis of ITS, GADPH, RPB2, TEF1, Alt a 1, EndoPG and OPA10-2 gene sequences, which are close to A. baoshanensis (Li et al. 2023) from Curcubita moschata (Cucurbitaceae), A. koreana (Romain et al. 2022) from Atractylodes ovata (Compositae), A. orobanches (Li et al. 2023) from Orobanche sp. (Orobanchaceae), and A. ovoidea (Li et al. 2022) from Dactylis glomerata (Poaceae). Three genes, GAPDH, RPB2, and OPA10-2, provide more informative data for the classification of the current species.

Small-spored Alternaria species have been frequently reported on Cucurbitaceae plants worldwide, including A. alternata (Chen et al. 1993; Zhao et al. 2016a, 2016b; Ma et al. 2021), A. baoshanensis (Li et al. 2023), A. caudata (Simmons 2007), A. gaisen (Ma et al. 2021), A. infecotria (Ma et al. 2021), A. peponicola (Zhang 2003; Simmons 2007), and A. tenuissima (Chen et al. 1993; Zhao et al. 2016a, 2016b; Ma et al. 2021). The present two small-spored species, A. jingzhouensis sp. nov. and A. momordicae sp. nov., were first found on C. lanatus and M. charantia, respectively, in China. Pathogenicity tests were performed on detached and living leaves for the two new species, which showed weak pathogenicity (data not shown). However, they did exhibit a certain level of aggressiveness on cucurbit plants. The two species, A. jingzhouensis sp. nov. and A. momordicae sp. nov., were found to be non-pathogenic to their host plants, possibly due to their saprophytic or weakly pathogenic nature when encountering resistance from C. lanatus and M. charantia. These findings provide valuable insights into Alternaria leaf diseases in Cucurbitaceae.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study is financed by the National Natural Science Foundation of China (32270022).

Author contributions

Sein LLA conceived and designed the study; Sein LLA, Liu FY, Gou YN, Zin MN, Yu ZH, conducted the experiments; Sein LLA, Deng JX wrote the manuscript and revised.

Author ORCIDs

Sein Lai Lai Aung https://orcid.org/0009-0006-2738-5598

Feng-Yin Liu https://orcid.org/0000-0003-3114-603X

Ya-Nan Gou https://orcid.org/0009-0005-1740-4065

Zin Mar Nwe https://orcid.org/0009-0000-6376-8306

Zhi-He Yu https://orcid.org/0000-0001-9477-4135

Jian-Xin Deng https://orcid.org/0000-0001-7304-5603

Data availability

All of the data that support the findings of this study are available in the main text.

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