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Research Article
Seven new species of Alternaria (Pleosporales, Pleosporaceae) associated with Chinese fir, based on morphological and molecular evidence
expand article infoJiao He‡, De-Wei Li§, Wen-Li Cui‡, Lin Huang‡
‡ Nanjing Forestry University, Nanjing, China
§ The Connecticut Agricultural Experiment Station Valley Laboratory, Windsor, United States of America

Corresponding author: Lin Huang ( lhuang@njfu.edu.cn )

Academic editor: Ajay Kumar Gautam
© 2024 Jiao He, De-Wei Li, Wen-Li Cui, Lin Huang.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation: He J, Li D-W, Cui W-L, Huang L (2024) Seven new species of Alternaria (Pleosporales, Pleosporaceae) associated with Chinese fir, based on morphological and molecular evidence. MycoKeys 101: 1-44. https://doi.org/10.3897/mycokeys.101.115370
Open Access

Abstract

Chinese fir (Cunninghamia lanceolata) is a special fast-growing commercial tree species in China and has significant ecological and economic value. However, it experienced damage from leaf blight caused by pathogenic fungi of the genus Alternaria. To determine the diversity of Alternaria species associated with leaf blight of Chinese fir in China, infected leaves were collected from five major cultivation provinces (Fujian, Henan, Hunan, Jiangsu and Shandong provinces). A total of 48 fungal strains of Alternaria were obtained. Comparison of morphology and phylogenetic analyses, based on nine loci (ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2) of the representative isolates as well as the pairwise homoplasy index tests, revealed that the fungal strains belonged to seven undescribed taxa of Alternaria, which are described here and named as Alternaria cunninghamiicola sp. nov., A. dongshanqiaoensis sp. nov., A. hunanensis sp. nov., A. kunyuensis sp. nov., А. longqiaoensis sp. nov., A. shandongensis sp. nov. and A. xinyangensis sp. nov. In order to prove Koch’s postulates, pathogenicity tests on detached Chinese fir leaves revealed significant pathogenicity amongst these species, of which A. hunanensis is the most pathogenic to Chinese fir. This study represents the first report of A. cunninghamiicola, A. dongshanqiaoensis, A. hunanensis, A. kunyuensis, A. longqiaoensis, A. shandongensis and A. xinyangensis causing leaf blight on Chinese fir. Knowledge obtained in this study enhanced our understanding of Alternaria species causing leaf blight on Chinese fir and was crucial for the disease management and the further studies in the future.

Key words

Alternaria, Cunninghamia lanceolata, diversity, leaf blight, new species, pathogenicity

Introduction

Alternaria is a genus (Pleosporaceae, Pleosporales, Ascomycota) (Seifert et al. 2011), which originally was described in 1816 by Nees (1816), typified with A. tenuis Nees. Since then, more than 900 epithets and varieties/f. spp. have been published in Alternaria (MycoBank 2023). At present, there are over 360 species (Wijayawardene et al. 2020). Alternaria is a ubiquitous fungal genus that includes saprobic, endophytic and pathogenic species (Li et al. 2023). For example, Alternaria species have been recorded as endophytes in grasses, angiosperms, rice and other herbaceous plants and shrubs (Fisher and Petrini 1992; Schulz et al. 1993; Rosa et al. 2009; Polizzotto et al. 2012) and have been also isolated from soil (Hong and Pryor 2004). Many Alternaria species are saprobes on a variety of plant tissues in different habitats (Thomma 2003; Liu et al. 2015b; Wanasinghe et al. 2018). Some Alternaria species, such as A. alternata, produce host-specific toxins (Hyde et al. 2018). Several taxa are also important postharvest pathogens, for example, A. alternata and A. solani (El-Goorani and Sommer 1981; Reddy et al. 2000), or airborne fungal allergens/pathogens-causing upper respiratory tract infections and asthma in humans (Mitakakis et al. 2001; Woudenberg et al. 2015; Hyde et al. 2018). Due to the significant negative health effects of Alternaria on humans and their surroundings, a correct and rapid identification of Alternaria species would be of great significance to researchers, plant pathologists, medical mycologists, other biological professionals and the public alike (Woudenberg et al. 2013).

The taxonomy of Alternaria species especially small-spored species within the alternata species group are particularly challenging because few morphological characters are able to clearly differentiate taxa and these characters are strongly influenced by the environment. Morphological characteristics, such as colour, size, shape of conidia and sporulation patterns have been used for the identification and classification of Alternaria species (Simmons 1992). Wiltshire (1945) divided Alternaria into three major sections, Brevicatenatae, Longicatenatae and Noncatenatae, based on conidial catenation. However, this division is unreliable as some of these characters overlap amongst species and vary depending on the cultural conditions, such as temperature and substrate (Simmons and Roberts 1993). Simmons (1992, 1995) arranged several species groups within Alternaria based on the morphological similarity amongst species. Some other genera, such as Stemphylium (Wallroth, 1833) and Ulocladium (Preuss, 1852) also produce phaeodictyospores and are morphologically similar to Alternaria, and this has further led to taxonomic complications (Bigelow 2003). Simmons (2007) revised Alternaria taxonomy, based on morphology and 275 species were recognised. At the same time, Simmons (2007) proposed three new genera Alternariaster, Chalastospora and Teretispora for some species that were previously described in Alternaria.

However, molecular phylogeny has revealed polyphyletic taxa within Alternaria and Alternaria species clades, which do not always correlate with morphological species-groups (Inderbitzin et al. 2006; Runa et al. 2009; Lawrence et al. 2012). Pryor and Gilbertson (2000) elucidated relationships amongst Alternaria, Stemphylium and Ulocladium based on ITS and SSU sequence data and revealed that Stemphylium species were phylogenetically distinct from Alternaria and Ulocladium species. Most Alternaria and Ulocladium clustered together in a large Alternaria/Ulocladium clade (Pryor and Gilbertson 2000). Chou and Wu (2002) confirmed that filament-beaked Alternaria species constitute a monophyletic group distinct from the other members in this genus and hypothesised that this group is evolutionarily distinct, based on phylogenies of ITS sequence. Two new species groups, A. panax and A. gypsophilae were introduced by Lawrence et al. (2013) with phylogenetic evidence and they accepted eight well supported asexual species-sections within Alternaria, while the taxa with known sexual morphs, the A. infectoria species-groups, were not given the similar rank. Woudenberg et al. (2013) delineated taxa within Alternaria and allied genera, based on SSU, LSU, ITS, GAPDH, RPB2 and TEF1 sequence data. The generic circumscription of Alternaria was emended and 24 internal clades in the Alternaria complex were treated as sections, together with six monotypic lineages (Woudenberg et al. 2013; Gannibal et al. 2022). Woudenberg et al. (2013) also demoted the genera Allewia, Brachycladium, Chalastospora, Chmelia, Crivellia, Embellisia, Lewia, Nimbya, Sinomyces, Teretispora, Ulocladium, Undifilum and Ybotromyces to synonymy with Alternaria. Therefore, the use of DNA sequence data is very important in resolving Alternaria taxonomy.

The DNA-based classification of the genus Alternaria has, so far, relied on over ten gene/region loci, including the nuclear small subunit (SSU) rRNA, large subunit (LSU) rRNA, internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II 2nd largest subunit (RPB2), translation elongation factor 1-α (TEF1), Alternaria major allergen (Alt a1), endopolygalacturonase (endoPG), anonymous gene region (OPA10-2), calmodulin (CAL) and eukaryotic orthologous group (KOG) (Lawrence et al. 2013; Woudenberg et al. 2013; Woudenberg et al. 2015; Ghafri et al. 2019; Jayawardena et al. 2019a, 2019b). Several studies have shown that multilocus phylogenetic identification can classify or segregate Alternaria species. For instance, Li et al. (2023) used sequences of ITS, LSU, TEF1, RPB2, GAPDH and Alt a1 loci and described 18 new species in sect. Alternaria, sect. Infectoriae, sect. Porri and sect. Radicina. Aung et al. (2020) reported the first case of small-spored A. alternata associated with Koerle pear (Pyrus × sinkiangensis T.T. Yu) in Korea, based on a multigene phylogeny of GAPDH, RPB2 and Alt a1 genes. Chen et al. (2018) used the multilocus phylogenetic analyses of ITS, GAPDH and β-tubulin genes/region to characterise A. alternata, a causal agent of black spots of tea plant (Camellia sinensis (L.) Kuntze), in the Chongqing city of China. Kgatle et al. (2018) recently showed that the multi-locus phylogeny of Alt a1, RPB2, GAPDH, TEF1 and ITS genes/region successfully identified A. alternata causing leaf blight on sunflower (Helianthus annuus L.) in South Africa. Lawrence et al. (2015) provided a comprehensive taxonomic treatment of Alternaria with multi-locus phylogeny and accepted 27 sections in Alternaria, but later revised it to 28 accepted sections (Ghafri et al. 2019; Gannibal et al. 2022; Li et al. 2023). Recently, Ghafri et al. (2019) and Gannibal et al. (2022) introduced two new sections (i.e. sects. Helianthiinficiens and Omanenses) of Alternaria and thus, 29 sections were accepted at present (Ghafri et al. 2019; Gannibal et al. 2022; Li et al. 2023).

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) is an important fast-growing timber species in China and its afforestation area and timber volume rank first amongst forest plantations; it plays an important role in forest carbon sequestration, increasing farmers’ income and rural revitalisation (Yan 2020). Average timber volume is estimated at 500–800 m3/ha and in China, Chinese fir contributes 40% of the total commercial timber production (Zheng et al. 2016). However, Chinese fir is often damaged by many diseases and insects (Lan et al. 2015). Previous studies reported that Alternaria sp., Bartalinia cunninghamiicola Tak. Kobay. & J.Z. Zhao, Bipolaris oryzae (Breda de Haan) Shoemaker, Bi. Setariae Shoemaker, Colletotrichum cangyuanense Z.F. Yu, C. fructicola Prihast., L. Cai & K.D. Hyde, C. gloeosporioides (Penz.) Penz. & Sacc., C. karsti You L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai, C. siamense Prihast., L. Cai & K.D. Hyde, Curvularia spicifera (Bainier) Boedijn, Cur. muehlenbeckiae Madrid, K.C. Cunha, Gené, Guarro & Crous, Ceratocystis collisensis F.F. Liu, M.J. Wingf. & S.F. Chen, Diaporthe anhuiensis H. Zhou & C.L. Hou, Dia. citrichinensis F. Huang, K.D. Hyde & Hong Y. Li, Discosia pini Heald, Fusarium oxysporum f. pini (R. Hartig) W.C. Snyder & H.N. Hansen, Fusarium sp., Lophodermium uncinatum Darker, Nigrospora sphaerica (Sacc.) E.W. Mason and Rhizoctonia solani J.G. Kühn have been identified as pathogens on Chinese fir (Anonymous 1976; Kobayashi and Zhao 1987; Wang et al. 1995; Chen 2002; Lan et al. 2015; Liu et al. 2015a; Xu and Liu 2017; Huang et al. 2018; Tian et al. 2019; Zhou and Hou 2019; Cui et al. 2020a, b; He et al. 2022). However, there is a lack of comprehensive study on Alternaria causing leaf blight disease on Chinese fir including diversity, occurrence and pathogenicity of the pathogens.

Surveys of fungal diseases on foliage of Chinese fir in its main cultivation regions in China were conducted from 2016 to 2020, 48 isolates of Alternaria spp. were collected and examined. The main aims of the present study were to determine the Alternaria spp. associated with leaf blight disease on Chinese fir using a polyphasic approach of fungal morphology and phylogenetic analyses, based on multi-locus sequences of ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2.

Materials and methods

Isolation of the potential fungal pathogen

A total of 48 isolates of Alternaria spp. were isolated from leaf blight samples of Chinese fir, which were collected in five provinces (Fujian, Henan, Hunan, Jiangsu and Shandong) in China (Suppl. material: table S1). Small pieces (2 × 3 mm) were cut from the margins of infected tissues and surface sterilised in 75% alcohol for 30 s, then in 1% sodium hypochlorite (NaOCl) for 90 s, followed by three rinses with sterile water (Huang et al. 2016), then blotted dry with sterilised filter paper, placed on 2% potato dextrose agar (PDA) Petri plates with 100 mg/l ampicillin and then cultured for 3 days at 25 °C in the dark. Fungal isolates were purified with the monosporic isolation method described by Li et al. (2007). Single-spore isolates were maintained on PDA plates. The obtained isolates were stored in the Forest Pathology Laboratory of Nanjing Forestry University. Holotype specimens of new species from this study were deposited at the China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing, China.

DNA extraction, PCR amplification and sequencing

Genomic DNA of 48 isolates was extracted using a modified CTAB method (Damm et al. 2008). The fungal plugs of each isolate were grown on the PDA plates for 5 days and then collected in a 2 ml tube. Then, 500 µl of chloroform and 500 µl of hexadecyltrimethyl ammonium bromide (CTAB) extraction buffer (0.2 M Tris, 1.4 M NaCl, 20 mM EDTA, 0.2 g/l CTAB) were added into the tubes, which were placed in a shaker at 25 °C at 200 rpm for 2 h. The mixture was centrifuged at 15,800 × g for 5 min. Three hundred µL of the supernatant was transferred into a new tube and 600 µl of 100% ethanol was added. The suspension was centrifuged at 15,800 × g for 5 min. Then, 600 µl of 70% ethanol was added into the precipitate. The suspension was centrifuged at 15,800 × g for 5 min and the supernatant was discarded. The DNA pellet was dried and resuspended in 30 µl ddH2O.

Whole or partial region/genes of nine loci were amplified. ITS and SSU were amplified with primers ITS1/ITS4 and NS1/NS4 (White et al. 1990), LSU with primers LROR/LR5 (Crous et al. 2009a), GAPDH with primers gpd1/gpd2 (Berbee et al. 1999), RPB2 with primers RPB2-5f2/fRPB2-7cr (Liu et al. 1999; Sung et al. 2007), TEF1 with primers 983F/2218R (Sung et al. 2007), Alt a1 with primers Alt-for/Alt-rev (Hong et al. 2005), endoPG and OPA10-2 with primers PG3/PG2b and OPA10-2L/OPA10-2R (Andrew et al. 2009). The information on primer pairs used are listed in Suppl. material: table S2.

The polymerase chain reaction (PCR) amplification was conducted as described by Woudenberg et al. (2015). PCR was performed in a 30 µl reaction volume containing 2 µl of genomic DNA (ca. 200 ng/µl), 15 µl of 2× Taq Plus Master Mix (Dye Plus) (Vazyme P212-01), 1 µl of 10 μM forward primer, 1 µl of 10 μM reverse primer and 11 µl of ddH2O. The PCR conditions consisted of an initial denaturation step of 4 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C for ITS, GAPDH and endoPG, 35 cycles of 30 s at 94 °C, 30 s at 62 °C and 45 s at 72 °C for OPA10-2 and Alt a1, and 35 cycles of 30 s at 94 °C, 30 s at 59 °C and 60 s at 72 °C for RPB2, TEF1, LSU and SSU, and a final elongation step of 10 min at 72 °C. All DNA sequencing was performed at Shanghai Sangon Biotechnology Company (Nanjing, China). Sequences generated in this study were deposited in GenBank (Table 1).

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

Species name and strain number1,2 Locality, host / substrate GenBank accession numbers3
SSU LSU ITS GAPDH TEF1 RPB2 Alta1 endoPG OPA10-2
Alternaria alternantherae (Outgroup) CBS 124392; HSAUP2798 China, Solanum melongena KC584251 KC584506 KC584179 KC584096 KC584633 KC584374 KP123846 – –
A. alstroemeriae CBS 118808; E.G.S. 50.116R USA, Alstroemeria sp. KP124917 KP124447 KP124296 KP124153 KP125071 KP124764 KP123845 KP123993 KP124601
A. alternata CBS 130254 India, human sputum KP125007 KP124537 KP124383 KP124235 KP125161 KP124853 KP123931 KP124087 KP124696
CBS 130255 India, human sputum KP125008 KP124538 KP124384 KP124236 KP125162 KP124854 KP123932 KP124088 KP124697
CBS 130258 India, human sputum KP125009 KP124539 KP124385 KP124237 KP125163 KP124855 KP123933 KP124089 KP124698
A. angustiovoidea CBS 195.86; E.G.S. 36.172; DAOM 185214T Canada, Euphorbia esula KP124939 KP124469 KP124317 KP124173 KP125093 KP124785 JQ646398 KP124017 KP124624
A. arborescens CBS 102605; E.G.S. 39.128T USA, Solanum lycopersicum KC584509 KC584253 AF347033 AY278810 KC584636 KC584377 AY563303 AY295028 KP124712
CBS 124281 Denmark, Triticum sp. KP125037 KP124567 KP124414 KP124265 KP125192 KP124883 KP123961 KP124118 KP124728
CBS 124282 Denmark, Hordeum vulgare KP125038 KP124568 KP124415 KP124266 KP125193 KP124884 KP123962 KP124119 KP124729
CPC 25266 Austria, Pyrus sp. KP125041 KP124571 KP124418 KP124269 KP125196 KP124887 KP123965 KP124122 KP124732
A. astragali CBS 127672; E.G.S. 52.122T USA, Astragalus bisulcatus KP125006 KP124536 KP124382 KP124234 KP125160 KP124852 KP123930 KP124086 KP124695
A. betae-kenyensis CBS 118810; E.G.S. 49.159; IMI 385709T Kenya, Beta vulgaris var. cicla KP125042 KP124572 KP124419 KP124270 KP125197 KP124888 KP123966 KP124123 KP124733
A. brassicinae CBS 118811; E.G.S. 35.158T USA, Brassica oleracea KP124978 KP124508 KP124356 KP124210 KP125132 KP124824 KP123904 KP124057 KP124667
A. broussonetiae CBS 121455; E.G.S. 50.078T China, Broussonetia papyrifera KP124992 KP124522 KP124368 KP124220 KP125146 KP124838 KP123916 KP124072 KP124681
A. burnsii CBS 108.27 Unknown, Gomphrena globosa KC584601 KC584343 KC584236 KC584162 KC584727 KC584468 KP123850 KP123997 KP124605
CBS 107.38; E.G.S. 06.185T India, Cuminum cyminum KP125043 KP124573 KP124420 JQ646305 KP125198 KP124889 KP123967 KP124124 KP124734
CBS 130264 India, human sputum KP125048 KP124578 KP124425 KP124275 KP125203 KP124894 KP123972 KP124129 KP124739
A. caudata CBS 121544; E.G.S. 38.022R USA, Cucumis sativus KP124995 KP124525 KP124371 KP124223 KP125149 KP124841 KP123919 KP124075 KP124684
A. citri CBS 107.27; ATCC 24463; QM 1736ET USA, Citrus limonium KP124921 KP124451 KP124300 KP124157 KP125075 KP124768 KP123849 KP123996 KP124604
A. cinerariae CBS 612.72; DSM 62012ET Germany, Senecio cineraria KP124930 KP124460 KP124308 KP124165 KP125084 KP124777 KP123861 KP124008 KP124615
A. citrimacularis CBS 102596; E.G.S. 45.090T USA, Citrus jambhiri KP124950 KP124480 KP124328 KP124183 KP125104 KP124796 KP123877 KP124030 KP124637
A. citriarbusti CBS 102598; E.G.S. 46.141T USA, Minneola tangelo KP124951 KP124481 KP124329 KP124184 KP125105 KP124797 KP123878 KP124031 KP124638
A. citricancri CBS 119543; E.G.S. 12.160T USA, Citrus paradisi KP124985 KP124515 KP124363 KP124215 KP125139 KP124831 KP123911 KP124065 KP124674
A. cunninghamiicola DSQ3-2 China, Cunninghamia lanceolata leaf OR229504 OR229647 OR229442 OR252424 OR233910 OR252520 OR252376 OR252472 OR233862
DSQ3-2-1 China, Cu. lanceolata leaf OR229505 OR229648 OR229443 OR252425 OR233911 OR252521 OR252377 OR252473 OR233863
DSQ3-2-2 China, Cu. lanceolata leaf OR229506 OR229649 OR229444 OR252426 OR233912 OR252522 OR252378 OR252474 OR233864
DSQ3-2-3 China, Cu. lanceolata leaf OR229507 OR229650 OR229445 OR252427 OR233913 OR252523 OR252379 OR252475 OR233865
DSQ3-2-4 China, Cu. lanceolata leaf OR229508 OR229651 OR229446 OR252428 OR233914 OR252524 OR252380 OR252476 OR233866
A. daucifolii CBS 118812; E.G.S. 37.050T USA, Daucus carota KC584525 KC584269 KC584193 KC584112 KC584652 KC584393 KP123905 KP124058 KP124668
A. destruens CBS 121454; E.G.S. 46.069T USA, Cuscuta gronovii KP124991 KP124521 – AY278812 KP125145 KP124837 JQ646402 KP124071 KP124680
A. dongshanqiaoensis DSQ2-2 China, Cu. lanceolata leaf OR229495 OR229638 OR229433 OR252415 OR233901 OR252511 OR252367 OR252463 OR233853
DSQ2-2-1 China, Cu. lanceolata leaf OR229496 OR229639 OR229434 OR252416 OR233902 OR252512 OR252368 OR252464 OR233854
DSQ2-2-2 China, Cu. lanceolata leaf OR229497 OR229640 OR229435 OR252417 OR233903 OR252513 OR252369 OR252465 OR233855
DSQ2-2-3 China, Cu. lanceolata leaf OR229498 OR229641 OR229436 OR252418 OR233904 OR252514 OR252370 OR252466 OR233856
HN43-6-1 China, Cu. lanceolata leaf OR229499 OR229642 OR229437 OR252419 OR233905 OR252515 OR252371 OR252467 OR233857
HN43-6-1-1 China, Cu. lanceolata leaf OR229500 OR229643 OR229438 OR252420 OR233906 OR252516 OR252372 OR252468 OR233858
HN43-6-1-2 China, Cu. lanceolata leaf OR229501 OR229644 OR229439 OR252421 OR233907 OR252517 OR252373 OR252469 OR233859
HN43-6-1-3 China, Cu. lanceolata leaf OR229502 OR229645 OR229440 OR252422 OR233908 OR252518 OR252374 OR252470 OR233860
HN43-6-1-4 China, Cu. lanceolata leaf OR229503 OR229646 OR229441 OR252423 OR233909 OR252519 OR252375 OR252471 OR233861
A. dumosa CBS 102604; E.G.S. 45.007T Israel, Minneola tangelo KP124956 KP124486 KP124334 AY562410 KP125110 KP124802 AY563305 KP124035 KP124643
A. eichhorniae CBS 489.92; ATCC 22255; ATCC 46777; IMI 121518T India, Eichhornia crassipes KP125049 KP124579 KC146356 KP124276 KP125204 KP124895 KP123973 KP124130 KP124740
A. gaisen CBS 632.93; E.G.S. 90.512R Japan, Pyrus pyrifolia KC584531 KC584275 KC584197 KC584116 KC584658 KC584399 KP123974 AY295033 KP124742
CBS 118488; E.G.S. 90.391R Japan, Pyrus pyrifolia KP125051 KP124581 KP124427 KP124278 KP125206 KP124897 KP123975 KP124132 KP124743
CPC 25268 Portugal, unknown KP125052 KP124582 KP124428 KP124279 KP125207 KP124898 KP123976 KP124133 KP124744
A. godetiae CBS 117.44; E.G.S. 06.190; VKM F-1870T Denmark, Godetia sp. KP124925 KP124455 KP124303 KP124160 KP125079 KP124772 KP123854 KP124001 KP124609
A. gossypina CBS 104.32T Zimbabwe, Gossypium sp. KP125054 KP124584 KP124430 JQ646312 KP125209 KP124900 JQ646395 KP124135 KP124746
A. grisea CBS 107.36T Indonesia, soil KP125055 KP124585 KP124431 JQ646310 KP125210 KP124901 JQ646393 KP124136 KP124747
A. herbiphorbicola CBS 119408; E.G.S. 40.140T USA, Euphorbia esula KP124984 KP124514 KP124362 JQ646326 KP125138 KP124830 JQ646410 KP124064 KP124673
A. hunanensis HN43-10-2 China, Cu. lanceolata leaf OR229486 OR229629 OR229424 OR252406 OR233892 OR252502 OR252358 OR252454 OR233844
HN43-10-2-1 China, Cu. lanceolata leaf OR229487 OR229630 OR229425 OR252407 OR233893 OR252503 OR252359 OR252455 OR233845
HN43-10-2-2 China, Cu. lanceolata leaf OR229488 OR229631 OR229426 OR252408 OR233894 OR252504 OR252360 OR252456 OR233846
HN43-10-2-3 China, Cu. lanceolata leaf OR229489 OR229632 OR229427 OR252409 OR233895 OR252505 OR252361 OR252457 OR233847
HN43-10-2-4 China, Cu. lanceolata leaf OR229490 OR229633 OR229428 OR252410 OR233896 OR252506 OR252362 OR252458 OR233848
A. interrupta CBS 102603; E.G.S. 45.011T Israel, Minneola tangelo KP124955 KP124485 KP124333 KP124188 KP125109 KP124801 KP123882 KP124034 KP124642
A. iridiaustralis CBS 118486; E.G.S. 43.014T Australia, Iris sp. KP125059 KP124589 KP124435 KP124284 KP125214 KP124905 KP123981 KP124140 KP124751
CBS 118487; E.G.S. 44.147R Australia, Iris sp. KP125060 KP124590 KP124436 KP124285 KP125215 KP124906 KP123982 KP124141 KP124752
A. jacinthicola CBS 133751; MUCL 53159T Mali, Eichhornia crassipes KP125062 KP124592 KP124438 KP124287 KP125217 KP124908 KP123984 KP124143 KP124754
CPC 25267 Unknown, Cucumis melo var. inodorus KP125063 KP124593 KP124439 KP124288 KP125218 KP124909 KP123985 KP124144 KP124755
A. kikuchiana CBS 107.53; DSM 3187; IFO 5778HT Japan, Pyrus pyrifolia KP124927 KP124457 KP124305 KP124162 KP125081 KP124774 KP123858 KP124005 KP124613
A. koreana SPL2-1 Korea, Atractylodes ovata – – LC621613 LC621647 LC621715 LC621681 LC631831 LC631844 LC631857
A. koreana SPL2-4 Korea, Atractylodes ovata – – LC621615 LC621649 LC621717 LC621683 LC631832 LC631845 LC631858
A. kunyuensis XXG21 China, Cu. lanceolata leaf OR229515 OR229658 OR229453 OR252435 OR233921 OR252531 OR252387 OR252483 OR233873
XXG22 China, Cu. lanceolata leaf OR229516 OR229659 OR229454 OR252436 OR233922 OR252532 OR252388 OR252484 OR233874
XXG26-2 China, Cu. lanceolata leaf OR229517 OR229660 OR229455 OR252437 OR233923 OR252533 OR252389 OR252485 OR233875
XXG31 China, Cu. lanceolata leaf OR229518 OR229661 OR229456 OR252438 OR233924 OR252534 OR252390 OR252486 OR233876
XXG30 China, Cu. lanceolata leaf OR229519 OR229662 OR229457 OR252439 OR233925 OR252535 OR252391 OR252487 OR233877
XXG12-2 China, Cu. lanceolata leaf OR229520 OR229663 OR229458 OR252440 OR233926 OR252536 OR252392 OR252488 OR233878
A. lini CBS 106.34; E.G.S. 06.198; DSM 62019; MUCL 10030T Unknown, Linum usitatissimum KP124924 KP124454 Y17071 JQ646308 KP125078 KP124771 KP123853 KP124000 KP124608
A. limoniasperae CBS 102595; E.G.S. 45.100T USA, Citrus jambhiri KC584540 KC584284 FJ266476 AY562411 KC584666 KC584408 AY563306 KP124029 KP124636
A. longipes CBS 113.35 Unknown, Nicotiana tabacum KP125064 KP124594 KP124440 KP124289 KP125219 KP124910 KP123986 KP124145 KP124756
CBS 917.96 USA, Nicotiana tabacum KP125066 KP124596 KP124442 KP124291 – KP124912 KP123988 KP124148 KP124759
A. longqiaoensis HN43-14 China, Cu. lanceolata leaf OR229491 OR229634 OR229429 OR252411 OR233897 OR252507 OR252363 OR252459 OR233849
HN43-14-1 China, Cu. lanceolata leaf OR229492 OR229635 OR229430 OR252412 OR233898 OR252508 OR252364 OR252460 OR233850
HN43-14-2 China, Cu. lanceolata leaf OR229493 OR229636 OR229431 OR252413 OR233899 OR252509 OR252365 OR252461 OR233851
HN43-14-3 China, Cu. lanceolata leaf OR229494 OR229637 OR229432 OR252414 OR233900 OR252510 OR252366 OR252462 OR233852
A. mali CBS 106.24; E.G.S. 38.029; ATCC 13963T USA, Malus sylvestris KP124919 KP124449 KP124298 KP124155 KP125073 KP124766 KP123847 AY295020 JQ800620
A. malvae CBS 447.86 Marocco, Malva sp. KP124940 KP124470 KP124318 JQ646314 KP125094 KP124786 JQ646397 KP124018 KP124625
A. palandui CBS 121336; E.G.S. 37.005; ATCC 11680T USA, Allium sp. KP124987 KP124517 KJ862254 KJ862255 KP125141 KP124833 KJ862259 KP124067 KP124676
A. pellucida CBS 479.90; E.G.S. 29.028T Japan, Citrus unshiu KP124941 KP124471 KP124319 KP124174 KP125095 KP124787 KP123870 KP124019 KP124626
A. perangusta CBS 102602; E.G.S. 44.160T Turkey, Minneola tangelo KP124954 KP124484 KP124332 KP124187 KP125108 KP124800 KP123881 AY295023 KP124641
A. platycodonis CBS 121348; E.G.S. 50.070T China, Platycodon grandiflflorus KP124990 KP124520 KP124367 KP124219 KP125144 KP124836 KP123915 KP124070 KP124679
A. postmessia CBS 119399; E.G.S. 39.189T USA, Minneola tangelo KP124983 KP124513 KP124361 JQ646328 KP125137 KP124829 KP123910 KP124063 KP124672
A. pulvinifungicola CBS 194.86; E.G.S. 04.090; QM 1347T USA, Quercus sp. KP124938 KP124468 KP124316 KP124172 KP125092 KP124784 KP123869 KP124016 KP124623
A. rhadina CBS 595.93T Japan, Pyrus pyrifolia KP124942 KP124472 KP124320 KP124175 KP125096 KP124788 JQ646399 KP124020 KP124627
A. sanguisorbae CBS 121456; E.G.S. 50.080; HSAUP 9600197T China, Sanguisorba offificinalis KP124993 KP124523 KP124369 KP124221 KP125147 KP124839 KP123917 KP124073 KP124682
A. seleniiphila CBS 127671; E.G.S. 52.121T USA, Stanleya pinnata KP125005 KP124535 KP124381 KP124233 KP125159 KP124851 KP123929 KP124085 KP124694
A. septorioides CBS 175.80 Italy, unknown KP124935 KP124465 KP124313 JQ646324 KP125089 KP124781 KP123866 KP124013 KP124620
A. shandongensis SDHG12 China, Cu. lanceolata leaf OR229509 OR229652 OR229447 OR252429 OR233915 OR252525 OR252381 OR252477 OR233867
SDHG12-1 China, Cu. lanceolata leaf OR229510 OR229653 OR229448 OR252430 OR233916 OR252526 OR252382 OR252478 OR233868
SDHG12-2 China, Cu. lanceolata leaf OR229511 OR229654 OR229449 OR252431 OR233917 OR252527 OR252383 OR252479 OR233869
A. shandongensis SDHG12-3 China, Cu. lanceolata leaf OR229512 OR229655 OR229450 OR252432 OR233918 OR252528 OR252384 OR252480 OR233870
SDHG12-4 China, Cu. lanceolata leaf OR229513 OR229656 OR229451 OR252433 OR233919 OR252529 OR252385 OR252481 OR233871
LY15 China, Cu. lanceolata leaf OR229514 OR229657 OR229452 OR252434 OR233920 OR252530 OR252386 OR252482 OR233872
A. soliaegyptiaca CBS 103.33; E.G.S. 35.182; IHEM 3319T Egypt, soil KP124923 KP124453 KP124302 KP124159 KP125077 KP124770 KP123852 KP123999 KP124607
A. tenuis CBS 126910 USA, soil KP125003 KP124533 KP124379 KP124231 KP125157 KP124849 KP123927 KP124083 KP124692
A. tenuissima CBS 620.83; ATCC 15052ET USA, Nicotiana tabacum KP124937 KP124467 KP124315 KP124171 KP125091 KP124783 KP123868 KP124015 KP124622
A. tomato CBS 103.30 Unknown, Solanum lycopersicum KP125069 KP124599 KP124445 KP124294 KP125224 KP124915 KP123991 KP124151 KP124762
CBS 114.35 Unknown, Solanum lycopersicum KP125070 KP124600 KP124446 KP124295 KP125225 KP124916 KP123992 KP124152 KP124763
A. tomaticola CBS 118814; E.G.S. 44.048T USA, Solanum lycopersicum KP124979 KP124509 KP124357 KP124211 KP125133 KP124825 KP123906 KP124059 KP124669
A. toxicogenica CBS 102600; E.G.S. 39.181; ATCC 38963T USA, Citrus reticulata KP124953 KP124483 KP124331 KP124186 KP125107 KP124799 KP123880 KP124033 KP124640
A. turkisafria CBS 102599; E.G.S. 44.166T Turkey, Minneola tangelo KP124952 KP124482 KP124330 KP124185 KP125106 KP124798 KP123879 KP124032 KP124639
A. vaccinii CBS 118818; E.G.S. 31.032T USA, Vaccinium sp. KP124981 KP124511 KP124359 KP124213 KP125135 KP124827 KP123908 KP124061 KP124671
A. xinyangensis ZLS1 China, Cu. lanceolata leaf OR229521 OR229664 OR229459 OR252441 OR233927 OR252537 OR252393 OR252489 OR233879
ZLS1-1 China, Cu. lanceolata leaf OR229522 OR229665 OR229460 OR252442 OR233928 OR252538 OR252394 OR252490 OR233880
ZLS1-2 China, Cu. lanceolata leaf OR229523 OR229666 OR229461 OR252443 OR233929 OR252539 OR252395 OR252491 OR233881
ZLS1-3 China, Cu. lanceolata leaf OR229524 OR229667 OR229462 OR252444 OR233930 OR252540 OR252396 OR252492 OR233882
ZLS1-4 China, Cu. lanceolata leaf OR229525 OR229668 OR229463 OR252445 OR233931 OR252541 OR252397 OR252493 OR233883
XYXY06 China, Cu. lanceolata leaf OR229526 OR229669 OR229464 OR252446 OR233932 OR252542 OR252398 OR252494 OR233884
XYXY8-2 China, Cu. lanceolata leaf OR229527 OR229670 OR229465 OR252447 OR233933 OR252543 OR252399 OR252495 OR233885
XYXY16 China, Cu. lanceolata leaf OR229528 OR229671 OR229466 OR252448 OR233934 OR252544 OR252400 OR252496 OR233886
XYXY15 China, Cu. lanceolata leaf OR229529 OR229672 OR229467 OR252449 OR233935 OR252545 OR252401 OR252497 OR233887
XYXY15-1 China, Cu. lanceolata leaf OR229530 OR229673 OR229468 OR252450 OR233936 OR252546 OR252402 OR252498 OR233888
XYXY15-2 China, Cu. lanceolata leaf OR229531 OR229674 OR229469 OR252451 OR233937 OR252547 OR252403 OR252499 OR233889
XYXY15-3 China, Cu. lanceolata leaf OR229532 OR229675 OR229470 OR252452 OR233938 OR252548 OR252404 OR252500 OR233890
XYXY15-4 China, Cu. lanceolata leaf OR229533 OR229676 OR229471 OR252453 OR233939 OR252549 OR252405 OR252501 OR233891
A. yali-inficiens CBS 121547; E.G.S. 50.048T China, Pyrus bretschneideri KP124996 KP124526 KP124372 KP124224 KP125150 KP124842 KP123920 KP124076 KP124685

1 ATCC: American Type Culture Collection, Manassas, VA, USA; CBS: Culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CPC: Personal collection of P.W. Crous, Utrecht, The Netherlands; DAOM: Canadian Collection of Fungal Cultures, Ottawa, Canada; DSM: German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany; E.G.S.: Personal collection of Dr. E.G. Simmons; HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; HSAUP: Department of Plant Pathology, Shandong Agricultural University, China; IFO: Institute for Fermentation Culture Collection, Osaka, Japan; IHEM: Biomedical Fungi and Yeast Collection of the Belgian Co-ordinated Collections of Micro-organisms (BCCM), Brussels, Belgium; IMI: Culture collection of CABI Europe UK Centre, Egham UK; LCP: Laboratory of Cryptogamy, National Museum of Natural History, Paris, France; MAFF: MAFF Genebank Project, Ministry of Agriculture, Forestry and Fisherie, Tsukuba, Japan; MUCL: (Agro)Industrial Fungi and Yeast Collection of the Belgian Co-ordinated Collections of Micro-organisms (BCCM), Louvain-la-Neuve, Belgium; QM: Quarter Master Culture Collection, Amherst, MA, USA; VKM: All-Russian Collection of Microorganisms, Moscow, Russia. T: ex-type isolate; ET: ex-epitype isolate; HT: ex-holotype isolate; R: representative isolate. 3 Bold accession numbers are generated in other studies; np: no product.

Phylogenetic analyses

The sequences generated in this study were compared against nucleotide sequences in GenBank using BLAST to determine closely-related taxa. Alignments of different loci, including the sequences obtained from this study and the ones downloaded from GenBank, were initially performed with the MAFFT v.7 online server (https://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013) and then manually adjusted in MEGA v. 10 (Kumar et al. 2018). The post-alignment sequences of multiple loci were concatenated in PhyloSuite software (Zhang et al. 2020). Maximum-Likelihood (ML) and Bayesian Inference (BI) were run in PhyloSuite software using IQ-TREE ver. 1.6.8 (Nguyen et al. 2015) and MrBayes v. 3.2.6 (Ronquist et al. 2012), respectively. ModelFinder was used to carry out statistical selection of best-fit models of nucleotide substitution using the corrected Akaike information criterion (AIC) (Kalyaanamoorthy et al. 2017). For ML analyses, the default parameters were used, and bootstrap support (BS) was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included two parallel runs of 2,000,000 generations, with the stop rule option and a sampling frequency set to each 1,000 generations. The 50% majority rule consensus trees and posterior probability (PP) values were calculated after discarding the first 25% of the samples as burn-in. Phylogenetic trees were visualised in FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) (Rambaut 2014).

Phylogenetically-related, but ambiguous species were analysed using the genealogical concordance phylogenetic species recognition (GCPSR) model by performing a pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. (2014). The PHI test was performed in SplitsTree4 (Huson 1998; Huson and Bryant 2006) in order to determine the recombination level within phylogenetically closely-related species using a concatenated multi-locus dataset (ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2). If the pairwise-homoplasy index results were below a 0.05 threshold (Фw < 0.05), it indicates significant recombination present in the dataset. The relationship amongst the closely-related species was visualised by constructing splits graphs.

Morphological study

One representative isolate was randomly selected from each Alternaria species for morphological research according to the method of Simmons (2007). Mycelial plugs (5 mm) of purified cultures were transferred from the growing edge of 5-d-old cultures to the centre of 7-mm-diameter potato carrot agar (PCA) plates (Crous et al. 2009b) in triplicate at 25 °C. Colony diameters were measured from 3 to 6 days to calculate mycelial growth rates (mm/d). Colony colour, size and density were also recorded. The morphology and size of conidial chains were studied and recorded using a Zeiss stereo microscope (SteRo Discovery v.20). The shape, colour and size of conidiophores and conidia were observed using a ZEISS Axio Imager A2m microscope (ZEISS, Germany) with differential interference contrast (DIC) optics. At least 30 measurements per structure were performed using Carl Zeiss Axio Vision software to determine their sizes, unless no or fewer individual structures were produced.

Pathogenicity tests

Seven representative isolates (ZLS1, DSQ2-2, SDHG12, XXG21, HN43-10-2, HN43-14 and DSQ3-2) of Alternaria species were selected for the pathogenicity test on detached leaves of Chinese fir collected from 1-year-old Chinese fir plants on the campus of Nanjing Forestry University, Jiangsu, China.

For in-vitro inoculation, detached leaves were surface-sterilised with 75% ethanol, washed three times with sterile water and air-dried on sterile filter paper. A 10 µl aliquot of conidial suspension (1.0 × 106 conidia/ml) was transferred to a sterile plastic tube (20 × 6 mm), in which a leaf was placed so that the base of the leaf was immersed in the conidial suspension. The control was treated with the same amount of double-distilled water. Leaves in the tubes were then placed in plastic trays (40 × 25 cm), covered with a piece of plastic wrap to maintain relative humidity at 99% and incubated at 25 °C in the dark for 5 days. Each treatment had twelves replicates and the experiment was conducted three times. Symptom development on each detached leaf was evaluated by determining the means of lesion lengths at 5 days post-inoculation (dpi). The data were analysed by analysis of variance (ANOVA) using SPSS v. 18 software. LSD’s range test was used to determine significant differences amongst or between different treatments. Origin v. 8.0 software was used to draw histograms (Li et al. 2020). Pathogens were re-isolated from the resulting lesions and identified as described above.

Results

Phylogenetic analyses

A total of 48 Alternaria isolates from Chinese fir were subjected to multi-locus phylogenetic analyses for Alternaria spp. with concatenated sequences of ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2. The data matrix contained a total of 5460 characters with gaps (Alt a1: 1–453, GAPDH: 454–952, ITS: 953–1462, LSU: 1463–2349, OPA10-2: 2350–3013, endoPG: 3014–3414, RPB2: 3415–4170, SSU: 4171–5167, TEF1: 5168–5460). Alternaria alternantherae Holcomb & Antonop. CBS 124392 was used as the out-group. The Maximum-likelihood (ML) and Bayesian Inference (BI) phylogenetic analyses showed that 48 isolates clustered into seven clades distantly from any known species (Fig. 1). Of these, 13 isolates clustered distantly from any known species with high support (ML-BS/BI-PP = 100/1) and closely related to A. dongshanqiaoensis sp. nov. (this study, DSQ2-2), A. citri (Penz.) Mussat (ex-epitype, CBS 107.27), A. cinerariae Hori & Enjoji (ex-type, CBS 612.72) and A. kikuchiana S. Tanaka (ex-type, CBS 107.53), are herein described as a new taxon, namely A. xinyangensis sp. nov. (Fig. 1). The results showed that nine isolates clustered in a distinct clade with high support (ML-BS/BI-PP = 100/1), which was distinct from all other known species and closely related to A. xinyangensis sp. nov. (this study, ZLS1), A. citri (ex-epitype, CBS 107.27), A. cinerariae (ex-type, CBS 612.72) and A. kikuchiana (ex-type, CBS 107.53), namely A. dongshanqiaoensis sp. nov. (Fig. 1). When applying the GCPSR concept to these isolates, the concatenated sequence dataset of nine-loci (ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2) was subjected to the PHI test and the result showed that no significant recombination was detected amongst these isolates/taxa (Φw = 0.1647) (Fig. 2A). It was a solid support for the proposition that these isolates belonged to six distinct taxa.

Figure 1. 

Phylogenetic relationships of 116 isolates of the Alternaria species complex with related taxa with concatenated sequences of the SSU, LSU, ITS, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2 loci using Bayesian inference (BI) and Maximum-likelihood (ML) methods. Bootstrap support values from ML ≥ 70% and BI posterior values ≥ 0.9 are shown at nodes (ML/BI). Alternaria alternantherae CBS 124392 was the outgroup. * and red font indicates strains of this study. T indicates the ex-type strains, ET indicates the ex-epitype strains, HT indicates the ex-holotype strains.

Figure 2. 

Splitgraphs showing the results of the pairwise homoplasy index (PHI) test of newly described taxa and closely-related species using both LogDet transformation and splits decomposition A the PHI of Alternaria xinyangensis sp. nov. and A. dongshanqiaoensis sp. nov. with their phylogenetically related isolates or species B the PHI of A. shandongensis sp. nov., A. kunyuensis sp. nov., A. hunanensis sp. nov. and A. longqiaoensis sp. nov. with their phylogenetically related isolates or species C the PHI of A. cunninghamiicola sp. nov. with their phylogenetically-related isolates or species. PHI test value (Φw) < 0.05 indicate significant recombination within a dataset. * indicates strains of this study. T indicates the ex-type strains, ET indicates the ex-epitype strains, HT indicates the ex-holotype strains.

The ML/BI phylogenetic analyses also showed that A. shandongensis (six isolates, ML-BS/BI-PP = 98/1), A. kunyuensis (six isolates, ML-BS/BI-PP = 100/1), A. hunanensis (five isolates, ML-BS/BI-PP = 100/1) and A. longqiaoensis (four isolates, ML-BS/BI-PP = 100/1) clustered in four distinct clades, which were distinct from all other known species and closely related to A. vaccinii E.G. Simmons (ex-type, CBS 118818), A. platycodonis Z.Y. Zhang & H. Zhang (ex-type, CBS 121348), A. rhadina E.G. Simmons (ex-type, CBS 595.93), A. citriarbusti E.G. Simmons (ex-type, CBS 102598) and A. tomaticola E.G. Simmons & Chellemi (ex-type, CBS 118814) (Fig. 1). When applying the GCPSR concept to these isolates, the concatenated sequence dataset of nine-loci (ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2) was subjected to the PHI test and showed that no significant recombination was detected amongst these isolates/taxa (Φw = 0.3502) (Fig. 2B). It was a solid support for the proposition that these isolates belonged to nine distinct taxa.

Phylogenetic analyses also showed that the five isolates (DSQ3-2, DSQ3-2-1, DSQ3-2-2, DSQ3-2-3 and DSQ3-2-4) clustered in a distinct clade with high support (ML-BS/BI-PP = 100/0.99), which was distinct from all other known species and a sister clade to the clades of A. broussonetiae T.Y. Zhang, W.Q. Chen & M.X. Gao (ex-type, CBS 121455), A. yali-inficiens R.G. Roberts (ex-type, CBS 121547), A. seleniiphila Wangeline & E.G. Simmons (ex-type, CBS 127671) and A. lini P.K. Dey (ex-type, CBS 106.34), namely A. cunninghamiicola sp. nov. (Fig. 1). When applying the GCPSR concept to these isolates, the concatenated sequence dataset of nine-loci (ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2) was subjected to the PHI test, and the result showed that no significant recombination was detected amongst these isolates/taxa (Φw = 0.2087) (Fig. 2C). It was a solid support for the proposition that these isolates belonged to five distinct taxa.

Taxonomy

Based on morphology and multi-locus sequence data, a total of 48 obtained isolates from Chinese fir were assigned to seven species of Alternaria, which represented seven undescribed taxa and were described below.

Alternaria cunninghamiicola Lin Huang, Jiao He & D.W. Li, sp. nov.

Index Fungorum: IF901036
Fig. 3

Holotype

China, Jiangsu Province, Nanjing City, Dongshanqiao Forest Farm, 31°51'11"N, 118°46'12"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, (holotype: CFCC 59358). Holotype specimen is a living specimen being maintained via lyophilisation at the China Forestry Culture Collection Center (CFCC). Ex-type (DSQ3-2) is maintained at the Forest Pathology Laboratory, Nanjing Forestry University.

Etymology

The specific epithet refers to the genus of the host plant (Cunninghamia lanceolata).

Host/distribution

From C. lanceolata in Dongshanqiao Forest Farm, Nanjing City, Jiangsu Province, China.

Description

Mycelium superficial on the PCA, composed of septate, branched, smooth, thin-walled, pale white to grey hyphae. Conidiophores macronematous, mononematous, solitary, subcylindrical, branched or unbranched, straight or geniculate, thin-walled, 2–10 septate, (18.3–)25.3–68.4(–93.8) × (3.0–)3.3–4.2(–4.8) μm, (mean ± SD = 46.9 ± 21.6 × 3.7 ± 0.5 μm, n = 32), arising mostly at right angles from undifferentiated hyphae, with conspicuous scars after conidia have seceded. Conidiogenous cells apical or subapical, cylindrical, light brown, smooth, (5.2–)7.3–14.0(–18.1) × (2.5–)3.0–4.2(–5.0) μm, (mean ± SD = 10.7 ± 3.3 × 3.6 ± 0.6 μm, n = 45), mono- or polytretic, with conspicuous scars at the loci of sporulating after conidia have seceded. Each conidiogenous locus bears a primary chain of 3–5 conidia with rarely lateral branches or occasionally a sole secondary conidium. Conidia pale brown to brown, shape varied, ovoid or ellipsoid, pyriform or obclavate, usually smooth; conidial bodies (12.2–)18.1–35.4(–51.6) × (7.5–)10.4–15.5(–18.7) μm, (mean ± SD = 26.6 ± 8.6 × 12.9 ± 2.6 μm, n = 53), with 1–5 transverse and 0–2 longitudinal septate. Secondary conidia directly (but rarely) produced by conidia through an inconspicuous apical conidiogenous locus or (commonly) by means of a short apical or lateral secondary conidiophore with 1–2 cells in length. Secondary conidiophores (false beaks) with one or a few conidiogenous loci, (4.5–)5.2–22.5(–32.7) × (2.7–)3.2–4.2(–4.7) μm, (mean ± SD = 13.8 ± 8.7 × 3.7 ± 0.5 μm, n = 31). Beakless conidia mostly with a conical cell at the apex. Chlamydospores not observed.

Culture characteristics

Colonies on PCA incubated at 25 °C in the dark growing at 9.3 ± 0.1 mm/d; aerial hypha cottony, white to pale grey; reverse centre dark green to black; sporulation sparse; diffusible pigment absent.

Additional materials examined

China, Jiangsu Province, Nanjing City, Dongshanqiao Forest Farm, 31°51'11"N, 118°46'12"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, DSQ3-2-1, DSQ3-2-2, DSQ3-2-3, DSQ3-2-4.

Notes

The isolates of A. cunninghamiicola were phylogenetically close to A. broussonetiae (ex-type, CBS 121455), A. yali-inficiens (ex-type, CBS 121547), A. seleniiphila (ex-type, CBS 127671) and A. lini (ex-type, CBS 106.34) (Fig. 2). Between A. cunninghamiicola isolates and A. broussonetiae (ex-type, CBS 121455), there were 1/453 differences in Alt a1, 4/510 in ITS and 1/664 in OPA10-2. Between A. cunninghamiicola isolates and A. yali-inficiens (ex-type, CBS 121547), there were 1/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS and 1/401 in endoPG. Between A. cunninghamiicola isolates and A. seleniiphila (ex-type, CBS 127671), there were 1/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS, 1/401 in endoPG and 6/757 in RPB2. Between A. cunninghamiicola isolates and A. lini (ex-type, CBS 106.34), there were 1/453 differences in Alt a1, 2/499 in GAPDH, 4/510 in ITS, 1/887 in LSU, 1/664 in OPA10-2 and 6/757 in RPB2. The PHI analysis showed that there was no significant recombination between A. cunninghamiicola isolates and its related species (Φw = 0.2087) (Fig. 2C). Distinguishing characteristics of this new species and other related species of Alternaria spp. are shown in Table 2. Morphologically, conidia in chains of the A. cunninghamiicola isolates were less than those of A. broussonetiae CBS 121455 (ex-type) (3–5 vs. 8–15 conidia) (Zhang et al. 1999) and A. yali-inficiens CBS 121547 (ex-type) (3–5 vs. 8–18 conidia) (Roberts 2005). Conidiophores of the A. cunninghamiicola isolates were shorter than those of A. seleniiphila CBS 127671 (ex-type) (25.3–68.4 × 3.3–4.2 μm vs. 80–250 × 4–5 μm) (Wangeline and Reeves 2007). Conidia of the A. cunninghamiicola isolates were shorter and wider than those of A. lini CBS 106.34 (ex-type) (18.1–35.4 × 10.4–15.5 μm vs. 42–60 × 3–7 μm) (Dey 1933). Thus, the phylogenetic and morphological evidence support this fungus being a new species within the Alternaria alternata species complex.

Figure 3. 

Alternaria cunninghamiicola (DSQ3-2) A colony on PCA after 6 days at 25 °C in the dark B sporulation patterns C, D conidiophores and conidiogenous cell E, F conidium. Scale bars: 50 μm (B); 10 μm (C–F).

Table 2.
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Distinguishing characteristics of the new species and similar known species of Alternaria spp. under growth conditionsa.

Species Conidiophores (μm)b Conidiogenous cells (μm)c Chain Size (μm)d Conidia Transverse septa Longitudinal or oblique septa Beak or secondary conidiophores (false beaks) (μm)e Reference
Alternaria broussonetiae (ex-type, CBS 121455) np np 8–15 conidia 25–38 × 9–12 5–6 0–1 beakless secondary conidiophore single hyaline cell 3–4 × 3–5 a well-differentiated up to ca 25–50 × 3–4 Zhang et al. (1999)
A. cinerariae (ex-epitype, CBS 612.72) 25–196 × 6–11 np 2–5(–9) conidia 18–295 × 8–63 1–14 up to 10 80–159 × 5–9 (Nishikawa and Nakashima 2020)
A. citri (ex-epitype, CBS 107.27) np np 3–6 conidia 10–22 × 8–15 (in early stages) 25–40 × 15–25 (Mature) (3–)4–6 one or more np (Pierce 1902)
A. citriarbusti (ex-type, CBS 102598) 200 × 5 np 5–8 conidia 30–60 × 8–12 6–11 0–1 beakless secondary conidiophores single cell 3–5 × 4 elongate but not filiform extension up to 25–35 × 2–3 (Simmons 1999)
A. cunninghamiicola (DSQ3-2) 25.3–68.4 × 3.3–4.2 7.3–14.0 × 3.0–4.1 3–5 conidia 18.1–35.4 × 10.4–15.5 1–6 0–5 beakless secondary conidiophores (false beaks) 5.2–22.5 × 3.2–4.2 this study
A. dongshanqiaoensis (DSQ2-2) 16.4–60.2 × 3.2–4.6 5.2–13.7 × 3.5–4.6 5–9 conidia 21.1–32.9 × 11.4–16.8 1–4 1–4 beakless, secondary conidiophores (false beaks) 2.2–9.4 × 2.8–4.0 this study
A. hunanensis (HN43-10-2) 18.4–41.8 × 3.7–4.7 4.6–9.5 × 3.0–4.5 3–7 conidia; one secondary chain of 1–2 conidia. 16.7–28.8 × 8.2–12.6 1–4 0–2 beakless, secondary conidiophores (false beaks) 2.9–21.7 × 2.8–4.3 this study
A. kikuchiana (ex-holotype, CBS 107.53) np np 6–9 conidia 10–70 × 6–22 1–3 1–10 np (Nishikawa and Nakashima 2019)
A. kunyuensis (XXG21) 21.4–53.5 × 3.3–4.0 5.2–11.1 × 3.2–4.2 3–8 conidia; one secondary chain of 2–4 conidia. 20.5–29.8 × 9.4–13.5 1–5 0–3 beakless, secondary conidiophores (false beaks) 2.9–20.0 × 2.8–3.9 this study
A. lini (ex-type, CBS 106.34) 26–80 × 3–7 np np 42–60 × 3–7 2–7 1–4 beakless (Dey 1933)
A. longqiaoensis 19.6–51.0 × 3.3–4.2 4.3–9.6 × 2.9–4.5 4–8 conidia; 1 to 3 secondary chains of 3–4 16.0–28.2 × 7.0–12.6 1–5 0–2 beakless, secondary conidiophores (false beaks) 3.3–11.6 × 2.9–3.9 this study
A. platycodonis (ex-type, CBS 121348) np np 8–10 conidia 25–45 × 8–12 4–7 0 beaklesssecondary conidiophore single hyaline cell 3–4 × 3–5 well-differentiated up to 20 × 3–4 (Zhang 2003)
A. rhadina (ex-type, CBS 595.93) 60–110 × 3–4 np 9–15 conidia 35–45 × 8–9 (narrow ovoid) 4–7 1 20–45 (tapered beak)
A. seleniiphila (ex-type, CBS 127671) 80–250 × 4–5 np 3–6 conidia 20–40 × 8–12 1–7 0–1 beakless secondary conidiophores (false beaks) 3–30 × 3 (Wangeline and Reeves 2007)
A. shandongensis (SDHG12) 23.6–51.1 × 3.4–4.3 4.8–9.6 × 3.2–4.3 9–13 conidia 20.1–31.2 × 9.3–14.1 2–7 0–3 beakless, secondary conidiophores (false beaks) 2.7–10.3 × 2.3–3.1 this study
A. tenuissima (ex-epitype, CBS 620.83) np np 6–10 conidia 32–45 × 11–13 (only transverse septa) 32–45 × 14–18 (ovoid muriformly septate) np np narrow-taper beak is near 64(–72) (Wiltshire 1933)
A. tomaticola (ex-epitype, CBS 118814) 50–80 × 3–5 np 10–15 conidia 30–40 × 9–12 (larger conidia) 6–7 (larger) 1–2 (larger) beakless secondary conidiophores 15–50 (Simmons 2007)
12–25 × 7–13 (smaller conidia) 1–4 (smaller) 0–1 (smaller)
A. vaccinii (ex-epitype, CBS 118818) 100–200 × 3–4 np 8–10 conidia 15–50 × 7–9 1–8 np beakless secondary conidiophores 65–150 × 3–4 (Simmons 2007)
A. xinyangensis (ZLS1) 15.3–54.9 × 3.7–4.8 5.3–9.6 × 3.3–4.9 2–7 conidia 19.9–31.8 × 8.6–12.9 1–6 1–5 beakless, secondary conidiophores (false beaks) 5.3–16.0 × 2.8–4.1 this study
A. yali-inficiens (ex-type, CBS 121547) 80–120 × 4–5 np 8–18 conidia 20–30 × 10–12 3–4 1–2 np (Roberts 2005)

a New species in this study are printed in bold. bcde Dimensions of conidiophores, Conidiogenous cells, conidia, and beaks (μm, mean ± SD for length × width). np: no product.

Alternaria dongshanqiaoensis Lin Huang, Jiao He & D.W. Li, sp. nov.

Index Fungorum: IF901037
Fig. 4

Holotype

China, Jiangsu Province, Nanjing City, Dongshanqiao Forest Farm, 31°51'11"N, 118°46'12"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, (holotype: CFCC 59353). Holotype specimen is a living specimen being maintained via lyophilisation at the China Forestry Culture Collection Center (CFCC). Ex-type (DSQ2-2) is maintained at the Forest Pathology Laboratory, Nanjing Forestry University.

Etymology

Epithet is after Dongshanqiao Forest Farm, Nanjing City, Jiangsu Province where the type specimen was collected.

Host/distribution

from C. lanceolata in Dongshanqiao Forest Farm, Nanjing City, Jiangsu Province, China.

Description

Mycelium superficial on the PCA, composed of septate, branched, smooth, thin-walled, white to pale brown hyphae. Conidiophores macronematous, mononematous, solitary and relatively short, pale brown, smooth, 1–3 septate, (8.1–)16.4–60.2(–100.5) × (2.4–)3.2–4.6(–5.6) μm, (mean ± SD = 38.3 ± 21.9 × 3.9 ± 0.7 μm, n = 30), arising mostly at right angles from undifferentiated hyphae. Conidiogenous cells apical or subapical, cylindrical, light brown, smooth, (3.8–)5.2–13.7(–20.2) × (2.8–)3.5–4.6(–5.2) μm, (mean ± SD = 9.4 ± 4.2 × 4.0 ± 0.5 μm, n = 36), mono- or di-tretic, with conspicuous scars at the loci of sporulating after conidia have seceded. Each conidiogenous locus bears a primary chain of 5–9 conidia; rarely with lateral branches or occasionally a sole secondary conidium. Conidial bodies brown to dark brown, ellipsoid to obclavate, smooth to verruculose, (16.4–)21.1–32.9(–40.1) × (10.2–)11.4–16.8(–22.2) μm, (mean ± SD = 27.0 ± 5.9 × 14.1 ± 2.7 μm, n = 48), with 1–4 (mostly 3) transverse and 1–4 longitudinal septate. Secondary conidia commonly produced by means of a short apical or lateral secondary conidiophore, but rarely by conidia through an inconspicuous apical conidiogenous locus. Secondary conidiophores (false beaks) at the apical end and median of conidium, short, mostly single-celled, (1.4–)2.2–9.4(–20.0) × (1.9–)2.8–4.0(–5.2) μm, (mean ± SD = 5.8 ± 3.6 × 3.4 ± 0.6 μm, n = 33). Beakless conidia mostly with a conical cell at the apex. Chlamydospores not observed.

Figure 4. 

Alternaria dongshanqiaoensis (DSQ2-2) A colony on PCA after 6 days at 25 °C in the dark B, C sporulation patterns D conidiophore and conidiogenous cell E conidia. Scale bars: 50 μm (B, C); 10 μm (D, E).

Culture characteristics

Colonies on PCA incubated at 25 °C in the dark growing at 7.8 ± 0.2 mm/d; aerial hyphae cottony, greyish-green, with grey margins; reverse centre black, with white margins.

Additional materials examined

China, Jiangsu Province, Nanjing City, Dongshanqiao Forest Farm, 31°51'11"N, 118°46'12"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, DSQ2-2-1, DSQ2-2-2, DSQ2-2-3, DSQ2-2-4; Hunan Province, Yiyang City, Longqiao Town, 28°27'24"N, 112°29'7"E, isolated from leaf spots of C. lanceolata, May 2017, Wen-Li Cui, HN43-6-1, HN43-6-1-1, HN43-6-1-2, HN43-6-1-3, HN43-6-1-4.

Notes

The isolates of A. dongshanqiaoensis were phylogenetically close to A. citri (ex-epitype, CBS 107.27), A. cinerariae (ex-epitype, CBS 612.72), A. kikuchiana (ex-holotype, CBS 107.53) and A. tenuissima (Kunze) Wiltshire (ex-epitype, CBS 620.83) (Fig. 2). Between A. dongshanqiaoensis isolates and A. citri (ex-epitype, CBS 107.27), there were 2/453 differences in Alt a1, 4/510 in ITS, 2/401 in endoPG, 1/757 in RPB2 and 2/996 in SSU. Between A. dongshanqiaoensis isolates and A. cinerariae (ex-epitype, CBS 612.72), there were 2/453 differences in Alt a1, 4/510 in ITS, 2/401 in endoPG, 1/757 in RPB2 and 2/996 in SSU. Between A. dongshanqiaoensis isolates and A. kikuchiana (ex-type, CBS 107.53), there were 2/453 differences in Alt a1, 4/510 in ITS, 8/664 in OPA10-2, 3/401 in endoPG, 2/757 in RPB2 and 2/996 in SSU. Between A. dongshanqiaoensis isolates and A. tenuissima (ex-epitype, CBS 620.83), there were 1/453 differences in Alt a1, 6/510 in ITS, 8/664 in OPA10-2, 3/401 in endoPG, 1/757 in RPB2 and 6/996 in SSU. The PHI analysis showed that there was no significant recombination between A. dongshanqiaoensis isolates and its related species (Φw = 0.1647) (Fig. 2A). Distinguishing characteristics of this new species and other related species of Alternaria spp. are shown in Table 2. Morphologically, conidia in chains of the A. dongshanqiaoensis isolates were more than those of A. citri CBS 107.27 (ex-epitype) (5–9 conidia vs. 3–6 conidia) (Pierce 1902). Conidia of the A. dongshanqiaoensis isolates were significantly different from those of A. cinerariae CBS 612.72 (ex-epitype) (21.1–32.9 × 11.4–16.8 μm vs. 18–295 × 8–63 μm) (Nishikawa and Nakashima 2020). Longitudinal septa of conidia of the A. dongshanqiaoensis isolates were less than those of A. kikuchiana CBS 107.53 (ex-holotype) (1–4 vs. 1–10 longitudinal or oblique septa) (Nishikawa and Nakashima 2019). Conidia of the A. dongshanqiaoensis isolates were different from those of A. tenuissima CBS 620.83 (ex-epitype) (beakless vs. with a narrow-taper beak) (Wiltshire 1933). In conclusion, the phylogenetic and morphological evidence support this fungus as being a new species within the Alternaria alternata species complex.

Alternaria hunanensis Lin Huang, Jiao He & D.W. Li, sp. nov.

Index Fungorum: IF901038
Fig. 5

Holotype

China, Hunan Province, Yiyang City, Longqiao Town, 28°27'24"N, 112°29'7"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, (holotype: CFCC 59356). Holotype specimen is a living specimen being maintained via lyophilisation at the China Forestry Culture Collection Center (CFCC). Ex-type (HN43-10-2) is maintained at the Forest Pathology Laboratory, Nanjing Forestry University.

Etymology

Epithet is after Longqiao Town, Yiyang City, Hunan Province where the type specimen was collected.

Host/distribution

From C. lanceolata in Longqiao Town, Yiyang City, Hunan Province, China.

Description

Mycelium superficial on the PCA medium, composed of septate, branched, smooth, thin-walled, white to light brown hyphae. Conidiophores macronematous, mononematous, solitary, subcylindrical, branched or unbranched, straight or geniculate, (12.7–)18.4–41.8(–65.0) × (2.5–)3.3–4.7(–5.2) μm, (mean ± SD = 30.1 ± 11.7 × 4.0 ± 0.7 μm, n = 45). Each conidiogenous locus bears a primary chain of 3–7 conidia; each chain usually has a secondary chain of 1–2 conidia. Conidiogenous cells apical or subapical, cylindrical, light brown, smooth, (2.9–)4.6–9.5(–13.6) × (1.8–)3.0–4.5(–6.3) μm, (mean ± SD = 7.0 ± 2.5 × 3.8 ± 0.8 μm, n = 46), mono- or polytretic. Newly developed conidia subhyaline or pale greyish, ellipsoidal or subacute, thin-walled, with few or no protuberance. Mature conidia pale brown to brown, ovoid or ellipsoid to long-ellipsoid, pyriform, usually smooth. Conidial bodies (10.0–)16.7–28.8(–39.3) × (5.9–)8.2–12.6(–14.8) μm, (mean ± SD = 22.7 ± 6.0 × 10.4 ± 2.2 μm, n = 49), with 1–4 transverse and 0–2 longitudinal septa. Secondary conidia commonly produced by means of a short apical or lateral secondary conidiophore, but rarely by conidia through an inconspicuous apical conidiogenous locus. Secondary conidiophores (false beaks) at the apical end and median of conidium, short, mostly single-celled, (2.8–)2.9–21.7(–41.7) × (2.5–)2.8–4.3(–6.2) μm, (mean ± SD = 12.3 ± 9.4 × 3.5 ± 0.7 μm, n = 37). Conidial beakless mostly with a conical cell at the apex. Chlamydospores not observed.

Figure 5. 

Alternaria hunanensis (HN43-10-2) A colony on PCA after 6 days at 25 °C in the dark B, C sporulation patterns D, E conidiophores and conidiogenous cells F conidia. Scale bars: 50 μm (B, C); 10 μm (D–F).

Culture characteristics

Colonies on PCA incubated at 25 °C in the dark growing at 7.8 ± 0.1 mm/d; aerial hypha cottony, pale gray to greyish-green, with white to pale grey margins; reverse centre brownish to dark green with pale grey margins; sporulation sparse; diffusible pigment absent.

Additional materials examined

China, Hunan Province, Yiyang City, Longqiao Town, 28°27'24"N, 112°29'7"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, HN43-10-2-1, HN43-10-2-2, HN43-10-2-3, HN43-10-2-4.

Notes

The isolates of A. hunanensis were phylogenetically close to A. longqiaoensis (this study, HN43-14), A. vaccinii (ex-type, CBS 118818), A. platycodonis (ex-type, CBS 121348), A. rhadina E.G. Simmons (ex-type, CBS 595.93), A. citriarbusti (ex-type, CBS 102598) and A. tomaticola (ex-type, CBS 118814) (Fig. 2). Between A. hunanensis isolates and A. longqiaoensis HN43-14, there were 2/453 differences in Alt a1, 3/510 in ITS, 2/401 in endoPG, 2/757 in RPB2 and 18/996 in SSU. Between A. hunanensis isolates and A. vaccinii (ex-type, CBS 118818), there were 4/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS and 3/401 in endoPG. Between A. hunanensis isolates and A. platycodonis (ex-type, CBS 121348), there were 1/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS and 2/401 in endoPG. Between A. hunanensis isolates and A. rhadina (ex-type, CBS 595.93), there were 1/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS and 2/401 in endoPG. Between A. hunanensis isolates and A. citriarbusti (ex-type, CBS 102598), there were 1/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS and 2/401 in endoPG. Between A. hunanensis isolates and A. tomaticola (ex-type, CBS 118814), there were 3/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS and 2/401 in endoPG. The PHI analysis showed that there was no significant recombination between A. hunanensis isolates and its related species (Φw = 0.3502) (Fig. 2B). Distinguishing characteristics of this new species and other morphologically related species of Alternaria spp. are shown in Table 2. Morphologically, sporulation patterns of the A. hunanensis isolates were different from those of A. longqiaoensis HN43-14 (one secondary chain of 1–2 conidia vs. 1–3 further branching chains (secondary, tertiary and quaternary chains) of 3–4 conidia). Conidia in chains of the A. hunanensis isolates were less than those of A. vaccinii CBS 118818 (ex-type) (3–7 vs. 8–10 conidia) (Simmons 2007), A. platycodonis CBS 121348 (ex-type) (3–7 vs. 8–10 conidia) (Zhang 2003), A. rhadina CBS 595.93 (ex-type) (3–7 vs. 9–15 conidia) (Simmons 1993) and A. tomaticola CBS 118814 (ex-type) (3–7 vs. 10–15 conidia) (Simmons 2007). Transverse septa of conidia of the A. hunanensis isolates were less than those of A. citriarbusti CBS 102598 (ex-type) (1–4 vs. 6–11 transverse septa) (Simmons 1999). Thus, the phylogenetic and morphological evidence supports this fungus as being a new species within the Alternaria alternata species complex.

Alternaria kunyuensis Lin Huang, Jiao He & D.W. Li, sp. nov.

Index Fungorum: IF901039
Fig. 6

Holotype

China, Shandong Province, Yantai City, Kunyu Mountain, 37°15'22"N, 121°46'05"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, (holotype: CFCC 59355). Holotype specimen is a living specimen being maintained via lyophilisation at the China Forestry Culture Collection Center (CFCC). Ex-type (XXG21) is maintained at the Forest Pathology Laboratory, Nanjing Forestry University.

Etymology

Epithet is after Kunyu Mountain, Yantai City, Shandong Province where the type specimen was collected.

Host/distribution

From C. lanceolata in Kunyu Mountain, Yantai City, Shandong Province, China.

Description

Mycelium superficial on the PCA medium, composed of septate, branched, smooth, thin-walled, colourless to pale brown hyphae. Conidiophores short to long, straight or geniculate, simple or branched, pale brown, 1–5 septate, with one or several apical conidiogenous loci, (17.0–)21.4–53.5(–79.2) × (3.0–)3.3–4.0(–4.6) μm, (mean ± SD = 37.4 ± 16.0 × 3.6 ± 0.4 μm, n = 33). Each conidiogenous locus bears a primary chain of 3–8 conidia; each chain usually has one secondary chain of 2–4 conidia. Conidiogenous cells apical or subapical, cylindrical, light brown, smooth, (3.6–)5.2–11.1(–14.7) × (2.5–)3.2–4.2(–4.7) μm, (mean ± SD = 8.1 ± 2.9 × 3.7 ± 0.5 μm, n = 37), mono- or polytretic. Conidia ovoid to ellipsoid, pyriform, pale brown to brown, usually smooth; conidial bodies (16.1–)20.5–29.8(–36.3) × (7.7–)9.4–13.5(–15.8) μm, (mean ± SD = 25.1 ± 4.6 × 11.5 ± 2.0 μm, n = 43), 1–5 transverse and 0–3 longitudinal septate, slightly constricted at the median. Some septa darkened. Secondary conidia commonly produced via a short apical or lateral secondary conidiophore, but rarely by conidia through an inconspicuous apical conidiogenous locus. Secondary conidiophores (false beaks) at the apical end and median of conidium, short or long, multicellular or single cell, (2.9–)2.9–20.0(–37.3) × (2.3–)2.8–3.9(–4.6) μm, (mean ± SD = 11.5 ± 8.5 × 3.3 ± 0.6 μm, n = 33). Conidial beakless mostly with a conical cell at the apex. Chlamydospores not observed.

Figure 6. 

Alternaria kunyuensis (XXG21) A colony on PCA after 6 days at 25 °C in the dark B, C sporulation patterns D conidiophores bear conidiogenous cells E secondary conidiophores, conidiogenous cells and conidia F conidium. Scale bars: 50 μm (B); 10 μm (C–F).

Culture characteristics

Colonies on PCA incubated at 25 °C in the dark growing at 7.5 ± 0.2 mm/d; aerial hypha sparse, olive green to dark green; reverse centre grey; sporulation abundant; diffusible pigment absent.

Additional materials examined

China, Shandong Province, Yantai City, Kunyu Mountain, 37°15'22"N, 121°46'05"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, XXG12-2, XXG22, XXG26-2, XXG30, XXG31.

Notes

The isolates of A. kunyuensis were phylogenetically close to A. hunanensis (this study, HN43-10-2), A. longqiaoensis (this study, HN43-14), A. vaccinii (ex-type, CBS 118818), A. platycodonis (ex-type, CBS 121348), A. rhadina (ex-type, CBS 595.93), A. citriarbusti (ex-type, CBS 102598) and A. tomaticola (ex-type, CBS 118814) (Fig. 2). Between A. kunyuensis isolates and A. hunanensis HN43-10-2, there were 2/453 differences in Alt a1, 1/510 in ITS, 1/664 in OPA10-2, 5/401 in endoPG, 4/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. Between A. kunyuensis isolates and A. longqiaoensis HN43-14, there were 3/453 differences in Alt a1, 2/510 in ITS, 1/664 in OPA10-2, 3/401 in endoPG, 6/757 in RPB2, 19/996 in SSU and 3/293 in TEF1. Between A. kunyuensis isolates and A. vaccinii CBS 118818 (ex-type), there were 5/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS, 1/664 in OPA10-2, 4/401 in endoPG, 4/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. Between A. kunyuensis isolates and A. platycodonis CBS 121348 (ex-type), there were 2/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS, 1/664 in OPA10-2, 3/401 in endoPG, 4/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. Between A. kunyuensis isolates and A. rhadina CBS 595.93 (ex-type), there were 2/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS, 1/664 in OPA10-2, 3/401 in endoPG, 4/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. Between A. kunyuensis isolates and A. citriarbusti CBS 102598 (ex-type), there were 2/453 differences in Alt a1, 3/510 in ITS, 1/664 in OPA10-2, 3/401 in endoPG, 4/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. Between A. kunyuensis isolates and A. tomaticola CBS 118814 (ex-type), there were 4/453 differences in Alt a1, 3/510 in ITS, 1/664 in OPA10-2, 3/401 in endoPG, 4/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. The PHI analysis showed that there was no significant recombination between A. kunyuensis isolates and its related species (Φw = 0.3502) (Fig. 2B). Distinguishing characteristics of this new species and other related species of Alternaria spp. are shown in Table 2. Morphologically, sporulation patterns of the A. kunyuensis isolates were different from those of A. hunanensis HN43-10-2 (one secondary chain of 2–4 conidia vs. one secondary chain of 1–2 conidia.) and A. longqiaoensis HN43-14 (one secondary chain of 2–4 conidia vs. 1–3 branching chains of 3–4 conidia). Conidia in chains of the A. kunyuensis isolates were less than those of A. vaccinii CBS 118818 (ex-type) (3–8 conidia vs. 8–10 conidia) (Simmons 2007), A. platycodonis CBS 121348 (ex-type) (3–8 conidia vs. 8–10 conidia) (Zhang 2003) A. rhadina CBS 595.93 (ex-type) (3–8 conidia vs. 9–15 conidia) (Simmons 1993) and A. tomaticola CBS 118814 (ex-type) (3–8 conidia vs. 10–15 conidia) (Simmons 2007). Transverse septa of conidia of the A. kunyuensis isolates were less than those of A. citriarbusti CBS 102598 (ex-type) (1–5 transverse septa vs. 6–11 transverse septa) (Simmons 1999). Thus, the phylogenetic and morphological evidence supports this fungus being as a new species within the Alternaria alternata species complex.

Alternaria longqiaoensis Lin Huang, Jiao He & D.W. Li, sp. nov.

Index Fungorum: IF901040
Fig. 7

Holotype

China, Hunan Province, Yiyang City, Longqiao Town, 28°27'24"N, 112°29'7"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, (holotype: CFCC 59357). Holotype specimen is a living specimen being maintained via lyophilisation at the China Forestry Culture Collection Center (CFCC). Ex-type (HN43-14) is maintained at the Forest Pathology Laboratory, Nanjing Forestry University.

Etymology

Epithet is after Longqiao Town, Yiyang City, Hunan Province where the type specimen was collected.

Host/distribution

from C. lanceolata in Longqiao Town, Yiyang City, Hunan Province, China.

Description

Mycelium superficial on the PCA medium, composed of septate, branched, smooth, thin-walled, pale brown to brown hyphae. Conidiophores macronematous, mononematous, solitary, subcylindrical, unbranched or barely branched, straight or geniculate, 2–4 septa, (4.7–) 19.6–51.0 (–66.3) × (2.9–)3.3–4.2(–4.8) μm, (mean ± SD = 35.3 ± 15.7 × 3.8 ± 0.5 μm, n = 39). Each conidiogenous locus bears a primary chain of 4–8 conidia; each chain usually has 1–3 secondary chains of 3–4 conidia. Conidiogenous cells apical or subapical, cylindrical, light brown, smooth, (2.8–)4.3–9.6(–17.4) × (2.3–)2.9–4.5(–5.8) μm, (mean ± SD = 7.0 ± 2.7 × 3.7 ± 0.8 μm, n = 45), mono- or polytretic. Conidia pale brown to brown, ovoid or ellipsoid to long-ellipsoid, pyriform, smooth or verruculose. Conidial bodies (11.0–)16.0–28.2(–40.2) × (6.1–)7.0–12.6(–20.8) μm, (mean ± SD = 22.1 ± 6.1 × 9.8 ± 2.8 μm, n = 48), with 1–5 transverse and 0–2 longitudinal septate. Secondary conidia commonly produced via a short lateral secondary conidiophore, but rarely by conidia through an inconspicuous apical conidiogenous locus. Apically or laterally formed secondary conidiophores (false beaks) with one or several conidiogenous loci, short, mostly single-celled, (3.5–)3.3–11.6(–19.7) × (2.8–)2.9–3.9(–4.8) μm, (mean ± SD = 7.5 ± 4.2 × 3.4 ± 0.5 μm, n = 33). Conidial beakless mostly with a conical cell at the apex. Chlamydospores not observed.

Figure 7. 

Alternaria longqiaoensis (HN43-14) A colony on PCA after 6 days at 25 °C in the dark B, C sporulation patterns D, E conidiophore and conidiogenous cells F conidium. Scale bars: 50 μm (B, C); 10 μm (D–F).

Culture characteristics

Colonies on PCA incubated at 25 °C in the dark growing at 8.3 ± 0.4 mm/d; aerial hypha cottony, dark green to black, with pale green margins; reverse centre black with pale grey margins; sporulation abundant; diffusible pigment absent.

Additional materials examined

China, Hunan Province, Yiyang City, Longqiao Town, 28°27'24"N, 112°29'7"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, HN43-14-1, HN43-14-2, HN43-14-3.

Notes

The isolates of A. longqiaoensis were phylogenetically close to A. vaccinii (ex-type, CBS 118818), A. platycodonis (ex-type, CBS 121348), A. rhadina (ex-type, CBS 595.93), A. citriarbusti (ex-type, CBS 102598) and A. tomaticola (ex-type, CBS 118814) (Fig. 2). Between A. longqiaoensis isolates and A. vaccinii CBS 118818 (ex-type), there were 4/453 differences in Alt a1, 2/499 in GAPDH, 4/510 in ITS, 1/401 in endoPG, 2/757 in RPB2 and 18/996 in SSU. Between A. longqiaoensis isolates and ex-type of A. platycodonis CBS 121348, there were 1/453 differences in Alt a1, 2/499 in GAPDH, 4/510 in ITS, 2/757 in RPB2 and 18/996 in SSU. Between A. longqiaoensis isolates and A. rhadina CBS 595.93 (ex-type), there were 1/453 differences in Alt a1, 2/499 in GAPDH, 4/510 in ITS, 2/757 in RPB2 and 18/996 in SSU. Between A. longqiaoensis isolates and A. citriarbusti CBS 102598 (ex-type), there were 1/453 differences in Alt a1, 4/510 in ITS, 2/757 in RPB2 and 18/996 in SSU. Between A. longqiaoensis isolates and A. tomaticola CBS 118814 (ex-type), there were 3/453 differences in Alt a1, 4/510 in ITS, 2/757 in RPB2 and 18/996 in SSU. The PHI analysis showed that there was no significant recombination between A. longqiaoensis isolates and its related species (Φw = 0.3502) (Fig. 2B). Distinguishing characteristics of this new species and other morphologically-related species of Alternaria spp. are shown in Table 2. Morphologically, conidia in chains of the A. longqiaoensis isolates were less than those of A. vaccinii CBS 118818 (ex-type) (4–8 conidia vs. 8–10 conidia) (Simmons 2007), A. platycodonis CBS 121348 (ex-type) (4–8 conidia vs. 8–10 conidia) (Zhang 2003) A. rhadina CBS 595.93 (ex-type) (4–8 conidia vs. 9–15 conidia) (Simmons 1993) and A. tomaticola CBS 118814 (ex-type) (4–8 conidia vs. 10–15 conidia) (Simmons 2007). Transverse septa of conidia of the A. longqiaoensis isolates were less than those of A. citriarbusti CBS 102598 (ex-type) (1–5 vs. 6–11 transverse septa) (Simmons 1999). Thus, the phylogenetic and morphological evidence supports this fungus as being a new species within the Alternaria alternata species complex.

Alternaria shandongensis Lin Huang, Jiao He & D.W. Li, sp. nov.

Index Fungorum: IF901041
Fig. 8

Holotype

China, Shandong Province, Yantai City, Penglai District, Hougou village, 37°27'32"N, 120°46'48"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, (holotype: CFCC 59354). Holotype specimen is a living specimen being maintained via lyophilisation at the China Forestry Culture Collection Center (CFCC). Ex-type (SDHG12) is maintained at the Forest Pathology Laboratory, Nanjing Forestry University.

Etymology

Epithet is after Shandong Province where the type specimen was collected.

Host/distribution

From C. lanceolata in Hougou village, Penglai District, Yantai City, Shandong Province, China.

Description

Mycelium superficial on the PCA medium, composed of septate, branched, smooth, thin-walled, pale brown hyphae. Conidiophores solitary, emerging from aerial or creeping hyphae, straight or geniculate, simple or branched, with one or several apical conidiogenous loci, 1–5 septate, variable in length, (16.8–)23.6–51.1(–68.8) × (3.0–)3.4–4.3(–5.0) μm, (mean ± SD = 37.3 ± 13.8 × 3.8 ± 0.4 μm, n = 35). Each conidiogenous locus bears a primary chain of 9–13 conidia; each primary chain usually has 1–3 lateral branches (secondary chains) of 1–2 conidia. Conidiogenous cells apical or subapical, cylindrical, light brown, smooth, (3.9–)4.8–9.6(–17.3) × (2.5–)3.2–4.3(–4.8) μm, (mean ± SD = 7.2 ± 2.4 × 3.7 ± 0.6 μm, n = 46), mono- or polytretic. Conidial bodies ovoid to ellipsoid, brown to dark brown, (14.8–)20.1–31.2(–51.5) × (7.5–)9.3–14.1(–17.0) μm, (mean ± SD = 25.6 ± 5.6 × 11.7 ± 2.4 μm, n = 66), with 2–7 transverse and 0–3 longitudinal septa, mostly smooth to occasionally roughened. Secondary conidia commonly produced via a short lateral secondary conidiophore. Secondary conidiophores (false beaks) at the apical end and median of conidium, short, mostly single-celled, (2.9–)2.7–10.3(–23.5) μm × (2.0–)2.3–3.1(–3.7) μm, (mean ± SD = 6.5 ± 3.9 μm × 2.7 ± 0.4 μm, n = 34). Conidial beakless mostly with a conical cell at the apex. Chlamydospores not observed.

Figure 8. 

Alternaria shandongensis (SDHG12) A colony on PCA after 6 days at 25 °C in the dark B–D sporulation patterns E, F conidiophores and conidiogenous cells G conidia. Scale bars: 50 μm (B, C); 10 μm (D–G).

Culture characteristics

Colonies on PCA incubated at 25 °C in the dark growing at 7.6 ± 0.7 mm/d; aerial hypha sparse, dark green to black; reverse centre grey, sporulation abundant; diffusible pigment absent.

Additional materials examined

China, Shandong Province, Yantai City, Penglai District, Hougou village, 37°27'32"N, 120°46'48"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, SDHG12-1, SDHG12-2, SDHG12-3, SDHG12-4; China, Fujian Province, Longyan City, Lianfeng Town, 25°09'27"N, 117°01'50"E, isolated from leaf spots of C. lanceolata, May 2017, Wen-Li Cui, LY15.

Notes

The isolates of A. shandongensis were phylogenetically close to A. kunyuensis (this study, XXG21), A. hunanensis (this study, HN43-10-2), A. longqiaoensis (this study, HN43-14), A. vaccinii (ex-type, CBS 118818), A. platycodonis (ex-type, CBS 121348), A. rhadina (ex-type, CBS 595.93), A. citriarbusti (ex-type, CBS 102598) and A. tomaticola (ex-type, CBS 118814) (Fig. 2). Between A. shandongensis isolates and A. kunyuensis XXG21, there were 1/453 differences in Alt a1, 2/499 in GAPDH, 1/664 in OPA10-2, 5/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. Between A. shandongensis isolates and A. hunanensis HN43-10-2, there were 1/453 differences in Alt a1, 2/499 in GAPDH, 1/510 in ITS, 5/401 in endoPG and 1/757 in RPB2. Between A. shandongensis isolates and A. longqiaoensis HN43-14, there were 3/453 differences in Alt a1, 2/499 in GAPDH, 2/510 in ITS, 3/401 in endoPG, 1/757 in RPB2 and 18/996 in SSU. Between A. shandongensis isolates and A. vaccinii CBS 118818 (ex-type), there were 5/453 differences in Alt a1, 4/499 in GAPDH, 3/510 in ITS, 4/401 in endoPG and 1/757 in RPB2. Between A. shandongensis isolates and A. platycodonis CBS 121348 (ex-type), there were 2/453 differences in Alt a1, 4/499 in GAPDH, 3/510 in ITS, 3/401 in endoPG and 1/757 in RPB2. Between A. shandongensis isolates and A. rhadina CBS 595.93 (ex-type), there were 2/453 differences in Alt a1, 4/499 in GAPDH, 3/510 in ITS, 3/401 in endoPG and 1/757 in RPB2. Between A. shandongensis isolates and A. citriarbusti CBS 102598 (ex-type), there were 2/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS, 3/401 in endoPG and 1/757 in RPB2. Between A. shandongensis isolates and A. tomaticola CBS 118814 (ex-type), there were 4/453 differences in Alt a1, 2/499 in GAPDH, 3/510 in ITS, 3/401 in endoPG and 1/757 in RPB2. The PHI analysis showed that there was no significant recombination between A. shandongensis isolates and its related species (Φw = 0.3502) (Fig. 2B). Distinguishing characteristics of this new species and their related species of Alternaria are shown in Table 2. Morphologically, conidia in chains of the A. shandongensis isolates were more than those of A. kunyuensis XXG21 (9–13 conidia vs. 6–8 conidia), A. hunanensis HN43-10-2 (9–13 conidia vs. 3–7 conidia), A. longqiaoensis HN43-14 (9–13 conidia vs. 4–8 conidia), A. citriarbusti CBS 102598 (ex-type) (9–13 conidia vs. 5–8 conidia) (Simmons 1999) and A. platycodonis CBS 121348 (ex-type) (9–13 conidia vs. 8–10 conidia) (Zhang 2003). Conidiophores of the A. shandongensis isolates were significantly shorter than those of A. vaccinii CBS 118818 (ex-type) (23.6–51.1 × 3.4–4.3 μm vs. 100–200 × 3–4 μm) (Simmons 2007), A. rhadina CBS 595.93 (ex-type) (23.6–51.1 × 3.4–4.3 μm vs. 60–110 × 3–4 μm) (Simmons 1993), A. citriarbusti CBS 102598 (ex-type) (23.6–51.1 × 3.4–4.3 μm vs. 200 × 5 μm) (Simmons 1999) and A. tomaticola CBS 118814 (ex-type) (23.6–51.1 × 3.4–4.3 μm vs. 50–80 × 3–5 μm) (Simmons 2007). In conclusion, the phylogenetic and morphological evidence supports this fungus as being a new species within the Alternaria alternata species complex.

Alternaria xinyangensis Lin Huang, Jiao He & D.W. Li, sp. nov.

Index Fungorum: IF901042
Fig. 9

Holotype

China, Henan Province, Xinyang City, Zhenlei Mountain, 32°04'51"N, 114°07'23"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, (holotype: CFCC 59352). Holotype specimen is a living specimen being maintained via lyophilisation at the China Forestry Culture Collection Center (CFCC). Ex-type (ZLS1) is maintained at the Forest Pathology Laboratory, Nanjing Forestry University.

Etymology

Epithet is after Xinyang City where the type specimen was collected.

Host/distribution

From C. lanceolata in Zhenlei Mountain, Xinyang City, Henan Province, China.

Description

Mycelium superficial on the PCA, composed of septate, branched, smooth, thin-walled, white to light brown hyphae. Conidiophores macronematous, mononematous, produced laterally or terminally on the hyphae, cylindrical, erect or ascending, simple or branched, geniculate, pale brown to dark brown, smooth, 1–7 septate, (9.4–)15.3–54.9(–80.4) × (2.9–)3.7–4.8(–5.2) μm, (mean ± SD = 35.1 ± 19.8 × 4.2 ± 0.6 μm, n = 40). Conidiogenous cells apical or subapical, cylindrical, brown, smooth, (3.9–)5.3–9.6(–12.9) × (2.4–)3.3–4.9(–5.5) μm, (mean ± SD = 7.5 ± 2.2 × 4.1 ± 0.8 μm, n = 39), mono- or polytretic, with conspicuous scars after conidia have seceded. Each conidiogenous locus bears a primary chain of 2–7 conidia; each primary chain usually has 1–3 branching chains of 1–3 conidia. Newly-developed conidia subhyaline or pale greyish, ellipsoidal or subacute, thin-walled, 1–3 septate, with few or no protuberance. Mature conidia brown to dark chocolate–brown, spheroidal or ellipsoid to long-ellipsoid, with 1–6 transverse septa and 1–5 longitudinal or oblique septa, (13.8–)19.9–31.8(–37.6) × (6.9–)8.6–12.9(–17.5) μm, (mean ± SD = 25.9 ± 6.0 × 10.7 ± 2.1 μm, n = 37) in size. Secondary conidia commonly produced by means of a short apical or lateral secondary conidiophore, but rarely by conidia through an inconspicuous apical conidiogenous locus. In addition, false beaks (secondary conidiophores), unbranched, short, blunted, pale brown, (3.0–)5.3–16.0(–24.4) × (2.4–)2.8–4.1(–5.1) μm, (mean ± SD = 10.6 ± 5.4 × 3.4 ± 0.7 μm, n = 31). Conidial beakless mostly with a conical cell at the apex. Chlamydospores not observed.

Figure 9. 

Alternaria xinyangensis (ZLS1) A colony on PCA after 6 days at 25 °C in the dark B, C sporulation patterns D conidiophores and conidiogenouse cells E conidium. Scale bars: 50 μm (B, C);10 μm (D, E).

Culture characteristics

Colonies on PCA incubated at 25 °C in the dark growing at 7.2 mm/d; aerial hyphae cottony, olive green, with white margins; reverse centre black to greyish; sporulation abundant; diffusible pigment absent.

Additional materials examined

China, Henan Province, Xinyang City, Zhenlei Mountain, 32°04'51"N, 114°07'23"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, ZLS1-1, ZLS1-2, ZLS1-3, ZLS1-4; China, Henan Province, Xinyang City, Xinyang University, 32°08'20"N, 114°02'06"E, isolated from leaf spots of C. lanceolata, May 2017, Wen-Li Cui, XYXY06, XYXY8-2, XYXY15, XYXY15-1, XYXY15-2, XYXY15-3, XYXY15-4, XYXY16.

Notes

The isolates of A. xinyangensis were phylogenetically close to A. dongshanqiaoensis (in this study, DSQ2-2), A. citri (ex-epitype, CBS 107.27), A. cinerariae (ex-epitype, CBS 612.72) and A. kikuchiana (ex-type, CBS 107.53) (Fig. 1). Between A. xinyangensis isolates and A. dongshanqiaoensis DSQ2-2, there were 1/453 differences in Alt a1, 1/510 in ITS, 8/664 in OPA10-2, 1/401 in endoPG, 1/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. Between A. xinyangensis isolates and A. citri (ex-epitype, CBS 107.27), there were 1/453 differences in Alt a1, 3/510 in ITS, 8/664 in OPA10-2, 1/401 in endoPG, 1/996 in SSU and 3/293 in TEF1. Between A. xinyangensis isolates and A. cinerariae (ex-epitype, CBS 612.72), there were 1/453 differences in Alt a1, 3/510 in ITS, 8/664 in OPA10-2, 1/401 in endoPG, 1/996 in SSU and 3/293 in TEF1. Between A. xinyangensis isolates and A. kikuchiana (ex-type, CBS 107.53), there were 3/453 differences in Alt a1, 3/510 in ITS, 2/401 in endoPG, 1/757 in RPB2, 1/996 in SSU and 3/293 in TEF1. The PHI analysis showed that there was no significant recombination between A. xinyangensis isolates and their related species (Φw = 0.1647) (Fig. 2A). Distinguishing characteristics of this new species and other similar species of Alternaria spp. are shown in Table 2. Morphologically, conidial number in chains of the A. xinyangensis isolates were less than those of A. dongshanqiaoensis DSQ2-2 (2–7 conidia vs. 5–9 conidia). Conidia of the A. xinyangensis isolates were smaller than those of A. citri CBS 107.27 (ex-epitype) (19.9–31.8 × 8.6–12.9 μm vs. 25–40 × 15–25 μm) (Pierce 1902). Secondary conidiophores of the A. xinyangensis isolates were significantly shorter than those of A. cinerariae CBS 612.72 (ex-epitype) (5.3–16.0 × 2.8–4.1 μm vs. 80–159 × 5–9 μm) (Nishikawa and Nakashima 2020). Conidia in chains of the A. xinyangensis isolates were less than those of A. kikuchiana CBS 107.53 (ex-type) (2–7 conidia vs. 6–9 conidia) (Nishikawa and Nakashima 2019). In conclusion, the phylogenetic and morphological evidence supports this fungus as being a new species within the Alternaria alternata species complex.

Pathogenicity assays

Pathogenicity was tested on detached Chinese fir leaves in vitro following Koch’s postulates for A. xinyangensis (ZLS1), A. kunyuensis (XXG21), A. cunninghamiicola (DSQ3-2), A. dongshanqiaoensis (DSQ2-2), A. longqiaoensis (HN43-14), A. shandongensis (SDHG12) and A. hunanensis (HN43-10-2). At five days’ post-inoculation, all the tested isolates caused leaf necrosis, with dark brown lesions. The control group remained symptom-less (Fig. 10A). After statistical analysis, these strains showed different levels of virulence. The virulence of A. hunanensis (HN43-10-2) was the strongest in all the Alternaria species studied, and its pathogenicity was significantly higher than those of A. xinyangensis (ZLS1), A. kunyuensis (XXG21) and A. cunninghamiicola (DSQ3-2) (P < 0.05), respectively, while there was no significant difference in pathogenicity amongst A. xinyangensis (ZLS1), A. dongshanqiaoensis (DSQ2-2), A. shandongensis (SDHG12), A. kunyuensis (XXG21), A. longqiaoensis (HN43-14) and A. cunninghamiicola (DSQ3-2) (P ≥ 0.05) (Fig. 10B).

Figure 10. 

Symptoms on detached Chinese fir leaves A inoculated with isolates: A. xinyangensis (ZLS1), A. kunyuensis (XXG21), A. cunninghamiicola (DSQ3-2), A. dongshanqiaoensis (DSQ2-2), A. longqiaoensis (HN43-14), A. shandongensis (SDHG12) and A. hunanensis (HN43-10-2) B lesion length on detached Chinese fir leaves inoculated with A. xinyangensis (ZLS1), A. kunyuensis (XXG21), A. cunninghamiicola (DSQ3-2), A. dongshanqiaoensis (DSQ2-2), A. longqiaoensis (HN43-14), A. shandongensis (SDHG12) and A. hunanensis (HN43-10-2). Error bars represent standard error and different letters indicate significant difference, based on LSD’s range test at P < 0.05 (n = 12). Scale bar: 10 mm (A).

The inoculated fungal isolates were re-isolated from the diseased spots on the inoculated leaves, but no fungus was isolated from the control leaves. Therefore, Koch’s postulates were satisfied and these isolates ZLS1, XXG21, DSQ3-2, DSQ2-2, HN43-14, SDHG12 and HN43-10-2 were determined to be the pathogens of leaf blight on C. lanceolata.

Discussion

This study represents the first reports of leaf blight disease of Chinese fir in China caused by Alternaria spp. Phylogenetic analyses of the combined polylocus data set and morphological study showed that the 48 isolates obtained in this study grouped within Section Alternaria. It is surprising that the diversity of Alternaria species was so abundant in Chinese fir. It includes seven new species: Alternaria cunninghamiicola sp. nov., A. dongshanqiaoensis sp. nov., A. hunanensis sp. nov., A. kunyuensis sp. nov., A. longqiaoensis sp. nov., A. shandongensis sp. nov. and A. xinyangensis sp. nov. The detached leaves of Chinese fir were selected for pathogenicity tests that confirmed the potential virulence. To our knowledge, it is the first comprehensive study on Alternaria species causing leaf blight disease on Chinese fir including diversity and pathogenicity of the pathogens.

Morphology was not the main means of identification, as Alternaria isolates could differ morphologically due to the different cultivating conditions and the overlap in the spore sizes of some species (Rahimloo and Ghosta 2015). Armitage et al. (2015) reported that the morphological characteristics used to delineate species in Alternaria sect. Alternata are phenotypically similar and may vary amongst many morpho-species. These characteristics may be deceptive in the identification of these small-spored Alternaria species and would require stringent identification via phylogenetic studies (Kgatle et al. 2018). In this study, the single-locus phylogenies showed unclear resolution because of the limited number of informative sites per locus. For example, the SSU distinguishes A. longqiaoensis effectively with other species, but there is little resolution to distinguish between other species. The TEF1 gene could be informative for A. xinyangensis, A. shandongensis, and A. kunyuensis but not for A. cunninghamiicola, A. dongshanqiaoensis, A. longqiaoensis and A. hunanensis. In addition, it is also noted that the ITS region is a good phylogenetic marker, which could be informative for these isolates in this study, while LSU gene for distinguishing these isolates has a little effect. Perhaps these loci evolve at various rates and have different effective ways of evolution at several phylogenetic scales. For instance, Lawrence et al. (2013) reported that TEF1 and RPB2 are slow-evolving genes used to resolve early divergences in Alternaria, while Alt a1 is fast-evolving and can be used to infer evolutionary relationships at lower phylogenetic scales (Aung et al. 2020). Combined analyses of all nine loci are, thus, the major approach to identify Alternaria species.

A previous multi-locus phylogenetic study Woudenberg et al. (2013) established the taxonomic conclusions of morpho-species known under A. alternata based on the multi-locus phylogenetic analysis. Subsequently, Woudenberg et al. (2015) used the same analysis to determine the discrete lineages of Alternaria spp. in section Alternaria, which showed a 97–98% genomic similarity, concluding that species, such as A. angustiovoide, A. citri, A. lini, A. mali (CBS 106.24), A. malvae and A. tenuissima (CBS 918.96) did not make discrete groupings, but all are synonymous with A. alternata sensu stricto. Although Woudenberg et al. (2015) assigned 35 morpho-species as synonyms of Alternaria alternata, their affinities are still unclear due to inconsistencies, lack of morphological details and a comparison of single nucleotide polymorphisms. However, further studies, based on combined multi-locus phylogeny, showed that recent A. alternata species may not constitute a monophyletic group in DNA sequence-based phylogenies (Li et al. 2023). Morphological characters and phylogenetic analyses of the nine loci showed all 48 Alternaria isolates clustered in the Sect. Alternata in the phylogenetic tree and divide into seven distinct clusters in the current study. We compared these strains, based on morphology and phylogeny. Interestingly, our phylogenetic analyses show that the morpho-species of A. alternata can be separated into different clades and our novel taxa from Chinese fir are both morphologically and phylogenetically distinct from the A. alternata complex and other species in Alternaria sect. Alternaria. Herein, based on these most recent classifications, these isolates from Chinese fir in this study are, thus, identified as the A. alternata complex including A. cunninghamiicola, A. dongshanqiaoensis, A. hunanensis, A. kunyuensis, A. longqiaoensis, A. shandongensis and A. xinyangensis.

The results of pathogenicity tests indicate that the seven new Alternaria species were pathogenic to Chinese fir. Alternaria hunanensis exhibited the strongest virulence in the Alternaria species from the present study, and A. xinyangensis, A. kunyuensis and A. cunninghamiicola with weaker virulence especially in shoots of Chinese fir. Nevertheless, compared with our previous study, Alternaria species showing weaker virulence than those of Colletotrichum spp. (He et al. 2022) and Fusarium spp. (unpublished) and the results may explain why most of Alternaria species are facultative parasites and their pathogenicities are not too strong. Alternaria spp. may prefer to be saprobes or secondary pathogens growing in senescent, near-dead or dead plant tissues. The diseases caused by these pathogens often attack senescent and diseased leaves before crop maturity or when the growth of the hosts is poor. In addition, according to previous studies, some Alternaria taxa carry out facultative parasitism life cycles mainly depending on the following three aspects: damaging the cell walls of their hosts by mechanical penetration and the degrading enzymes, producing mycotoxins that target the cytoplasmic membrane, mitochondria, chloroplast and influencing the activity of enzymes related metabolisms, and mediating pathogenicity through signal transduction (Thomma 2003; Kang et al. 2013). At present, there are few studies on the pathogenic mechanism of Alternaria species, without revealing the specific process of host infection. Therefore, the thorough study of its pathogenic mechanism is the basis and key to solving the damage from Alternaria.

Until now, over 360 species of Alternaria are reported as plant pathogens and saprobes, resulting in the decline of forest quality and fruit decay during storage and resulting in huge economic losses (Wijayawardene et al. 2020; Li et al. 2023). For example, A. citri caused orange brown spot disease (Peever et al. 2004); A. yali-inficiens caused black spots of Japanese pear (Roberts 2005); A. alternata, A. longipes (Ellis & Everh.) E.W. Mason and A. yali-inficiens caused tobacco brown spots (Wang et al. 2018); A. malicola caused fruit spot on apple in China (Dang et al. 2018); A. yunnanensis Z.Y. Cai, X.Y. Liu, Y.X. Liu & Y.P. Shi caused foliage spots of rubber tree in China (Cai et al. 2019); A. koreana O. Hassan, B.B.N.D. Romain, J.S. Kim & T. Chang caused leaf spots of ovate-leaf Atractylodes in South Korea (Romain et al. 2022) and A. capsicicola Nasehi, Kadir & Abed-Asht. [nom. inval., Art. F.5.1 (Shenzhen)] caused leaf spots of pepper in Malaysia (Nasehi et al. 2014). Surprisingly, A. alternata had been considered as a saprobic fungus and to be nonpathogenic on Chinese cabbage (Brassica rapa L. pekinensis group) (Liu and Ke 1992; Zhang et al. 1998). However, A. alternata had been confirmed to be pathogenic on Chinese cabbage (Shi et al. 2021). In addition, many recent studies reported various diseases caused by Alternaria species. For example, Xiang et al. (2023) reported the black spots caused by A. alternata on persimmon fruit in China. Yan et al. (2023) identified A. tenuissima causing leaf spots on Lonicera caerulea L. in Heilongjiang Province, China. Zhou et al. (2023) characterised A. alstroemeriae E.G. Simmons & C.F. Hill, a causal agent of grey spots on tobacco in China. Dantes et al. (2022) discovered A. cinerariae causing leaf blight on Farfugium japonicum (L.) Kitam. in South Carolina, USA. To our knowledge, however, so far, there is no detailed record that Alternaria spp. have been identified as pathogens on Chinese fir, except Alternaria sp. reported by Anonymous (1976).

In summary, our study provides the first systematic and polyphasic study from morphological, molecular and pathogenicity aspects to study Alternaria spp. associated with Chinese fir and reports seven novel species, A. cunninghamiicola, A. dongshanqiaoensis, A. hunanensis, A. kunyuensis, A. longqiaoensis, A. shandongensis and A. xinyangensis causing leaf blight on Chinese fir. However, more studies are necessary on these new taxa in order to elucidate their host range, specificity, mechanism of infection, and global distribution, as well as their potential impact on the Chinese fir industry.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was supported by the Nature Science Foundation of China (31870631), the National Key R & D Program of China (2017YFD0600102), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX23_1225), Qing Lan Project and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author contributions

LH designed research. WLC collected samples. JH and WLC isolated cultures and performed DNA isolation and PCR amplification. JH conducted the pathogenicity test and morphological analysis, and wrote the original draft. DWL and LH reviewed and edited the draft. All authors read and approved the final manuscript.

Author ORCIDs

Jiao He https://orcid.org/0000-0002-4146-2223

De-Wei Li https://orcid.org/0000-0002-2788-7938

Wen-Li Cui https://orcid.org/0009-0005-7515-7672

Lin Huang https://orcid.org/0000-0001-7536-0914

Data availability

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

References

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Supplementary material

Supplementary material 1 

Supplementary information

Jiao He, De-Wei Li, Wen-Li Cui, Lin Huang

Data type: docx

Explanation note: table S1. Fungal cultures isolated from Chinese fir in this study. table S2. Primers used for PCR amplification and DNA sequences.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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